WO2020163553A1 - Mda-7/il-24 pour le traitement du cancer et procédés de surveillance correspondant - Google Patents

Mda-7/il-24 pour le traitement du cancer et procédés de surveillance correspondant Download PDF

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WO2020163553A1
WO2020163553A1 PCT/US2020/016924 US2020016924W WO2020163553A1 WO 2020163553 A1 WO2020163553 A1 WO 2020163553A1 US 2020016924 W US2020016924 W US 2020016924W WO 2020163553 A1 WO2020163553 A1 WO 2020163553A1
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treatment
treatment level
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Paul B. Fisher
Anjan K. PRADHAN
Praveen BHOOPATHI
Swadesh DAS
Devanand Sarkar
Luni Emdad
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Virginia Commonwealth University
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • NS047463, CA016059, CA097318, CA108520, P01 CAI04177, and CA16059 awarded by the National Institutes of Health, and grant no. WSIXWTI- 14- 1-0409 awarded by the Department of Defense. The government has certain rights in the invention.
  • MDA-7/IL-24 Melanoma differentiation associated gene-7/Interleukin-24 displays broad spectrum anti-cancer activity without harming normal cells or tissues.
  • RNAi-mediated gene regulation is an evolutionary conserved pathway, which regulates gene expression in a sequence-specific manner.
  • microRNAs are noncoding 18-22 nucleotide long RNAs, which either degrade RNA or translationaliy block protein synthesis.
  • RNAs may play a role in evolution and disease development, including cancer.
  • Biogenesis of miRNAs is typically regulated by multiple steps including, DROSHA regulating nuclear generation of primary ' miRNA (pri-), Exportin 5 transferring pri- miRNA to the cytoplasm, and pri -miRNA maturation in the cy toplasm.
  • Conventional miRNA biogenesis consists of nuclear primary miRNA generation to synthesis of cytoplasmic mature miRNA.
  • RISC RNA-induced silencing complex
  • the disclosure provides methods of detecting a DICER level in a cancer patient, wherein the cancer patient has received a MDA-7 treatment, the method including: (i) obtaining a post treatment biological sample from the cancer patient; and (ii) detecting a post-treatment level of DICER in the post-treatment biological sample.
  • the disclosure provides methods of detecting a miRNA level in a cancer patient, wherein the cancer patient has received a MDA-7 treatment, the method including: (i) obtaining a post- treatment biological sample from the cancer patient; and (ii) detecting a post-treatment level of the miRNA in the post-treatment biological sample, wherein the miRNA is miR-674, miR-lOa, miR- 21, miR-501, let ⁇ 7d, miR-107, miR-672, miR ⁇ 34c, miR-34b-3p, miR-292-3p, miR-193, miR-202, miR-293, miR-365, miR-222, miR-339, miR-182, miR-125, or a combination of two or more thereof.
  • the method does not comprise detecting a miR-221 level.
  • the disclosure provides methods of detecting a miRNA level in a cancer patient, wherein the cancer patient has received a MDA-7 treatment, the method including: (i) obtaining a post treatment biological sample from the cancer patient; and (ii) detecting a post-treatment level of the miRNA in the post-treatment biological sample, wherein the miRNA is miR-221, miR-200c, miR- 320, miR-185, miR-17, let ⁇ 7c, miR-210, miR-200a, miR-103, let-7e, or a combination of two or more thereof.
  • the method does not comprise detecting a miR-221 level.
  • the disclosure provides methods of treating cancer in a subject in need thereof wherein the subject has a cancer expressing DICER (or a downstream target thereof, such as a miRNA regulated by DICER), the method including administering to the subject an effective amount of MDA-7.
  • the subject has a cancer expressing DICER (or a downstream target thereof such as a miRNA regulated by DICER) and not expressing MDA-7
  • FIGS. IA-1C MDA-7/IL-24 regulates miR-221.
  • FIG. 1 A MDA-MB-231 cells were infected with either Ad. null or Ad.mda-7. Seventy-two hours after infection miRNA fractions were isolated and real time PCR was done using different taqman probes, i.e., miR-221 or miR- 222. RNU44 was used as an endogenous control.
  • FIG. IB MDA-MB-231 cells were infected with increasing doses of Ad.mda-7 (500 vp, 1000 vp and 2000 vp per cell).
  • FIG. 1C The down regulation of miR-221 by mda-7/IL-24 wns temporal as confirmed by a time point kinetics study MTT assays were done to check the effect of mda ⁇ 7/IL-24 on the proliferation of cells. The level of MDA-7/IL-24 protein was checked by western blotting.
  • FIGS. 2A-2D MDA-7/IL-24 down regulates miR-221 in diverse cancer cell lines.
  • FIG. 2A Different breast cancer cells were infected with Ad. null or Ad.mda-7 (2000 vp/ee!l) for 72 hours. RQ-PCR was performed to check the level of miR-221.
  • FIG. 2B Indicated cells were infected with Ad. null or Ad.mda-7 (2000 vp/cell) for 72 hours. RQ-PCR was performed to check the level of miR-221.
  • FIG. 2C A549 and DU-145 cells were treated with His-MDA-7. RQ-PCR was performed to check the level of miR-221.
  • FIG. 2D A549 cells were transfected with IL-20R2 or IL-22R1 and treated with His-MDA-7. RQ-PCR was performed to check the level of miR-221.
  • FIGS. 3A-3E Over expression of miR-221 can rescue cells from mda-7/IL-24-mediated ceil death.
  • FIG. 3 A MDA-MB-231 cells were transfected with pCDNAS.l (vector) or miR-221 and then treated with Ad null or Ad.mda-7 After 72 hours cells were stained with Annexin-V and then analyzed by flow cytometer.
  • FIG. 3B Cells were treated as in FIG. 3 A and MTT assays were done to check the effect of miR-221 overexpression on mda-7/IL-24-mediated cell growth inhibition.
  • FIG. 3C Cells w ? ere treated as in FIG. 3 A and stained with live dead staining kit 72 hours after treatment.
  • FIG. 3D MDA-MB-231 cells w ⁇ ere stably transfected with pCDNA3.1 (vector) or miR-221. After selection, clones were checked for miR-221 expression.
  • FIG. 3E MDA-MB-231 cells stably overexpressing either pCDNA3. I (vector) or miR-221 were treated with Ad.null or Ad.mda-7. Two thousand cells were plated and after two weeks they were stained with crystal violet. Number of colonies was counted and the data were plotted in the graph.
  • FIGS. 4A-4C mda-7/IL-24 regulates miR-221 expression in a ROS-dependent manner.
  • FIG. 4 A MDA-MB-231 cells were infected with Ad.null or Ad.mda-7 (500 vp/cell) for 72 hours, N-acetyl cysteine pretreatment was for 12 hours. Arsenic trioxide (ATO) was added to cells for 12 hours as indicated RQ-PCR was performed to check the level of miR-221. The level of ROS was measured and presented below.
  • FIG. 4B MDA-MB-231 cells were treated as above in FIG. 4A. Cells were exposed to Pyocyanin for 12 hours as indicated. RQ-PCR was performed to check the level of miR-221.
  • FIG. 4C MDA-MB- 231 cells were treated as above in FIG. 4 A. Hydrogen peroxide was added to cells for 4 hours as indicated. RQ-PCR was performed to determine the level of miR-221 expression. The graphs showm below' represent the amount of ROS produced.
  • FIGS. 5A-5F Beclin-1 is a direct target of miR-221.
  • FIG. 5A MDA-MB-231 cells were transfected with control pCDNA3.1 (vector) or miR-221. Western blotting analysis was performed to show' the expression of Beclin- 1/LC3B/EF 1 a.
  • FIG. 5B MDA-MB-231 cells were transfected with increasing concentrations of miR-221, RNA was isolated 48-hours post-transfection and real time PCR was done to check the level of Beclin-1.
  • FIG. 5C Cells were transfected with a miR- 221 construct and then infected with either Ad. null or Ad.mda-7 virus (2000 vp/celi) for 72 hours.
  • FIG. 5D Reporter gene assays were done in HeLa cells using the 3’UTR Beclin-1 construct; miR-221 over expression significantly decreased the luciferase activity of the wt Beclin- 1 UTR.
  • FIG. 5E MDA-MB-231 cells were transfected with increasing concentrations of anti- miR-221, RNA w'as isolated after 48 hours and real time PCR was done to check the level of Beclin-1.
  • FIG. 5F Cells were transfected/treated with the indicated constructs and after 24 hours of transfection they were serum-starved by growth in serum-free medium for 24 hours. Cells were stained with acridine orange and then analyzed by flow ' cytometry'.
  • FIGS. 6A-6D Intratumoral injections of mda-7/IL-24 induces miR-221 -mediated cell death.
  • FIG. 6 A MDA-MB-231 human breast cancer cells, stably expressing a control pCDNA3.1 vector, miR-221 or miR-221 and Beclin-1, were subcutaneously implanted in both fl anks of nude mice. Left sided tumors were treated with 8 intratumoral injections of Ad.mda-7. Ad. null w'as used as control. A total of 5 mice were studied in each group. Once the control tumors reached maximum allowable limit, tumors were isolated from both flanks. A. Tumor volumes on both flanks were measured and results are presented in a graphical manner.
  • FIG. 6B Graphical representation of the weight of the tumors on both fl anks
  • FIG. 6C RNA was isolated from the tumor sections (injected tumors) and real time PCR was done to validate the level of miR-221.
  • FIG. 6D Immunohi stochemical analysis of MDA-7/IL-24 and Beclin-1 in tumor sections (injected tumors).
  • FIG. 7 Schematic representation of Ad. mda-7-induced cell death in cancer cells. MDA- 7/TL-24 down regulates miR-221 which in turn up regulates Beclin-1 to induce toxic autophagy and cell death in cancer cells. Additionally, the pathways that are regulated by mda-7/IL-24 are depicted here schematically.
  • FIG. 8 MDA-MB-231 cells were infected with either Ad. null or Ad.mda-7. miRNA fractions were isolated 72 hours after infection and real time PCR was done using different taqman probes RNU44 was used as endogenous control .
  • FIG. 9 Cells were infected with either Ad.null or Ad.mda-7. miRNA fractions were isolated 72 hours after infection and real time PCR was done using the miR-221 taqman probe. RNU44 was used as endogenous control
  • FIG. 10 Cells were treated with hydrogen peroxide (100 mM for 4 hours), Arsenic tri oxide (10 m.M for 12 hours) or pyocyanin (100 mM for 12 hours). RQ-PCR was performed to determine the level of miR-221 expression . The graph on right panel represents the amounts of ROS produced.
  • FIG. 11 Cells were transfected with increasing concentrations of miR-221 or anti -miR- 221. Cell lysates were probed against Beclin-1. EFla was used as an endogenous control.
  • FIG. 12 Cells were transfected with an anti -miR-221 construct and then infected with either Ad.null or Ad.mda-7 virus (2000 vp/cell) for 72 hours. Cell lysates were probed with Beclin-1, p27, and PUMA antibodies. EFla was used as an endogenous control.
  • FIG. 13 Cells were transfected with miR-221 construct and then treated with
  • Rapamyein Cell lysates were probed with beclin-1 antibody. EFla was used as an endogenous control.
  • FIG. 14 Quantification of MD A-7/IL-24 and Beclin-1 in immunohistochemistry images (FIG. 6D). This data is graphically represented.
  • FIG. 15 Immunohistochemical analysis of p27 and PUMA in tumor sections.
  • FIG. 16 Schematic representation of MDA-7/IL-24 protein with predicted and established domains and protein modification sites indicated. Cleavage of the 49-amino acid signal peptide allows for secretion of the MDA-7/1L-24 protein.
  • the IL-10 signature sequence is located between amino acid 101 and 121. N-glycosylation can occur at amino acids 85, 99 and 126.
  • Protein kinase C consensus phosphorylation sites are present at amino acids 88, 133 and 161.
  • Casein kinase II (CKII) consensus phosphorylation sites are present at amino acids 101, I l l and 161. Numbers indicate amino acids. Not drawn to scale. ( Figure reproduced from Menezes et ah, 2014).
  • FIG. 17 Schematic representation of the splice isoforms of MDA-7/IL-24. ( Figure reproduced from Whitaker et ah, 2011).
  • FIG. 18 Schematic representation of the pathways regulated by MDA-7/IL-24.
  • MDA- 7/IL-24 regulates both pro and anti-apoptotic molecules to induce tumor specific cell death. This involves a series of signaling events including down regulation of Mcl-1 and Bcl-xL and activation of tumor suppressors i.e. SARI, PUMA, AIF, PERP and others as shown in the figure. Also the cy tokine induces ER stress and regulates a number of genes/proteins to block invasion and metastasis. MDA-7/IL-24 also modulates the immune pathways by deregulating a number of cytokines, which in turn activates the immune system to induce cytotoxic cell death
  • FIG. 19 Model depicting the molecular mechanism of MDA-7/IL-24-mediated autophagy induction.
  • MDA-7/TL-24 regulates autophagy mediated through ER stress and ceramide production.
  • MDA-7/IL-24 down regulates miR-221, which in turn upregulates Beclin-1 to induce toxic autophagy leading to cell death.
  • the transition of protective to toxic autophagy is mediated by the cleavage of ATG5 by Calpain.
  • FIGS. 20A-20D mda-7/IL-24 downregulates mature miR-221, but not pri-miR-221.
  • FIGS. 21A-21B mda-7/IL-24 downregulates DICER protein. Effect of mda-7/IL-24 on DICER, DROSHA, DGCR8 (PASHA), Ago2, Beclin-1, and GRP78 protein levels determined by Western blotting.
  • RWPE-1 and DU-145 (Fig. 21 A), MDA-MB-231 and A549 (Fig. 21B) cells were infected with Ad.null or Ad.mda-7 (2000vp/cell) for 72 hrs. Cells were lysed and Western blotting was done.
  • EFl a was used as a loading control
  • FIGS. 22A-22B DICER overexpression rescues miRs regulated by mda-7/IL-24.
  • DU- 145 (Fig. 22 A) and MDA-MB-231 (Fig. 22B) cells were transfected with DICER overexpressing plasmid and infected with Ad.mda-7.
  • miR-221, mill-320, and mill-451 levels were monitored by RQ-PCR.
  • Treatment with Ad.mda-7 significantly downregulated miR-221 and miR-320, which were rescued in DIC Ell-transfected cells.
  • the level of miR-451 which is a DICER -independent miKNA, does not change with either Ad.mda-7 infection or DICER overexpression.
  • FIGS. 23A-23C ROS-dependent regulation of DICER by mda-7/IL ⁇ 24.
  • RWPE-l and DU-145 (Fig. 23 A), MDA-MB-231 and A549 (Fig. 23B) cells were pretreated with 10 mM N- Acetyl Cysteine (NAC) and infected with either Ad. null or Ad.mda-7 (2000 vp/cell for 72 hrs). Western blotting was done with DICER antibody EFl a was used as a loading control.
  • Fig. 23C Bar graph representation of quantitative ROS levels.
  • FIGS. 24A-24F DICER overexpression rescues cancer cells from mda-7/IL-24-mediated cell death.
  • DU-145 (Fig 24A) and MDA-MB-231 (Fig 24B) cells were transfected with pCDNA3.1 (control) or DICER and then infected with Ad. null or Ad.mda-7.
  • MTT assays showed that treatment with rada-7/IL-24 resulted in an inhibition in proliferation of DU- 145 and MDA- MB-231 cells which is less in DICER overexpressing cells.
  • DICER overexpression also rescues inhibition in colony formation in DU-145 (Fig. 24 A) and MDA-MB-231 (Fig. 24B) cells treated with Ad.mda-7.
  • DU-145 (Fig. 24C) and MDA-MB-231 (Fig. 24D) cells were transfected with pCDNA3.1 (control) or DICER and then infected with Ad. null or Ad.mda-7 (2000 vp/cell). After 72 hrs, cells were lysed and Western blotting was done with PARP, DICER, and MDA-7/IL-24 antibody. Actin was used as a loading control.
  • DU-145 (Fig. 24E) and MDA-MB-231 (Fig. 24F) cells were transfected with pCDNA3.1 (control) or DICER and then treated with Ad. null or Ad.mda-7. After 72 hrs cells were stained with Annexin-V/PI and then analyzed by flow cytometry.
  • FIGS. 25A-25C In vivo xenograft study showing DICER -mediated cell death by mda- 7/IL-24. DU-145 cells, stably expressing a control pCDNA3.1 vector or DICER, were
  • Fig 25 A Graphical representation of the tumor weight on both flanks.
  • Fig. 25B Tumor volumes on both flanks were measured and results are presented in a graphical manner.
  • Fig 25C Tumor volumes on both flanks were measured and results are presented in a graphical manner.
  • FIGS. 26A-26G mda-7/IL-24-mediated DICER regulation is controlled by the transcription factor MITF mda-7/IL ⁇ 24 downregulates MITF in different cancer cell lines (Fig 26A, Fig. 26B), but not in normal RWPE-1 cells (Fig. 26A).
  • DU-145 Fig. 26C
  • MDA-MB- 231 cells Fig. 26D
  • RNA was isolated 72 hrs post-infection and RQ-PCR was done to check the level of DICER.
  • DU-145 (Fig. 26E) and MDA-MB-231 cells Fig.
  • FIG. 26F Schematic representation of regulation of the miRNA processing enzyme DICER by mda-7/IL-24 MDA-7/IL-24 down regulates the transcription factor MITF in a ROS-dependent manner which in turn downregulates DICER.
  • FIG. 27 Quantification of the Western blotting shown in Figure 21.
  • FIG. 28 Cells were infected with either Ad. null or Ad.mda-7. RNA fractions were isolated 72 hrs after infection and real time PCR was done using DICER TaqMan probes. The GAPDH gene was used as an internal loading control.
  • FIGS. 29A-29B Cells were infected with increasing concentrations and for different time periods with Ad.mda-7. Cell lysates were probed with dICER antibody using Western blotting analysis. EFla was used as an internal loading control.
  • Fig. 29 A DU-145; Fig. 29B, MDA-MB-231. Quantification and graphical representation of the Western blotting is as shown.
  • FIGS. 30A-30B Fig 30L Role of IL-20/IL-22 surface receptors in defining DICER response to downregulation by MDA-7/IL-24.
  • A549, DU-145 and A549 ceils transfected with IL- 22R1 or IL-20R2 were treated with His-MDA-7 and RNA was collected.
  • RQ-PCR was performed to check the level of DICER.
  • Fig. 30B Quantification of the Western blotting shown in Figure 23 A and Figure 23B.
  • FIG. 31 Quantification of the Western blotting shown in Figures 24C and 24D.
  • FIG. 32 Temporal tumor volumes during therapeutic treatment are shown graphically.
  • FIG. 33 Relative quantification of the IHC data shown in Figure 25C.
  • FIGS. 34A-34B Quantification of the Western blotting shown in Fig. 26A.
  • Fig. 34B Cells were infected with Ad. null or Ad.mda-7, RNA was isolated 72 hrs post-infection and real time PCR was done to check the level of MITF.
  • FIG. 35 Cells were pretreated with 10 niM N- Acetyl Cysteine (NAC) and infected with either Ad. null or Ad.mda-7 (2000 vp/cell for 72 hrs). Western blotting was done with MITF antibody. Actin was used as an internal loading control. Quantification of the Western blots is represented below
  • FIGS. 36A-36B CRISPR-CAS9 mediated DU-145 mda-7/IL-24 stable knockout (KO) cells were generated. Both wild type and KO cells were treated with recombinant His-MDA-7 protein (10 pg/ml). RQ-PCR was done to check the level of MITF (Fig. 36 A) and mda-7/IL-24 (Fig. 36B).
  • FIG. 37 Quantification of the Western blotting data shown in Figures 26E and 26F.
  • FIG. 38 Reporter gene assays were done in DU-145 and MDA-MB-231 cells using the DICER promoter and MITF overexpression plasmid; treatment with mda-7/IL-24 decreased DICER promoter activity. Overexpression of MITF increased the lueiferase activity, which was again reduced upon mda-7/IL-24 treatment.
  • MDA-7/IL-24 downregulates DICER in a reactive oxygen species (ROS) ⁇ dependent manner. This results in deregulation of miRNAs that are controlled by MDA-7/IL-24. Overexpression of DICER partially rescues MDA ⁇ 7/IL-24 ⁇ mediated cell death in can cer cells. Moreover, MDA-7/IL-24-mediated DICER deregulation occurs through regulation of melanogenesis associated transcription factor (MITF).
  • MIMS reactive oxygen species
  • miRNAs are abundant in different cell types and in diverse organisms including plants, animals, and viruses. They resemble siRNAs and are part of the RNA interference pathway (30) However, they differ in that miRNAs are produced from RNA transcripts that fold on themselves forming hairpins, whereas siRNAs are generated from double-stranded RNAs. miRNAs target about 60% of genes in humans and other mammals (30). They are evolutionary conserved, implying their importance in many biological functions. Many mi RN A genes are located in both exons and introns and are transcribed by RNA Polymerase II (30). Our study reveals a previously unrecognized mechanism of DICER regulation by mda-7 /IL-24 and the melanoeytic transcription factor MITF. This regulation leads to noteworthy changes in downstream miRNAs. We also demonstrate that DICER plays a decisive role in mda ⁇ 7/lL-24 ⁇ mediated cell death that is cancer selective.
  • DICER is ubiquitously expressed in many cell types, its regulation of miRNA biogenesis in diverse and specific cellular and biological contexts requires clarification. DICER expression and its occurrence in cancer varies (31), with no clear correlation between DICER expression and disease stage or occurrence (32). DICER can function both as an oncogene and as a tumor suppressor gene in specific cancer indications. While DICER is upregulated in prostate cancer (33), it is downregulated in breast cancer (34). Also, contradicting studies suggest that DICER protein levels do not always correlate with mature miRNA production (35).
  • Maturation of miRNA involves several steps and three central molecules, DROSHA, DGCR8, and DICER, which play crucial roles in maturation (36).
  • DICER is recognized for its canonical role in the generation and maturation of miRNAs (37). This was traditionally thought to be a cytoplasmic process, however, recent evidences suggest that functional DICER can also localize to the nucleus (38).
  • a DICER-independent pathway was described for the precursor of miR-451. miR-451 bypasses DICER, is loaded into Ago2 directly (39) and uses Ago2 for its maturation instead of DICER (39).
  • mda-7/IL-24 does not regulate miR-451, but regulates other DICER-dependent mxRs emphasizing the specific role of mda-7/IL-24 on DICER -mediated miR regulation.
  • mda-7 /IL-24 is a tumor suppressor gene displaying selective tumor inhibitor ⁇ ' activity in a broad spectrum of cancer cells (12, 40).
  • This IL-10 gene family cytokine targets several anti- apoptotic proteins for suppression, e.g., Bcl-2, Bcl-xL (12), Mcl-1 , and AIF (41), while it activates the tumor suppressors SARI (24, 42), Beclin-1 (21, 23), p27 (21), and PUMA (21).
  • mda-7 /IL-24 also regulates specific microRNAs including miR-221 (21).
  • This immunomodulatory anticancer cytokine inhibits angiogenesis (43), and the secreted MDA-7/IL-24 protein has potent“bystander activity” targeting both local and distant tumors (19, 44)
  • MITF a micropthalmia family oncogenic transcription factor, transcriptionally activates many oncogenes and lysosomal genes (45).
  • the underlying mechanisms of regulation of MITF are not well understood. It is an evolutionary conserved transcription factor with homologues in C. elegans and Drosophila (46). MITF is mainly expressed in melanocytes and retinal pigment epithelial cells, but it is also expressed in other cell types (47). Genomic amplification of MITF is observed in cancer where it can function as an oncogene (48). MITF promotes cellular growth and oncogenesis through regulation of genes involved in survival, proliferation, motility, oxidative stress, and DNA repair (45).
  • mda-7/IL-24 can specifically downregulate MITF transcriptionally and translationally, which are mediated by ROS. Also, this regulation downregulates a target of MITF, DICER, culminating in a decrease in miRNA biogenesis (Fig. 260 ⁇ Wiesen and Tomasi have shown that ROS and HD AC inhibitors regulate DICER protein levels (51). As a result, the HD AC inhibitor, Panobinostat, downregulates DICER activity, which provides therapeutic opportunities in cancer. In these contexts, downregulating DICER can have direct therapeutic applications and may be an additional mechanism by which MDA-7/IL-24 exerts such potent anticancer activity in diverse cancers.
  • the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/ ⁇ 10% of the specified value. In embodiments, about means the specified value.
  • Nucleic acid refers to deoxyribonucieotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • polynucleotide refers to a linear sequence of nucleotides.
  • nucleotide typically refers to a single unit of a polynucleotide, i.e , a monomer. Nucleotides can be ribonucleotides, deoxyribonucieotides, or modified versions thereof.
  • nucleic acid as used herein also refers to nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and/or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid.
  • a nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid.
  • analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide- nucleic acids (PNAs).
  • DICER or“endoiibonuclease DICER” or“helicase with Rnase motif” refers to the RNAase III endonuclease molecule that provides key functions in miRNA processing. “DICER” includes homologs, isoforms, and functional fragments thereof. In embodiments,
  • DICER is substantially identical to the RNA identified by HGNC: 17098 or a variant, homolog, or isoform having substantial identity thereto. In embodiments, DICER is substantially identical to the nucleic acid sequence set forth in RefSeq NM_030621.4, or a variant, homolog, or isoform having substantial identity thereto. In embodiments, the nucleic acid sequence is the sequence known at the time of filing of the present application.
  • DICER has a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over the whole sequence or a portion of the sequence identified by HGNC: 17098 or RefSeq NM 030621.4, a variant, hornolog, or isoform having substantial identity thereto.
  • the term includes any recombinant or naturally-occurring form of DICER or variants, homologs, isoforms thereof that maintain DICER activity (e.g. within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wildtype DICER).
  • the variants, homologs, or isoforms have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring DICER RNA
  • RNA molecules e.g., 20 nucleotides in length
  • miRNAs accomplish these functions via base pairing with complementary sequences within RNA molecules (e.g., mRNA) resulting in cleavage of the bound RNA (e.g., mRNA), destabilization of the mRNA (e.g., mRNA) through shortening of the poly(A) tail, and/or decreasing translation efficiency of the RNA (e.g., mRNA) by ribosomes.
  • A“microRNA” or“miRNA,” is a single-stranded nucleic acid forming part of or derived from a double- stranded nucleic acid which includes complementary portions of substantial or complete identity also referred to as doublestranded hairpin structures. Upon intracellular processing of the hairpin structure the miRNA is released and able to bind its cellular target sequence which it completely or partially complementary' to.
  • a miRNA has the ability to reduce or inhibit expression of a gene or target gene when expressed in the same cell as the gene or target gene.
  • a miRNA refers to a nucleic acid that has substantial or complete identity to a target sequence.
  • the miRNA inhibits gene expression by interacting with a complementary cellular mRNA thereby interfering with the expression of the complementary mRNA.
  • the miRNA is at least about 15-50 nucleotides in length.
  • the length is 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • microRNA shown in Table A and Table B refer to a microRNA (including homologs, isoforms, and functional fragments thereof) with the activity associated with each, e.g., the ability of the miRNA to bind cellular sequences complementary' to that miRNA.
  • the microRNA shown in Table A and Table B are substantially identical to the mircoRNA identified by known nucleic acid sequences or a variant, homolog, or isoform having substantial identity thereto.
  • the nucleic acid sequence is the sequence known at the time of filing of the present application.
  • the microRNA in Table A and Table B have a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over the whole sequence or a portion of the nucleic acid sequence known in the art, a variant, homolog, or isoform having substantial identity thereto.
  • the term includes any recombinant or naturally-occurring form of the microRNA in Table A and Table B or variants, homologs, isoforms thereof that maintain the activity of the micrRNA (e.g. within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wikltype miRNA).
  • the variants, homologs, or isoforms have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to the naturally occurring microRNA.
  • the miRNAs in Table A and/or Table B are human miRNAs.
  • probe or “primer”, as used herein, is defined to be one or more nucleic acid fragments whose specific hybridization to a sample can be detected.
  • a probe or primer can be of any length depending on the particular technique it will be used for.
  • PCR primers e.g., real time PCR
  • nucleic acid probes for, e.g., a Southern blot can be more than a hundred nucleotides in length.
  • the probe may be unlabeled or labeled (e.g., with a deteactable moiety) as described below so that its binding to the target or sample can be detected.
  • the probe can be produced from a source of nucleic acids from one or more particular (preselected) portions of a chromosome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products.
  • PCR polymerase chain reaction
  • the length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure, and to provide the required resolution among different genes or genomic locations.
  • complementarity refers to the ability of a nucleic acid in a polynucleotide to form a base pair (e.g., hybridize) with another nucleic acid in a second polynucleotide.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.
  • a nucleic aicd that is complementary' to a target nucleic acid is capable of hybridizing to the target nucleic acid under stringent hybridation conditions.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T ni ) for the specific sequence at a defined ionic strength pH.
  • T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplar ⁇ -“moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1%
  • the term "gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • the leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene.
  • a “protein gene product” is a protein expressed from a particular gene.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical
  • polypeptide “peptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturaily occurring amino acid polymer.
  • amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5 ! -end). Due to deletions, insertions, truncations, fusions, and the like that may be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting fro the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence.
  • numbered with reference to or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue.
  • a selected residue in a selected protein corresponds to, for example, serine at position 101 of a human MDA-7 protein when the selected residue occupies the same essential spatial or other structural relationship as a serine at position 101 in human MDA-7 protein.
  • a selected protein is aligned for maximum homology with the human MDA-7 protein
  • the position in the aligned selected protein aligning with serine 101 is said to correspond to serine 101.
  • a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the human MDA-101 protein and the overall structures compared. In this case, an amino acid that occupies the same essential position as serine 101 in the structural model is said to correspond to the serine 101 residue.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every' position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations,” which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and allel es of the invention
  • the following eight groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Glycine (G), (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (7) Serine (S), Threonine (T); and (8) Cysteine (C), Methionine (M). (see, e.g., Creighton, Proteins (1984))
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence compari son algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity ' .
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well -known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970 ) Adv. Appl. Math.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W word length
  • E expectation
  • the BLAST algorithm also perfomis a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Set. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immuno!ogical!y cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • MDA-7 refers to a protein (including homologs, isoforms, and functional fragments thereof) with MDA-7 activity.
  • the term includes any recombinant or naturally-occurring form of MDA-7 or variants, homologs, or isoforms thereof that maintain MDA-7 activity (e.g within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wildtype MDA-7).
  • the variants, homologs, or isoforms have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring MDA-7 protein.
  • the MDA-7 protein is substantially identical to the protein identified by Accession No. NP 006841 or a variant or homolog having substantial identity thereto.
  • the MDA-7 protein is substantially identical to the protein identified by UniProt Q 13007 or a variant or homolog having substantial identity thereto.
  • the IL-24 gene is substantially identical to the nucleic acid sequence set forth in RefSeq (mRNA) NM 006850, or a variant or homolog having substantial identity thereto. In embodiments, the IL-24 gene is substantially identical to the nucleic acid sequence set forth in Ensembl reference number ENSG00000162892, or a variant or homolog having substantial identity thereto. In embodiments, the amino acid sequence or nucleic acid sequence is the sequence known at the time of filing of the present application.
  • a "cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA.
  • a cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring.
  • Cells may include prokaryotic and eukaryotic ceils.
  • Prokaryotic cells include but are not limited to bacteria.
  • Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.
  • A“detectable agent” or“detectable moiety” is a composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means.
  • useful detectable agents include l8 F, 3 2 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, &8 Ga, 77 As, 86 Y, 90 Y. 89 Sr, 89 Zr, 94 Tc, 94 Tc, 94 Tc,
  • fluorophore e.g. fluorescent dyes
  • electron-dense reagents enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetie iron oxide (“USPIQ”) nanoparticles, USPIO nanoparticle aggregates, superparamagnetie iron oxide (“ SPIO”) nanoparticles, SPK) nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate (“Gd- chelate”) molecules, Gadolinium, radioisotopes, radionuclides (e.g.
  • microbubbles e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.
  • iodinated contrast agents e.g.
  • a detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.
  • Radioactive substances e.g., radioisotopes
  • Radioactive substances that may be used as imaging and/or labeling agents in accordance with the embodiments of the discl osure include, but are not limi ted to, 18 F, 3 2 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y.
  • Paramagnetic ions that may be used as additional imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g.
  • metals having atomic numbers of 21-29, 42, 43, 44, or 57- 71 include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Hr. Tm, Yb and Lu.
  • expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
  • Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, immunoprecipitation, flow cytometry, immunofluorescence, immunohistochemistry, etc.) and/or nucleic acids (e.g., PCR (e.g., real time/quantitative, reverse transcriptase), in situ hybridization, including FISH; Southern blotting; Northern blotting, etc.).
  • protein e.g., ELISA, Western blotting, immunoprecipitation, flow cytometry, immunofluorescence, immunohistochemistry, etc.
  • nucleic acids e.g., PCR (e.g., real time/quantitative, reverse transcriptase), in situ hybridization, including FISH; Southern blotting; Northern blotting, etc.).
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry' techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.
  • the terms“disease’ or“condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein.
  • the disease may ⁇ be a cancer.
  • the disease may be an autoimmune disease.
  • the disease may be an inflammatory disease.
  • the disease may be an infectious disease.
  • cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non- Hodgkin’s lymphomas (e.g, Burkitt’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.
  • solid and lymphoid cancers including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine,
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas.
  • exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and Non-Hodgkin's Lymphomas
  • Exemplary cancers that may be treated with a compound or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples include, thyroid carcinoma, cholangio
  • adenocarcinoma rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insuianoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pan
  • leukemia refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Exemplar ⁇ ' leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hem oblastic leukemia.
  • hemocytoblastic leukemia histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastie leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.
  • the term“lymphoma” refers to a group of cancers affecting
  • Non-Hodgkin’s lymphomas can be classified based on the rate at which cancer grows and the type of cells involved. There are aggressive (high grade) and indolent (low- grade) types of NHL. Based on the type of cells involved, there are B-cell and T-cell NHLs.
  • Exemplary B-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, small lymphocytic lymphoma, Mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranoda! (MALT) lymphoma, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large cell B-lymphoma, Burkitt’s lymphoma,
  • T-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, cunateous T-ceil lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sar
  • the terra "melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas that may be treated with a compound or method provided herein include, for example, acral -lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullar ⁇ ' thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basal oid carcinoma, basosquamous ceil carcinoma, bronchi oalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, eholangioceliular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma du
  • intraepidermal carcinoma intraepithelial carcinoma, Krompecher's carcinoma, KuJchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, !ymphoepithefial carcinoma, carcinoma raedullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma
  • mucocelluiare mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet- ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma vi!iosum.
  • the terms "metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer.
  • a second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary ' tumor.
  • the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
  • lung cancer metastasizes to the breast the secondary ' tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells.
  • the secondary' tumor in the breast is referred to a. metastatic lung cancer.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary' tumor but not one or more secondary tumors.
  • metastatic lung cancer refers to a disease in a subject with or with a history' of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.
  • autoimmune disease refers to a disease or condition in which a subject’s immune system has an aberrant immune response against a substance that does not normally elicit an immune response in a healthy subject.
  • autoimmune diseases include Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic
  • Autoimmune inner ear disease AIED
  • Autoimmune myocarditis Autoimmune myocarditis
  • Autoimmune oophoritis Autoimmune pancreatitis.
  • Autoimmune retinopathy Autoimmune thrombocytopenic purpura (ATP)
  • Autoimmune thyroid disease Autoimmune urticaria, Axonal or neuronal neuropathies, Bafo disease, Behcet’s disease.
  • Optic neuritis Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Tumer syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardia!
  • infarction syndrome Postpericardiotomy syndrome, Progesterone dermatitis, Primary' biliary cirrhosis, Primary' sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter’s syndrome.
  • Vasculitis, Vesiculobullous dermatosis, Vitiligo, or Wegener’s granulomatosis i.e.,
  • the term“inflammatory' disease” refers to a disease or condition characterized by aberrant inflammation (e.g. an increased level of inflammation compared to a control such as a healthy person not suffering from a disease).
  • inflammatory' diseases include traumatic brain injury', arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SEE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1 , Guillain-Barre syndrome, Hashimoto’s encephalitis,
  • infectious disease refers to a disease or condition that can be caused by organisms such as a bacterium, virus, fungi or any other pathogenic microbial agents.
  • the infectious diseases is a viral infection (e.g., HIV, SARS, HPV, influenza), or bacterial colonization in the human gastrointestinal tract (e.g , pathenogenic bacterial colonization).
  • the infectious disease is associated with elevated expression of mda-9.
  • the infectious di sease is characterized by the presence of virus shedding (e.g., HIV viral shedding or Herpes viral shedding).
  • the infectious disease is a bacterial infection.
  • the infectious disease is a gram -positive bacterial infection. In embodiments, the infectious disease is a Staphylococcus aureus infection. In embodiments, the infectious disease is Gram-positive or Gram-negative bacterial infection. In embodiments, the infectious disease is an infection associated with S aureus, E. facium, E.
  • the infectious disease is a S. aureus, E. facium, E. faecalis, K pneumonoiaea, H. influenzaea, or P. aeruginosa infection.
  • cardiovascular disease refers to a disease or condition wherein blood vessels of the cardiovascular system are blocked, narrowed, or calcified, thereby increasing the likelihood of heart attack, heart failure, angina (chest pain), peripheral artery disease, aneurysm, sudden cardiac arrest, and/or stroke. Cardiovascular disease also includes diseases or conditions affecting heart muscles, valves, or rhythm.
  • the cardiovascular disease is coronary artery disease.
  • the cardiovascular disease is high blood pressure.
  • the cardiovascular disease is cardiac arrest.
  • the cardiovascular disease is congestive heart failure.
  • the cardiovascular disease is arrhythmia.
  • the cardiovascular disease is stroke.
  • the cardiovascular disease is peripheral artery disease.
  • the cardiovascular disease is congenital heart disease. In embodiments, the cardiovascular disease is cardiomyopathy. In embodiments, the cardiovascular disease is premature coronary artery' disease (CAD). In embodiments, the cardiovascular disease is subcli ni cal atheroscl erosi .
  • Patient or “subject in need thereof' refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound, composition, or pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
  • a patient is human.
  • Biological sample refers to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy (e.g., tumor biopsy) and autopsy samples, and frozen sections taken for histological purposes.
  • Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, circulating tumor cells, and the like), lymph, sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc.
  • bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, circulating tumor cells, and the like), lymph, sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells
  • tumor biopsy refers to tumor tissue sample taken by appropriate means, such as via fine needle biopsy, core needle biopsy, excisional or incisional biopsy, endoscopic biopsy, laparscopic biopsy, thorascopic mediastrinoscopic biopsy, laparotomy, thoracotomy, skin biopsy, and sentinel lymph node mapping and biopsy. Any suitable method for obtaining a tissue sample of a tumor may be used in conjunction with the methods as provided herein
  • the term“circulating tumor cell” refers to a cancer cell derived form (e.g. that has detached from) a tumor (e.g. primary tumor).
  • the circulating tumor cell may be circulating in the bloodstream and/or lymphatic system of the subject having the tumor.
  • a biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human, cow; dog; cat, a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
  • a biological sample is obtained from a patient.
  • a biological samples is obtained from a normal (non-disease) individual. In embodiments, the sample is obtained from a human
  • a biological sample is obtained from a subject (patient) prior to administering a treatment to the subject.
  • the term“pre-treatment biological sample” refers to a biological sample taken from a patient prior to the patient receiving a treatment (e.g., AIDA-7 treatment).
  • the pre-treatment sample may be obtained at any time point prior to the patient receiving a treatment (e.g., MDA-7 treatment).
  • the pre-treatment biological sample is obtained 5, 4, 3, 2, or 1 year prior to the patient receiving a treatment.
  • the pre-treatment biological sample is obtained 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month prior to the patient receiving a treatment.
  • the pre-treatment biological sample is obtained 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to the patient receiving a treatment.
  • the pre-treatment biological sample is obtained 23, 22, 21 , 20, 15, 10, 9, 8, 7, 6, 5,
  • the pre-treatment biological sample is obtained 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute prior to the patient receiving a treatment.
  • a biological sample is obtained from a subject (patient) after
  • the term“post-treatment biological sample” refers to a biological sample taken from a patient after the patient has received a treatment (e.g., MDA-7 treatment).
  • the post-treatment sample may be obtained at any time point after the patient has received a treatment.
  • the post-treatment biological sample is taken 5, 4, 3, 2, or 1 year after the patient has received a treatment.
  • the post-treatment biological sample is obtained 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month after the patient has received a treatment.
  • the post-treatment biological sample is obtained 30, 25, 20, 15, 10, 9,
  • the post treatment biological sample is obtained 23, 22, 21, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour after the patient has received a treatment.
  • the post-treatment biological sample is obtained 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, to 1 minute after the patient has received a treatment.
  • pre-treatment refers to the time period prior to a treatment and can refer to, for example, a biological sample, level of protein, level of mRNA, level of microRNA, obtained or detected 5, 4, 3, 2, or 1 year prior to the patient receiving a treatment; 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month prior to the patient receiving a treatment; 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to the patient receiving a treatment; 23, 22, 21, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour prior to the patient receiving a treatment; 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, to 1 minute prior to the patient receiving a treatment.
  • post-treatment refers to the time period after a treatment and can refer to, for example, a biological sample, level of protein, level of mRNA, level of microRNA, obtained or detected 5, 4, 3, 2, or 1 year after the patient has received a treatment; 11, 10, 9, 8, 7,
  • A“control” or "standard control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample.
  • a test sample can be taken from a test condition, e.g, in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control).
  • a test sample can be taken from a patient suspected of having a given disease (e.g., cancer) and compared to samples from a known disease patient (e.g., cancer patient), or a known normal (non-disease, healthy) individual (e.g., standard control).
  • a control can also represent an average value gathered from a number of tests or results.
  • the average value is attained from testing a population of non disease individuals.
  • the average value is attained from testing a population of disease patients.
  • the average value is attained from testing a population of disease patients prior to the patients receiving a treatment.
  • the average value is attained from testing a population of disease patients after the patients have received a treatment.
  • controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects).
  • a control value can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to disease, or prior to treatment (e.g., pre-treatment).
  • controls can be designed for assessment of any number of parameters.
  • One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.
  • A“level of DICER” refers to a level (e.g. an expression level or an activity level) of DICER detected in a biological sample.
  • the DICER level e.g., amount of DICER molecules, or amount of molecules indicative of DICER activity (e.g , an amount of DICER-regulated iniRNA)
  • a disease e.g., cancer, inflammatory disease, infectious disease, autoimmune disease, cardiovascular disease.
  • the level of DICER is detected in a pre-treatment or post-treatment biological sample taken from a patient.
  • the level of DICER is a pre-treatment DICER level or a post-treatment DICER level.
  • the level of DICER is detected in a control sample.
  • the control sample is derived from a healthy individual.
  • the pre treatment or post-treatment level of DICER may be compared to a DICER level in a control sample obtained from, for example, a non-disease individual.
  • the pre-treatment or post-treatment level of DICER may be greater than the level of DICER detected in the control sample obtained from the non-diseased individual.
  • the pre-treatment level of DICER is at least
  • the post-treatment level of DICER is at least 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 13,
  • a standard control may be a level of DICER detected in a non-diseased tissue from the same patient.
  • the pre-treatment level of DICER is detected in a biological sample from diseased tissue from the same patient.
  • the post-treatment level of DICER is detected in a biological sample (e.g., tumor biopsy) from diseased tissue from the same patient.
  • a standard control may be a level of DICER detected in a population of non-diseased individuals. In embodiments, the level of DICER is greater than the mean of the level of DICER detected in the non-disease population. In embodiments, the level of DICER is greater than the median of the level of DICER detected in the non-disease population.
  • a standard control may be a level of DICER detected in a population of patients (e.g., patients suffering from a DICER associated disease).
  • the standard control may be a level of DICER detected in a population of patients suffering from cancer.
  • the level of DICER detected in a biological sample is compared to the mean of the level of DICER detected in the cancer patient population.
  • the level of DICER detected in a biological sample is compared to the median of the level of DICER detected in the cancer patient population.
  • a standard control may be a level of DICER detected in a population of patients suffering from the same disease (e.g., cancer (e.g., lung cancer, prostate cancer, melanoma, neuroblastoma, etc.) as the subject.
  • the level of DICER detected in a biological sample is compared to the mean of the level of DICER detected in the cancer patient population having the same type of cancer as the subject.
  • the level of DICER detected in a biological sample is compared to the median of the level of DICER detected in the cancer patient population having the same type of cancer as the subject.
  • A“level of miRNA in Table A or Table B” refers to a level (e.g. an expression level) of the miRNA shown in Table A and/or Table B (e.g., amount of miRA molecules) detected in a biological sample.
  • one or more of the miRNA from Table A and/or Table B e.g., amount of miRNA molecules
  • is a miRNA level detected in a subject suffering from a disease e.g., cancer, inflammatory disease, infectious disease, autoimmune disease, cardiovascular disease.
  • the level of one or more of the miRNA from Table A and/or Table B is detected in a pre-treatment or post-treatment biological sample taken from a patient.
  • the level of one or more of the miRNA from Table A and/or Table B is a pre treatment miRNA level or a post-treatment miRNA level.
  • the level of one or more of the miRNA from Table A and/or Table B is detected in a control sample.
  • the control sample is derived from a healthy individual.
  • the pre-treatment or post-treatment level of one or more of the miRNA from Table A and/or Table B may be compared to the level of one or more of the miRNA from Table A and/or Table B in a control sample obtained from, for example, a non-disease individual.
  • the pre-treatment or post-treatment level of the one or more of the miRNA from Table A and/or Table B may be greater than the level of one or more of the miRNA from Table A and/or Table B detected in the control sample obtained from the non- diseased individual.
  • the pre-treatment level of one or more of the miRNA from Table A and/or Table B is at least 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,
  • the post-treatment level of one or more of the miRNA from Table A and/or Table B is at least 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3 5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,
  • a standard control may be a level of one or more of the miRNA from Table A and/or Table B detected in a non-diseased tissue from the same patient.
  • the pre treatment level of one or more of the miRNA from Table A and/or Table B is detected in a biological sample from diseased tissue from the same patient.
  • the post-treatment level of one or more of the miRNA from Table A and/or Table B is detected in a biological sample (e.g., tumor biopsy) from diseased tissue from the same patient.
  • a standard control may be a level of one or more of the miRNA from Table A and/or Table B detected in a population of non-diseased individuals.
  • the level of one or more of the miRNA from Table A and/or Table B is greater than the mean of the level of one or more of the miRNA from Table A and/or Table B detected in the non-disease population.
  • the level of one or more of the miRNA from Table A and/or Table B is greater than the median of the level of one or more of the miRNA from Table A and/or Table B detected in the non-disease population
  • a standard control may be a level of one or more of the miRNA from Table A and/or Table B detected in a population of patients (e.g., patients suffering from a miRNA-associated disease).
  • the standard control may be a level of one or more of the miRNA from Table A and/or Table B detected in a population of patients suffering from cancer.
  • the level of one or more of the miRNA from Table A and/or Table B detected in a biological sample is compared to the mean of the level of one or more of the miRNA from Table A and/or Table B detected in the cancer patient population. In embodiments, the level of one or more of the miRNA from Table A and/or Table B detected in a biological sample is compared to the median of the level of one or more of the miRNA from Table A and/or Table B detected in the cancer patient population.
  • a standard control may be a level of one or more of the miRNA from Table A and/or Table B detected in a population of patients suffering from the same disease (e.g., cancer (e.g., lung cancer, prostate cancer, melanoma, neuroblastoma, etc.) as the subject.
  • cancer e.g., lung cancer, prostate cancer, melanoma, neuroblastoma, etc.
  • the level of one or more of the miRNA from Table A and/or Table B detected in a biological sample is compared to the mean of the level of one or more of the miRNA from Table A and/or Table B detected in the cancer patient population having the same type of cancer as the subject.
  • the level of one or more of the miRNA from Table A and/or Table B detected in a biological sample is compared to the median of the level of one or more of the miRNA from Table A and/or Table B detected in the cancer patient population having the same type of cancer as the subject.
  • a cancer“not expressing MDA-7” refers to a cancer which has a reduced expression (e.g. lacks expression) of detectable levels (amounts of protein or RNA) of MDA-7 relative to a standard control.
  • the level of MDA-7 in a sample deemed as not expressing MDA-7 is 0 8, 0 7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 0 09, 0 08, 0 7, 0 06, 05, 0 04, 0 03, 0 02, 0.01,
  • a standard control may be a level of MDA-7 obtained from non-diseased tissue from the patient.
  • the level of MDA-7 detected in a pre-treatment biological sample may be compared to the level of MDA-7 detected in the healthy tissue taken from the same patient.
  • a standard control may be a level of MDA-7 obtained from a population of non-disease individuals.
  • the standard control is the mean or median level of MDA-7 detected in the non-diseased patient population.
  • a disease not expressing MDA-7 has a level less than the mean of the level of MDA-7 detected in the non-disease population.
  • a disease not expressing MDA-7 has a level less than the median of the level of MDA-7 detected in the non-disease population.
  • a standard control may be a level of MDA-7 detected in a population of patients (e.g , patients suffering from a DICER associated disease).
  • the standard control may be a level of MDA-7 detected in a population of patients suffering from cancer.
  • the level of MDA-7 detected in a biological sample is compared to the mean of the level of MDA-7 detected in the cancer patient population .
  • the level of MDA-7 detected in a biological sample is compared to the median of the level of MDA-7 detected in the cancer patient population.
  • a standard control may be a level of MDA-7 detected in a population of patients suffering from the same disease (e.g., cancer (e.g , lung cancer, prostate cancer, melanoma, neuroblastoma, etc.) as the subject.
  • the level of MDA-7 detected in a biological sample is compared to the mean of the level of MDA-7 detected in the cancer patient population having the same type of cancer as the subject.
  • the level of MDA-7 detected in a biological sample is compared to the median of the level of MDA-7 detected in the cancer patient population having the same type of cancer as the subject.
  • the term“associated” or“associated with” in the context of a substance or substance activity, function, or level (e.g., amount of substance) associated with a disease e.g. a DICER protein associated disease, a cancer associated with DICER protein activity, a DICER protein associated cancer, a DICER protein associated disease (e.g., cancer, inflammatory disease, autoimmune disease, infectious disease, or cardiovascular disease)
  • a disease e.g. a DICER protein associated disease, a cancer associated with DICER protein activity, a DICER protein associated cancer, a DICER protein associated disease (e.g., cancer, inflammatory disease, autoimmune disease, infectious disease, or cardiovascular disease)
  • the disease e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease, or cardiovascular disease
  • a symptom of the disease is caused by (in whole or in part) the substance or substance activity, function, or level (e.g., amount of substance).
  • a cancer associated with DICER activity, function, or level may be a cancer that results (entirely or partially) from aberrant DICER function (e.g. enzyme activity, protein-protein interaction, signaling pathway) or a cancer wherein a particular symptom of the disease is caused (entirely or partially) by aberrant DICER activity, function, or level (e.g., amount, or amount of one or more downstream targets of DICER).
  • aberrant DICER function e.g. enzyme activity, protein-protein interaction, signaling pathway
  • aberrant DICER activity, function, or level e.g., amount, or amount of one or more downstream targets of DICER
  • a cancer associated with DICER activity, function, or level e.g., amount of substance
  • a DICER associated disease e.g., cancer, inflammatory disease, autoimmune disease, cardiovascular disease, or infectious disease
  • MDA-7 MDA-7
  • aberrant DICER activity, function (e.g. signaling pathway activity), or level e.g., amount of substance
  • causes the disease e.g , cancer, inflammatory disease, autoimmune disease, or infectious disease
  • aberrant refers to different from normal.
  • activity, function, or level e.g., amount of substance
  • Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease- associated amount (e.g. by administering a compound or using a method as described herein), results in reduction of the disease or one or more disease symptoms.
  • Aberrant activity may refer to a level of a substance (e.g., amount of substance) that results in a disease, wherein returning the aberrant level to a normal or non-disease-associated level (e.g. by administering a compound or using a method as described herein), results in reduction of the disease or one or more disease symptoms.
  • DICER level is upreguiated in the DICER associated disease.
  • DICER level is downregulated in the DICER associated disease.
  • a level e.g., amount of substance (e.g., DICER, miRNA from Table A or Table B) may be detected (e.g., identified and/or quantified) using methods of detecting nucleic acids (e.g., microRNA, mRNA) and proteins well known in the art.
  • nucleic acids may be detected using nucleic acid hybridization methods that make use of complementary probes or primers that hybridize to specific nucleic acid sequences.
  • Nucleic acid hybridization methods can be used to identify small amounts of DNA or RNA (e.g., after reverse transcription to create DNA from the RNA) in PCR (e.g., real-time PCR, also known as quantitative PCR (qPCR); single cell PCR).
  • hybridizing probes including probes that include a detectable moiety (e.g., fluorescently labeled, radioactively labeled) can be used to detect DNA in Southern blotting, for the detection of genes or to detect RNA in Northern blotting. Detection can be made in biological samples, for example, tumor biopsy or blood samples. Detection of DNA or RNA using hybridizing probes in an intact cell (e.g., tumor cell, circulating tumor cell) or tissue sample (e.g., tumor biopsy) can be accomplished via in situ hybridization.
  • a detectable moiety e.g., fluorescently labeled, radioactively labeled
  • probes including a detectable moieties are allowed to hybridize with nucleic acids (e.g., RNA, DNA) residing in an intact ceil (e.g., tumor cell, circulating tumor cell) or tissue sample (e.g., tumor biopsy) and subsequently analyzed (e.g., quantified) by microscope examination.
  • nucleic acids e.g., RNA, DNA
  • tissue sample e.g., tumor biopsy
  • Non-limiting examples for performing in situ hybridization are disclosed in McFadden Meth in Cell Biol, 1995, Jensen E. The Anatomical Review, 2014; Ratan et ak, Cureus 2017, which are incorporated by reference in their entirety.
  • a biological sample may be further processed to produce, for example, cellular extracts including RNA, DNA, protein.
  • Cellular extracts may be further purified to isolate DNA, RNA, or protein.
  • Isolated DNA can be used in Southern blotting analysis while, RNA can be used in Northern blotting analysis to determine the presence and amount of the DNA or RNA of interest.
  • small amounts of isolated DNA or RNA for example amount of DNA or RNA taken from a single cell, can be identified using PCR, for example real time PCR which allows quantification of the amount of DNA or RNA in the sample.
  • PCR for example real time PCR which allows quantification of the amount of DNA or RNA in the sample.
  • Non-limiting examples of methods for performing real time PCR are disclosed in Held et ak, Genome Res, 1996; US 2009/00537261 At, which are hereby incorporated by reference in their entirety.
  • Non-limiting examples of methods for detecting total protein in a sample include absorbance measures, Bradford protein assay, Biuret test derived assays including bicinchoninic acid assay and Lowry protein assay, fiuorescamine, ami do black, colloidal gold, and nitrogen detection methods including the Kjeldahl method and Dumas method.
  • Non-limiting examples of methods for detecting a single protein in a sample include spectrometry methods, including high- performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS); antibody dependent methods, including enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, Immunoelectrophoresis, protein immunostaining, and Western blotting analysis.
  • Western blotting analysis is useful for detecting and quantifying a single protein.
  • Non-limiting examples for performing Western blotting analysis are disclosed in Kurien BT and Scofield RH, Methods 2006; Mahmood T and Yang P-C, N Am J Med Sci 2012, which are incorporated by reference in their entirety.
  • protein levels may be determined by use of PCR, e.g., real time PCR, by isolating mRNA and converting the mRNA to DNA via reverse transcriptase.
  • a level is detected in a biological sample obtained from a patient prior to the patient being administered a treatment (i.e. , a pre-treatment biological sample). In this case, the level is referred to as a“pre-treatment level.”
  • a level is detected in biological samples obtained from a population of patients prior to the patients receiving a treatment.
  • a level is detected in a biological sample obtained from a patient after the patient has been administered a treatment (i.e., a post-treatment biological sample).
  • a level is referred to as a“post-treatment level.”
  • a level is detected in biological samples obtained from a population of patients after the patients have received a treatment.
  • a level is detected in a biological sample obtained from a non-disease individual.
  • a level is detected in biological samples obtained from a population of non-disease individuals.
  • the terms“inhibitor,”“repressor,”“antagonist” or“downregu!ator” interchangeably refer to a substance capable of delectably decreasing the expression or activity of a given gene or protein relative to the absence of the“inhibitor,”“repressor,”“antagonist” or“downregulator”.
  • the antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist.
  • the term“signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.
  • deliver ⁇ ' of an MDA-7 treatment may act to modulate DICER, e.g., downregulate DICER or upregulate DICER.
  • MDA-7 treatment may act on DICER to modulate downstream effectors or signaling pathway components (e.g., one or more miRNA shown in Table B, miR-221, miR-200c, miR-320, mill- 185, miR-17, let-7c, mill- 210, TIMP3, BMP2, secreted uPAR isoform2, MMP), resulting in changes in ceil growth, proliferation, or survival.
  • downstream effectors or signaling pathway components e.g., one or more miRNA shown in Table B, miR-221, miR-200c, miR-320, mill- 185, miR-17, let-7c, mill- 210, TIMP3, BMP2, secreted uPAR isoform2, MMP
  • a pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery ' , depending on the route of administration.
  • a dose refers to the amount of active ingredient given to an individual at each administration.
  • the dose will generally refer to the amount of pulmonary ' disease treatment, anti inflammatory agent, agonist or antagonist.
  • the dose w ll vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration.
  • the dose can be modified depending on the above factors or based on therapeutic progress
  • “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration.
  • a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.
  • Treatment can refer to any delay in onset, reduction in the frequency or severity of symptoms, amelioration of symptoms, improvement in patient comfort and/or respiratory function, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving a given treatment, or to the same patient prior to, or after cessation of, treatment.
  • Treating or“treatment” as used herein (and as well -understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • treatment as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring;
  • Treating” and “treatment” as used herein include prophylactic treatment.
  • Treatment methods include administering to a subject a therapeutically effective amount of an active agent.
  • the administering step may consist of a single administration or may include a series of administrations.
  • the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof.
  • the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.
  • chronic administration may be required.
  • the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.
  • treating or treatment does not include prophylactic treatment.
  • MDA-7 treatment refers to administering of an effective amount of MDA-7 to a subject, such as patient in need thereof, a cancer patient or a patient having a disease associated with aberrant DICER (e.g , cancer, inflammatory disease, infectious disease, autoimmune disease, or cardiovascular disease).
  • DICER e.g , cancer, inflammatory disease, infectious disease, autoimmune disease, or cardiovascular disease.
  • Anti-cancer agent is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cancer cells.
  • an anti-cancer agent is a chemotherapeutic.
  • an anti-cancer agent is an agent identified herein having utility in methods of treating cancer.
  • an anti-cancer agent is an agent approved by the FDA or similar regulator ⁇ ' agency of a country other than the USA, for treating cancer.
  • compositions described herein can be used in combination with one another, with other active agents known to be useful in treating a cancer such as anti-cancer agents.
  • anti-cancer agents include, but are not limited to, radiation, MEK (e.g MEK 1, MEK2, or MEK 1 and MEK2) inhibitors (e.g. XL518, Cl- 1040, PD035901, selumetinib/ AZD6244, GSKl 120212/ trametinib, GDC -0973.
  • MEK e.g MEK 1, MEK2, or MEK 1 and MEK2
  • XL518, Cl- 1040 e.g. XL518, Cl- 1040, PD035901, selumetinib/ AZD6244, GSKl 120212/ trametinib, GDC -0973.
  • ARRY-162, ARRY-300, AZD8330 e.g., AZD8330,
  • alkylating agents e.g , cyclophosphamide, ifosfamide, chlorambucil, busulfan, meiphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan), ethyienimine and methylmel amines (e.g., hexamethlymel amine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti-metabolites
  • alkylating agents e.g , cyclophosphamide, ifosfamide, chlorambucil, busulfan, me
  • methotrexate methotrexate
  • pyrimidine analogs e.g , fiuorouracil, floxouridine, Cytarahine
  • purine analogs e.g., mereaptopurine, thioguanine, pentostatin
  • plant alkaloids e.g, vincristine, vinblastine, vinorelbine, vindesine, podophyTlotoxin, paclitaxel, docetaxel, etc.
  • topoisomerase inhibitors e.g., irinotecan, topotecan, amsacrine, etoposide (VP 16), etoposide phosphate, teniposide, etc.
  • antitumor antibiotics e.g, doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.
  • platinum-based compounds
  • hydroxyurea methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g.,
  • daunorubicin, doxorubicin, bleomycin enzymes (e.g., L-asparaginase), inhibitors of mitogen- activated protein kinase signaling (e.g. U0126, PD98059, PD 184352, PD0325901, AHRY- 142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies (e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, all trans- retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleeve
  • anastrozole andrographolide; angiogenesis inhibitors; antagonist D; antagonist G, antarelix; anti- dorsalizing morphogenetic protein- 1 ; antiandrogen, prostatic carcinoma; antiestrogen;
  • antineoplaston antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;
  • combretastatin A4 comb reta statin analogue
  • conagenin crambescidin 816
  • crisnatol
  • cryptophycin 8 cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
  • dehydrodidemnin B deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin;
  • diphenyl spiromustine diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;
  • duocarmycin SA ebselen
  • ecomustine edelfosine
  • edrecoiomab eflornithine, elemene
  • emitefur epirubicin
  • epristeride estramustine analogue
  • estrogen agonists estrogen antagonists
  • etanidazole etoposide phosphate, exemestane; fadrozole; trasrabine; fenretinide; filgrastim; finasteride; flavopiridol; flezeJastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; foraiestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
  • galocitabine ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexam ethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
  • idramantone ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists, interferons; interleukins;
  • iobenguane iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole;
  • isohomohalicondrin B itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate, lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor;
  • leukocyte alpha interferon leuprolide+estrogen+progesterone; leuprorelin; !evamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds;
  • iissoclinamide 7 lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin;
  • loxoribine lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A, marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;
  • naloxone+pentazocine napavin; naphterpin; nartograstim; nedaplatin, nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitruilyn; 06 ⁇ benzylguanine, octreotide; okicenone; oligonucleotides; onapristone; ondansetron, ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid, panaxytriol; panomifene; parabactin;
  • pazelliptine pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
  • tamoxifen methiodide tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin, triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turoster
  • vitaxin vitaxin
  • vorozole zanoterone
  • zeniplatin zilascorb
  • zinostatin stimalamer Adriamycin
  • cytarabine dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;
  • hydrochloride elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbuiozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; trasrabine; fenretinide;
  • mitindomide mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride, mycophenolic acid; nocodazoie; nogalaraycin, ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
  • piposulfan piroxantrone hydrochloride; plicamycin; plomestane; porflmer sodium; porfiromycin, prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; pumprazene, sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfm; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; torem
  • vapreotide verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate;
  • vinepidine sulfate vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, agents that arrest cells in the G2-M phases and/or modulate the formation or stability of microtubules, (e.g. Taxol.TM (i.e. paclitaxel), Taxotere.TM, compounds comprising the taxane skeleton, Erbuiozole (i.e. R-55104), Dolastatin 10 (i.e.
  • DLS-10 and NSC-376128 Mivobulin isethionate (i.e. as CI- 980), Vincristine, NSC-639829, Discodermolide (i.e as NVP-XX-A-296), ABT-751 (Abbott, i.e E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.
  • Epothilone E Epothilone F
  • Epothilone B N-oxide Epothilone A N-oxide
  • 16-aza-epothilone B Epothilone B
  • 21-aniinoepothilone B i.e. BMS-310705
  • 21 -hydroxy epothilone D i.e. Desoxyepothilone F and dEpoF
  • 26- fluoroepothilone i.e. NSC-654663
  • Soblidotin i.e. TZT-1027
  • Cryptophycin 52 i.e.
  • LY-355703 Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-106969), Oncocidin A1 (i.e. BTO-956 and DIME), Fijianolide B, Lauiimalide, Narcosine (also known as NSC-5366), Nascapine, Vanadocene acetylacetonate, T-l 38026 (Tularik), Monsatrol, lnanocine (i.e.
  • Eleutherobins such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin
  • Caribaeoside Caribaeolin
  • Halichondrin B Diazonaniide A
  • Taccalonolide A Diozostatin
  • (-)-Phenylahistin i.e. NSCL-96F037)
  • Myoseverin B
  • Resverastatin phosphate sodium e.g., steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide,
  • adrenocorticosteroids e.g., prednisone
  • progestins e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate
  • estrogens e.g., diethlystilbestrol, ethinyl estradiol
  • antiestrogen e.g , tamoxifen
  • androgens e.g , testosterone propionate, fluoxymesterone
  • antiandrogen e.g , flutamide
  • immunostimulants e.g, Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.
  • monoclonal antibodies e.g., anti-CD20, anti- HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies
  • immunotoxins e.g., anti-CD33 monoclonal antibody-
  • EGFRVtargeted therapy or therapeutic e.g gefitinib (IressaTM), erlotinib (TarcevaTM), cetuximab (ErbituxTM), lapatinib (TykerbTM), panitumumab (VectibixTM), vandetanib
  • compositions herein may be used in combination with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent
  • “Chemotherapeutic” or “chemotherapeutic agent” is used in accordance with its plain ordinary' meaning and refers to a chemical composition or compound having antineoplastic properties or the ability to inhibit the growth or proliferation of cells.
  • An“ROS inducer” refers to compounds or compositions useful for increasing reactive oxygen species.
  • a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above.
  • a therapeutically effective amount will show' an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as“-fold” increase or decrease.
  • a therapeutically effective amount can have at least a 1 2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • co-administer it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or
  • the compounds of the invention can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or can be administered alone or
  • Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • compositions of the present invention may additionally include components to provide sustained release and/or comfort.
  • Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841,
  • compositions of the present invention can also he delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel
  • the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, e.g., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis.
  • compositions of the present invention can also be delivered as nanoparticles.
  • a pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant ad verse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringers solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, faty acid esters, hydroxymethyceliulose, polyvinyl pyrrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents
  • pharmaceutically acceptable salt refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • preparation is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • the pharmaceutical preparation is optionally in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • the unit dosage form can be of a frozen dispersion.
  • a method of detecting a DICER level or DICER activity in a cancer patient wherein the cancer patient has received a MDA-7 treatment, the method including; (i) obtaining a post-treatment biological sample from the cancer patient; and (ii) detecting a post-treatment level of DICER or DICER activity in the post-treatment biological sample.
  • the method is for detecting a DICER level.
  • the method is for detecting DICER activity.“MDA-7 treatment” as provided herein refers to administering to a subject in need thereof a therapeutically effective amount of a MDA-7 protein or vector encoding the same.
  • the post-treatment biological sample is a tumor biopsy.
  • the post-treatment biological sample is a blood sample.
  • the post treatment biological sample includes a circulating tumor cell.
  • the post-treatment biological sample is a circulating tumor cell.
  • the detecting includes performing real-time PCR.
  • the detecting includes performing in situ hybridization.
  • the detecting includes performing Western blotting analysis.
  • detecting a level of DICER activity comprises detecting a level of one or more proteins or endopeptidases regulated by DICER, including TIMP3 (metalloproteainase inhibitor 3), BMP2 (bone morphogenetic protein 2), secreted uPAR isoform2, MMP (matrix m etalloproteinase), or a combination of two or more thereof.
  • TIMP3 metaloproteainase inhibitor 3
  • BMP2 bone morphogenetic protein 2
  • secreted uPAR isoform2 secreted uPAR isoform2
  • MMP matrix m etalloproteinase
  • detecting a level of DICER activity comprises detecting a level of one or more miRNA regulated by DICER.
  • miRNAs regulated by DICER see for example: Krill et al., Mol Endocrinol. 2013 May 27(5):754 ⁇ 68; and Ueda et al., Proc Natl Acad Sci U S A. 2009 Jun 30; 106(26): 10746-51.
  • detecting DICER activity comprises detecting a level of one or more miRNA selected from Table A. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-221. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-200c. In embodiments, detecting a level of DICER activity compri ses detecting a level of miR-320. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-185. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-501. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-17.
  • detecting a level of DICER activity comprises detecting a level of let-7c. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-210. In embodiments, detecting DICER activity does not comprise detecting a level of one or more miRNA selected from Table A. In embodiments, detecting DICER activity does not comprise detecting a level of miR-221.
  • detecting DICER activity comprises detecting a level of one or more miRNA selected from Table B. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-674. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-lOa. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-21. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-501. In embodiments, detecting a level of DICER activity comprises detecting a level of let-7d. In embodiments, detecting a level of DICER activity comprises detecting a level of mlR-107.
  • detecting a level of DICER activity comprises detecting a level of miR-672. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-34c. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-34b-3p. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-292-3p. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-193. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-202. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-293.
  • detecting a level of DICER activity comprises detecting a level of miR-365. In embodiments, detecting a level of DICER activity comprises detecting a level of mlR-222. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-339. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-182. In embodiments, detecting a level of DICER activity comprises detecting a level of miR-125.
  • detecting a post-treatment level of DICER includes detecting a post- treatment level of one or more miRNA selected from Table A in the post-treatment biological sample.
  • the miRNA from Table A is miR-221.
  • the miRNA from Table A is i R- 200c.
  • the miRNA from Table A is miR-320.
  • the miRNA from Table A is miR-185.
  • the miRNA from Table A is miR-17.
  • the miRNA from Table A is let-7c.
  • the miRNA from Table A is miR-210.
  • detecting a post-treatment level of DICER includes detecting a post- treatment level of one or more miRNA selected from Table B in the post-treatment biological sample.
  • the miRNA from Table B is miR-674.
  • the miRNA from Table B is miR-lOa.
  • the miRNA from Table B is miR-21.
  • the miRNA from Table B is miR-501.
  • the miRNA from Table B is let-7d.
  • the miRNA from Table B is miR-107
  • the miRNA from Table B is miR-672.
  • the miRNA from Table B is miR-34c.
  • the miRNA from Table B is miR-34b-3p.
  • the miRNA from Table B is miR-292-3p. In aspects, the miRNA from Table B is miR-193. In aspects, the miRNA from Table B is miR-202. In aspects, the miRNA from Table B is miR-293. In aspects, the miRNA from Table B is miR-365. In aspects, the miRNA from Table B is miR-222. In aspects, the miRNA from Table B is miR-339. In aspects, the miRNA from Table B is miR-182. In aspects, the miRNA from Table B is mi R- 125
  • detecting a level of DICER may be accomplished by detecting a level of a downstream target of DICER (e.g., a target the expression of which is directly or indirectly regulated by DICER).
  • Downstream targets of DICER include without limitation selected miRNA shown in Table A (e.g., iniR-221, miR-200c, mill-320, miR-185, miR-17, let-7c, miR-210), the miRNA shown in Table B, MMP (i.e., the family of matrix metalloproteinases), TIMP3, BMP2, and secreted uPAR isoform2.
  • the detecting a post treatment level of DICER includes detecting a post-treatment level of one or more miRNA shown in Table A, a post-treatment level of one or more the miRNA shown in Table B, a post-treatment level of MMP, a post -treatment level of TIMP3, a post-treatment level of BMP2, a post-treatment level of secreted uPAR isoform2, or a combination thereof in the post-treatment biological sample.
  • the detecting a post-treatment level of DICER includes detecting a post treatment level of one or more miRNA shown in Table A, a post-treament level of one or more miRNA shown in Table B, a post-treatment level of MMP, a post-treatment level of TIMP3, a post-treatment level of BMP2, a post-treatment level of secreted uPAR isoform2, or a combination thereof in the post-treatment biological sample.
  • the method further includes (i) obtaining a pre-treatment biological sample from the cancer patient prior to the cancer patient receiving a MDA-7 treatment; and (ii) detecting a pre-treatment level of DICER in the pre-treatment biological sample.
  • the pre treatment level may be detected in a non-diseased tissue of the patient or in a diseased tissue.
  • the pre-treatment biological sample is a tumor biopsy.
  • the pre treatment biological sample is a blood sample.
  • the pre-treatment biological sample includes a circulating tumor cell.
  • the pre-treatment biological sample is a circulating tumor cell.
  • the detecting includes performing real-time PCR.
  • the detecting includes performing in situ hybridization.
  • the post-treatment level of DICER detected in the post-treatment biological sample is compared to the pre-treatment level of DICER detected in the pre-treatment biological sample. In embodiments, the post-treatment level of DICER is decreased relative to the pre-treatment level of DICER. In embodiments, the post-treatment level of DICER is increased relative to the pre-treatment level. In embodiments, the post-treatment level of DICER is essentially the same relative to the pre-treatment level of DICER.
  • the detecting a pre-treatment level of DICER includes detecting a pre treatment level of one or more miRNA selected from Table A in the pre-treatment biological sample.
  • the iRNA from Table A is miR-221.
  • the miRNA from Table A is mill-200c.
  • the miRNA from Table A is mill-320.
  • the miRNA from Table A is miR-185.
  • the miRNA from Table A is miR-17.
  • the miRNA from Table A is let-7e.
  • the miRNA from Table A is miR-210.
  • the post-treatment level of one or more miRNA selected from Table A detected in the post-treatment biological sample is compared to the pre-treatment level of one or more miRNA selected from Table A detected in the pre-treatment biological sample.
  • the post-treatment level of one or more miRN A selected from Table A is decreased relative to the pre-treatment level of one or more miRNA selected from Table A.
  • the post-treatment level of one or more miRNA selected from Table A is increased relative to the pre-treatment level
  • the post-treatment level of one or more miRNA selected from Table A is essentially the same relative to the pre-treatment level of one or more miRNA selected from Table A.
  • the miRNA from Table A is mill-221.
  • the miRNA from Table A is miR-200c. In aspects, the miRNA from Table A is miR-320. In aspects, the miRNA from Table A is miR-185. In aspects, the miRNA from Table A is miR-17. In aspects, the miRNA from Table A is let-7c. In aspects, the miRNA from Table A is miR-210.
  • detecting a pre-treatment level of DICER includes detecting a pre treatment level of one or more miRNA selected from Table B in the pre-treatment biological sample.
  • the miRNA from Table B is miR-674.
  • the miRNA from Table B is miR-lOa.
  • the miRNA from Table B is miR-21 In aspects, the miRNA from Table B is mill-501.
  • the miRNA from Table B is iet-7d.
  • the miRNA from Table B is miR-107.
  • the miRNA from Table B is miR-672.
  • the miRNA from Table B is miR-34c.
  • the miRNA from Table B is miR-34b-3p.
  • the miRNA from Table B is miR-292-3p. In aspects, the miRNA from Table B is miR-193. In aspects, the miRNA from Table B is miR-202. In aspects, the miRNA from Table B is miR-293. In aspects, the miRNA from Table B is miR-365. In aspects, the miRNA from Table B is i R. -222. In aspects, the miRNA from Table B is miR-339. In aspects, the miRNA from Table B is miR-182. In aspects, the miRNA from Table B is miR-125.
  • the post-treatment level of one or more miRNA selected from Table B detected in the post-treatment biological sample is compared to the pre-treatment level of one or more miRNA selected from Table B detected in the pre-treatment biological sample.
  • the post-treatment level of one or more miRNA selected from Table B is decreased relative to the pre-treatment level of one or more miRNA selected from Table B. In embodiments, the post-treatment level of one or more miRNA selected from Table B is increased relative to the pre-treatment level. In embodiments, the post-treatment level of one or more miRNA selected from Table B is essentially the same relative to the pre-treatment level of one or more miRNA selected from Table B.
  • the miRNA from Table B is miR-674. In aspects, the miRNA from Table B is mi R- 10a. In aspects, the miRNA from Table B is miR-21. In aspects, the miRNA from Table B is miR-501. In aspects, the miRNA from Table B is let-7d.
  • the miRNA from Table B is miR-107. In aspects, the miRNA from Table B is miR-672. In aspects, the miRNA from Table B is miR-34c. In aspects, the miRNA from Table B is miR-34b-3p. In aspects, the miRNA from Table B is miR-292-3p. In aspects, the miRNA from Table B is miR-193. In aspects, the miRNA from Table B is miR-202. In aspects, the miRNA from Table B is miR-293.
  • the miRNA from Table B is miR-365. In aspects, the miRNA from Table B is miR- 222. In aspects, the miRNA from Table B is miR-339. In aspects, the miRNA from Table B is miR-182. In aspects, the miRNA from Table B is miR-125.
  • the detecting a pre-treatment level of DICER includes detecting a pre- treatment level of MMP, a pre-treatment level of TIMP3, a pre-treatment level of BMP2, a pre treatment level of secreted uPAR isoform2, or any combination thereof in the pre-treatment biological sample.
  • the cancer patient has been further treated with an additional anti-cancer agent.
  • the additional anti-cancer agent is not MDA-7.
  • the additional anti -cancer agent is a ROS inducer.
  • ROS inducers include arsenic trioxide, hydrogen peroxide, or pyocyanin.
  • the ROS inducer is arsenic trioxide, hydrogen peroxide, or pyocyanin.
  • the ROS inducer is arsenic trioxide.
  • the ROS inducer is hydrogen peroxide.
  • the ROS inducer is pyocyanin.
  • the ROS inducer is delivered at a low dose.
  • a low dose is about 0.01 mM, 0 05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0 6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 m M, 85 mM, 90 mM, 95 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 hiM.
  • a low dose is about 0.01 mM In embodiments, a low dose is about 0.05 mM. In embodiments, a low dose is about 0.1 mM. In embodiments, a low 7 dose is about 0.2 mM. In embodiments, a low dose is about 0.3 mM. In embodiments, a low dose is about 0.4 mM. In embodiments, a low dose is about 0 5 mM. In embodiments, a low dose is about 0.6 mM. In embodiments, a low 7 dose is about 0.7 mM. In embodiments, a low dose is about 0.8 mM, 0.9 mM. In embodiments, a low 7 dose is about 1 mM.
  • a low dose is about 5 mM. In embodiments, a low dose is about 10 mM. In embodiments, a low dose is about 15 mM, 20 mM. In embodiments, a low dose is about 25 mM.
  • a low dose is about 30 mM. In embodiments, a low dose is about 35 mM In embodiments, a low dose is about 40 mM. In embodiments, a low 7 dose is about 45 mM. In embodiments, a low dose is about 50 mM In embodiments, a low dose is about 55 mM. In embodiments, a low dose is about 60 mM. In embodiments, a low dose is about 65 mM. In embodiments, a low dose is about 70 mM. In embodiments, a low dose is about 75 mM. In embodiments, a low dose is about 80 mM. In embodiments, a low 7 dose is about 85 mM.
  • a low dose is about 90 mM. In embodiments, a low dose is about 95 mM. In embodiments, a lo 7 dose is about 100 mM. In embodiments, a low dose is about 200 m M In embodiments, a low dose is about 300 mM. In embodiments, a low dose is about 400 mM. In embodiments, a low dose is about 500 mM. In embodiments, a low dose is about 600 mM. In embodiments, a low dose is about 700 mM. In embodiments, a low 7 dose is about 800 mM. In embodiments, a low dose is about 900 mM. In embodiments, a low dose is about 1 mM.
  • a low dose is 0.01 mM. In embodiments, a low dose is 0.05 mM. In embodiments, a low dose is 0.1 mM. In embodiments, a low 7 dose is 0.2 mM. In embodiments, a low dose is 0.3 mM. In embodiments, a low dose is 0.4 mM. In embodiments, a low dose is 0.5 m M In embodiments, a low dose is 0.6 mM. In embodiments, a low dose is 0.7 mM. In embodiments, a low dose is 0.8 mM, 0.9 mM. In embodiments, a low dose is 1 mM. In embodiments, a low dose is 0.05 mM. In embodiments, a low dose is 0.1 mM. In embodiments, a low 7 dose is 0.2 mM. In embodiments, a low dose is 0.3 mM. In embodiments, a low dose is 0.4 mM. In embodiments, a low dose is 0.5
  • a low dose is 5 m M In embodiments, a low dose is 10 mM. In embodiments, a low dose is 15 mM, 20 mM. In embodiments, a low dose is 25 mM. In embodiments, a low dose is 30 mM In embodiments, a low dose is 35 mM. In embodiments, a low dose is 40 mM. In
  • a low dose is 45 mM. In embodiments, a low dose is 50 mM. In embodiments, a low dose is 55 mM In embodiments, a low dose is 60 mM. In embodiments, a low dose is 65 mM. In embodiments, a low dose is 70 mM. In embodiments, a low dose is 75 mM. In embodiments, a low dose is 80 mM. In embodiments, a low dose is 85 mM. In embodiments, a low dose is 90 mM. In embodiments, a low dose is 95 mM. In embodiments, a low dose is 100 mM. In embodiments, a low dose is 200 mM.
  • a low dose is 300 mM. In embodiments, a low dose is 400 mM. In embodiments, a low dose is 500 mM. In embodiments, a low dose is 600 mM. In embodiments, a low dose is 700 mM. In embodiments, a low dose is 800 mM. In embodiments, a low dose is 900 mM. In embodiments, a low dose is 1 mM.
  • the MDA-7 treatment and the additional anti -cancer agent are administered in a combined synergistic amount.
  • a “combined synergistic amount” as used herein refers to the sum of a first amount of a first agent (e.g., an amount of MDA-7) and a second amount of a second agent (e.g., an anti- cancer agent (e.g., ROS inducer)), that results in a synergistic effect (i.e. an effect greater than an additive effect).
  • a first agent e.g., an amount of MDA-7
  • a second agent e.g., an anti- cancer agent (e.g., ROS inducer)
  • the terms “synergy”, “synergism”, “synergistic”, “combined synergistic amount”, and “synergistic therapeutic effect” which are used herein interchangeably, refer to a measured effect of compounds administered in combination where the measured effect is greater than the sum of the individual effects of each of the compounds administered alone as a single agent.
  • a combined synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
  • the amount of the first amount e.g., AIDA-7
  • the second amount e.g., an anti -cancer agent
  • a combined synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5,
  • the amount of the second amount e.g., an anti-cancer agent (e.g , ROS inducer)
  • the first amount e.g., MDA-7
  • the cancer patient has melanoma, prostate cancer, neuroblastoma, osteosarcoma, renal carcinoma, leukemia, epithelial cancer, pancreatic cancer, glioblastoma, thyroid papillary carcinoma, esophageal squamous cell carcinoma, breast cancer, hepatocellular carcinoma, liver cancer, or lung cancer.
  • the cancer patient has melanoma.
  • the cancer patient has prostate cancer.
  • the cancer patient has neuroblastoma.
  • the cancer patient has osteosarcoma.
  • the cancer patient has renal carcinoma.
  • the cancer patient has leukemia.
  • the cancer patient has epithelial cancer.
  • the cancer patient has pancreatic cancer. In embodiments, the cancer patient has glioblastoma. In embodiments, the cancer patient has thyroid papillary carcinoma. In embodiments, the cancer patient has esophageal squamous cell carcinoma. In embodiments, the cancer patient has breast cancer. In embodiments, the cancer patient has hepatocellular carcinoma. In embodiments, the cancer patient has liver cancer. In embodiments, the cancer patient has lung cancer. In embodiments, the cancer patient being treated has a metastatic cancer.
  • the post-treatment biological sample is a tumor biopsy.
  • the post-treatment biological sample is a blood sample.
  • the post treatment biological sample includes a circulating tumor cell.
  • the post-treatment biological sample is a circulating tumor cell.
  • the detecting includes performing real-time PCR.
  • the detecting includes performing Western blotting analysis.
  • the method further includes detecting a post-treatment level of DICER in the post-treatment biological sample.
  • the detecting includes performing real time PCR.
  • the detecting includes performing in situ hybridization.
  • the pre-treatment biological sample is a tumor biopsy. In embodiments, the pre-treatment biological sample is a blood sample. In embodiments, the pre-treatment biological sample includes a circulating tumor cell. In embodiments, the pre-treatment biological sample is a circulating tumor cell. In embodiments, the detecting includes performing real-time PCR. In embodiments, the detecting includes performing Western blotting analysis.
  • the post-treatment level of one or more miRNA selected from Table A detected in the post-treatment biological sample is compared to the pre-treatment level of one or more miRNA selected from Table A in the pre-treatment biological sample.
  • the post-treatment level of one or more miRNA selected from Table A is increased relative to the pre- treatment one or more miRNA selected from Table A level.
  • the post-treatment level of one or more miRNA selected from Table A is decreased relative to the pre-treatment one or more miRNA selected from Table A level.
  • the post-treatment level of one or more miRNA selected from Table A is essentially the same relative to the pre-treatment level one or more miRNA selected from Table A.
  • the miRNA from Table A is miR-221.
  • the miRNA from Table A is miR-200c. In aspects, the miRNA from Table A is miR-320. In aspects, the miRNA from Table A is miR-185. In aspects, the miRNA from Table A is miR-17. In aspects, the miRNA from Table A is let-7c. In aspects, the miRNA from Table A is miR-210.
  • the post-treatment level of one or more miRNA selected from Table B detected in the post-treatment biological sample is compared to the pre-treatment level of one or more miRNA selected from Table B in the pre-treatment biological sample.
  • the post-treatment level of one or more miRNA selected from Table B is increased relative to the pre treatment one or more miRNA selected from Table B level.
  • the post-treatment level of one or more miRNA selected from Table B is decreased relative to the pre-treatment one or more miRNA selected from Table B level.
  • the post-treatment level of one or more miRNA selected from Table B is essentially the same relative to the pre-treatment level one or more miRNA selected from Table B.
  • the miRNA from Table B is miR-674.
  • the miRNA from Table B is miR-lOa In aspects, the miRNA from Table B is miR-21. In aspects, the miRNA from Table B is miR-501. In aspects, the miRNA from Table B is iet-7d. In aspects, the miRNA from Table B is miR-107. In aspects, the miRNA from Table B is miR-672.
  • the miRNA from Table B is miR-34c. In aspects, the miRNA from Table B is miR- 34b-3p In aspects, the miRNA from Table B is miR-292 ⁇ 3p. In aspects, the miRNA from Table B is miR-193. In aspects, the miRNA from Table B is miR-202. In aspects, the miRNA from Table B is miR-293. In aspects, the miRNA from Table B is miR-365. In aspects, the miRNA from Table B is miR-222. In aspects, the miRNA from Table B is miR-339. In aspects, the miRNA from Table B is miR-182. In aspects, the miRNA from Table B is miR-125.
  • the cancer patient has been further treated with an additional anti-cancer agent.
  • the additional anti -cancer agent is not MDA-7.
  • the additional anti -cancer agent is a ROS inducer.
  • Non-limiting examples of in clinic ROS inducers include arsenic trioxide, hydrogen peroxide, or pyocyanin.
  • the ROS inducer is arsenic trioxide, hydrogen peroxide, or pyocyanin.
  • the ROS inducer is arsenic trioxide.
  • the ROS inducer is hydrogen peroxide.
  • the ROS inducer is pyocyanin.
  • the ROS inducer is delivered at a low dose.
  • a low dose may be about 0 01 mM, 0 05 mM, 0.1 mM, 0.2 mM, 0 3 mM, 0.4 mM, 0.5 mM, 0 6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1 ihM.
  • the MDA-7 treatment and the additional anti-cancer agent may be administered in a combined synergistic amount
  • the cancer patient has melanoma, prostate cancer, neuroblastoma, osteosarcoma, renal carcinoma, leukemia, epithelial cancer, pancreatic cancer, glioblastoma, thyroid papillary carcinoma, esophageal squamous cell carcinoma, breast cancer, hepatocellular carcinoma, liver cancer, or lung cancer.
  • the cancer patient has melanoma.
  • the cancer patient has prostate cancer.
  • the cancer patient has neuroblastoma.
  • the cancer patient has osteosarcoma.
  • the cancer patient has renal carcinoma.
  • the cancer patient has leukemia.
  • the cancer patient has epithelial cancer.
  • the cancer patient has pancreatic cancer. In embodiments, the cancer patient has glioblastoma. In embodiments, the cancer patient has thyroid papillary carcinoma. In embodiments, the cancer patient has esophageal squamous cell carcinoma. In embodiments, the cancer patient has breast cancer. In embodiments, the cancer patient has hepatocellular carcinoma. In embodiments, the cancer patient has liver cancer. In embodiments, the cancer patient has lung cancer. In embodiments, the cancer patient being treated has a metastatic cancer.
  • modulating (e.g., downregulating) DICER via MDA-7 treatment is useful for treating cancers expressing DICER.
  • a method of cancer in a subject in need thereof, wherein the subject has a cancer expressing DICER the method including administering to the subject an effective amount of MDA-7.
  • the cancer does not express beclin-1.
  • modulating (e.g., downregulating) DICER via MDA-7 treatment is useful for treating cancers expressing DICER and not expressing MDA-7.
  • a method of cancer in a subject in need thereof, wherein the subject has a cancer expressing DICER and not expressing MDA-7 the method including administering to the subject an effective amount of MDA-7.
  • the cancer does not express beclin-1.
  • the method further includes, prior to administering the effective amount of MDA-7: (i) obtaining a pre-treatment biological sample from the subject; and (ii) detecting a pre-treatment level of DICER in the pre-treatment biological sample.
  • the pre treatment biological sample is a tumor biopsy.
  • the pre-treatment biological sample is a blood sample.
  • the pre-treatment biological sample includes a circulating tumor cell.
  • the pre-treatment biological sample is a circulating tumor ceil.
  • the detecting includes performing real-time PCR.
  • the detecting includes performing in situ hybridization.
  • the pre-treatment level of DICER in the pre-treatment biological sample is compared to a standard control. In embodiments, the pre-treatment level of DICER is increased relative to the standard control. In embodiments, the pre-treatment level of DICER is decreased relative to the standard control. In embodiments, the pre-treatment level of DICER is essentially the same relative to the standard control. In embodiments, the standard control is a median level of DICER obtained from a population of non-disease individuals. In embodiments, the standard control is a level of DICER obtained from non-disease tissue obtained from the patient.
  • the standard control is a mean level of DICER obtained from a population of patients having a DICER and/or DICER associated disease (e.g., cancer). In embodiments, the standard control is a median level of DICER obtained from a population of patients having a DICER and/or DICER associated disease (e.g., cancer).
  • the detecting a pre-treatment level of DICER may include detecting a pre-treatment level of one or more miRNA selected from Table A in the pre-treatment biological sample.
  • the miRNA from Table A is miR-221.
  • the miRNA from Table A is miR-200c.
  • the miRNA from Table A is miR-320.
  • the miRNA from Table A is miR-185.
  • the miRNA from Table A is miR-17.
  • the miRNA from Table A is let-7c.
  • the miRNA from Table A is miR-210.
  • the detecting a pre-treatment level of DICER may include detecting a pre-treatment level of one or more miRNA selected from Table B in the pre-treatment biological sample.
  • the miRNA from Table B is miR-674.
  • the miRNA from Table B is miR-lOa.
  • the miRNA from Table B is miR-21.
  • the miRNA from Table B is miR-501.
  • the miRNA from Table B is let-7d.
  • the miRNA from Table B is miR-107.
  • the miRNA from Table B is mill-672.
  • the miRNA from Table B is miR ⁇ 34c. In aspects, the miRNA from Table B is miR-
  • the miRNA from Table B is miR-292-3p. In aspects, the miRNA from Table B is miR-193. In aspects, the miRNA from Table B is miR-202. In aspects, the miRNA from Table B is mill-293. In aspects, the miRNA from Table B is miR-365. In aspects, the miRNA from Table B is miR-222. In aspects, the miRNA from Table B is miR-339. In aspects, the miRNA from Table B is miR-182, In aspects, the miRNA from Table B is miR-125.
  • the pre-treatment level of one or more miRNA selected from Table A in the pre-treatment biological sample is compared to a standard control. In embodiments, the pre treatment level of one or more miRNA selected from Table A is decreased relative to the standard control. In embodiments, the pre-treatment level of one or more miRNA selected from Table A is increased relative to the standard control. In embodiments, the pre-treatment level of one or more miRNA selected from Table A is essentially the same relative to the standard control. In embodiments, the standard control is a median level of one or more miRNA selected from Table A obtained from a population of non-disease individuals. In embodiments, the standard control is a level of one or more miRNA selected from Table A obtained from non-disease tissue obtained from the patient.
  • the standard control is a mean level of one or more miRNA selected from Table A obtained from a population of patients having a diseae (e.g., cancer) associated with one or more miRNA selected from Table A.
  • the standard control is a median level of one or more miRNA selected from Table A obtained from a population of patients having a diseae (e.g., cancer) associated with one or more miRNA selected from Table A.
  • the miRNA from Table A is miR-221.
  • the miRNA from Table A is miR- 200c.
  • the miRNA from Table A is miR-320.
  • the miRNA from Table A is mxR-185.
  • the miRNA from Table A is miR-17.
  • the miRNA from Table A is let-7c.
  • the miRNA from Table A is miR-210.
  • the pre-treatment level of one or more miRNA selected from Table B in the pre-treatment biological sample is compared to a standard control.
  • the pre treatment level of one or more miRNA selected from Table B is decreased relative to the standard control.
  • the pre-treatment level of one or more miRNA selected from Table B is increased relative to the standard control.
  • the pre-treatment level of one or more miRNA selected from Table B is essentially the same relative to the standard control.
  • the standard control is a median level of one or more miRNA selected from Table B obtained from a population of non-disease individuals.
  • the standard control is a level of one or more miRNA selected from Table B obtained from non-disease tissue obtained from the patient.
  • the standard control is a mean level of one or more miRNA selected from Table B obtained from a population of patients having a diseae (e.g , cancer) associated with one or more miRNA selected from Table B.
  • the standard control is a median level of one or more miRNA selected from Table B obtained from a population of patients having a diseae (e.g., cancer) associated with one or more miRNA selected from Table B.
  • the miRNA from Table B is miR-674.
  • the miRNA from Table B is miR- 10a.
  • the miRNA from Table B is miR-21.
  • the miRNA from Table B is miR- 501.
  • the miRNA from Table B is let-7d.
  • the miRNA from Table B is miR- 107. In aspects, the miRNA from Table B is miR-672. In aspects, the miRNA from Table B is rmR-34c. In aspects, the miRNA from Table B is miR-34b-3p. In aspects, the miRNA from Table B is miR-292-3p. In aspects, the miRNA from Table B is miR-193. In aspects, the miRNA from Table B is miR-202. In aspects, the miRNA from Table B is miR-293. In aspects, the rniRNA from Table B is miR-365. In aspects, the miRNA from Table B is miR-222. In aspects, the miRNA from Table B is miR-339 In aspects, the miRNA from Table B is miR-182. In aspects, the miRNA from Table B is miR-125.
  • the detecting a pre-treatment level of DICER may include detecting a pre-treatment level of MMP, a pre-treatment level of TIMP3, a pre-treatment level of BMP2, a pre-treatment level of secreted uPAR isoform2, or any
  • administering the effective amount of MDA-7 reverses a multi drug chemoresistance.
  • multidrug chemoresistance refers to the mechanism by which cancers develop resistance to multiple chemotherapy drugs, resulting in the failure of
  • the method further includes administering to the subject an additional anti-cancer agent.
  • the additional anti-cancer agent is not MDA-7.
  • the cancer patient has been further treated with an additional anti-cancer agent.
  • the additional anti -cancer agent is a ROS inducer.
  • Non-limiting examples of in clinic ROS inducers include arsenic trioxide, hydrogen peroxide, or pyocyanin.
  • the ROS inducer is arsenic trioxide, hydrogen peroxide, or pyocyanin.
  • the ROS inducer is arsenic trioxide.
  • the ROS inducer is hydrogen peroxide.
  • the ROS inducer is pyocyanin.
  • a low' dose is about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1 ihM.
  • modulating e.g., downregulating or upregulating
  • DICER DICER via MDA-7 treatment
  • the method including administering to the subject an effective amount of AIDA-7.
  • the cancer expresses DICER. In embodiments, the cancer expresses one or more miRNA shown in Table A. In embodiments, the cancer expresses one or more miRNA shown in Table B.
  • the method further includes, prior to administering the effective amount of MDA-7: (i) obtaining a pre-treatment biological sample from the subject; and (ii) detecting a pre-treatment level of beclin-1 in the pre-treatment biological sample.
  • the pre treatment biological sample is a tumor biopsy.
  • the pre-treatment biological sample is a blood sample.
  • the pre-treatment biological sample includes a circulating tumor cell.
  • the pre-treatment biological sample is a circulating tumor cell.
  • the detecting includes performing real-time PCR.
  • the detecting includes performing Western blotting analysis.
  • the method further includes administering to the subject an additional anti-cancer agent.
  • the additional anti -cancer agent is not MDA-7.
  • the cancer patient has been further treated with an additional anti- cancer agent.
  • the additional anti-cancer agent is a ROS inducer as described herein (e.g., arsenic trioxide, hydrogen peroxide, or pyocyanin).
  • the MDA-7 treatment and the additional anti-cancer agent are administered in a combined synergistic amount.
  • the cancer patient has melanoma, prostate cancer, neuroblastoma, osteosarcoma, renal carcinoma, leukemia, epithelial cancer, pancreatic cancer, glioblastoma, thyroid papillary carcinoma, esophageal squamous cell carcinoma, breast cancer, hepatocellular carcinoma, liver cancer, or lung cancer.
  • the cancer patient has melanoma.
  • the cancer patient has prostate cancer.
  • the cancer patient has neuroblastoma.
  • the cancer patient has osteosarcoma.
  • the cancer patient has renal carcinoma.
  • the cancer patient has leukemia.
  • the cancer patient has epithelial cancer.
  • the cancer patient has pancreatic cancer. In embodiments, the cancer patient has glioblastoma. In embodiments, the cancer patient has thyroid papillary carcinoma. In embodiments, the cancer patient has esophageal squamous cell carcinoma. In embodiments, the cancer patient has breast cancer. In embodiments, the cancer patient has hepatocellular carcinoma. In embodiments, the cancer patient has liver cancer. In embodiments, the cancer patient has lung cancer. In embodiments, the cancer is a metastatic cancer.
  • the methods described herein may be effective in preventing or reducing cancer-associated angiogenesis. Therefore, in an aspect is provided a method of inhibiting cancer- associated angiogenesis in a subject in need thereof, the method including administering to the subject an effective amount of MDA-7.
  • a method of treating an autoimmune disease in a subject in need thereof including administering to the subject an effective amount of MDA-7.
  • the autoimmune disease is rheumatoid arthritis.
  • the subject has aberrant DICER levels and/or activity, including aberrant levels and/or activity of the one or more miRNA shown in Table A and/or Table.
  • a method of treating an infectious disease in a subject in need thereof including administering to the subject an effective amount of AIDA-7.
  • the infectious disease is tuberculosis.
  • the infectious disease is influenza.
  • the subject has aberrant DICER levels and/or activity, including aberrant levels and/or activity of the one or more miRNA shown in Table A and/or Table.
  • a method of treating an inflammatory disease in a subject in need thereof including administering to the subject an effective amount of AIDA-7.
  • the inflammatory disease is psoriasis.
  • the inflammatory disease is inflammatory bowel disease.
  • the subject has aberrant DICER levels and/or activity, including aberrant levels and/or activity of the one or more miRNA shown in Table A and/or Table
  • a treating a cardiovascular disease in a subject in need thereof including administering to the subject an effective amount of MDA-7.
  • the cardiovascular disease includes vascular calcification.
  • the cardiovascular disease is premature coronary artery disease.
  • the cardiovascular disease is subclinical atherosclerosis.
  • the subject has aberrant DICER levels and/or activity, including aberrant levels and/or activity of the one or more miRNA shown in Table A and/or Table
  • Example 1 mda-7/TL-24 Mediates Cancer Cell-Specific Death through Regulation of miR-221 and the beclin-1 Axis
  • mda-7/IL-24 displays broad-spectrum anti-cancer activity in vitro, in vivo in preclinical animal models and in a phase 1 11 clinical trial in patients with advanced cancers, without harming normal cells or tissues.
  • mda-7/IL-24 regulates a specific subset of miRNAs, including cancer- associated miR-221.
  • Both ectopic expression of mda-7/IL-24 or treatment with recombinant His- MDA-7 protein down regulate miR-221, while up regulating p27 and PUM A, in a panel of cancer cells culminating in cell death.
  • Mda-7/IL-24 -induced cancer cell death was dependent on reactive oxygen species induction and was rescued by over expressing miR-221.
  • Beclin-1 was identified as a new transcriptional target of miR-221 and mda-7/IL-24 regulated autophagy through a miR- 221/beclin-l feedback loop.
  • miR-221 overexpressing MDA-MB-231 clones were more aggressive and resistant to mda-7/IL-24-mediated ceil death than MDA-MB-231 parental clones.
  • mda-7/IL-24 (1) has potent anti-tumor activity in almost all types of cancers (2-6). mda-
  • 7/TL-24 is a member of the IL-IO-related cytokine gene family, which was cloned using subtraction hybridization and induction of terminal cancer cell differentiation in melanoma cells (1).
  • Extensive research has confirmed the ubiquitous anti-tumor properties of mda-7/IL-24 both in in vitro cell cultures and animal models (6).
  • mda-7/IL-24 displayed safety and efficacy in a Phase I/II clinical trial in patients with several advanced cancers (7, 8). Forced over expression of mda- 7/EL-24 inhibits angiogenesis (9, 10), sensitizes cancer cells to radiation or chemotherapy (3-6) and elicits potent‘bystander’ antitumor activity (11).
  • MicroRNAs are small noncoding RNAs, which degrade RNAs, negatively affect the stability of RNAs or block the translation of mR As (14-16). MicroRNAs are aberrantly expressed in many diseases including cancer (17, 18). MicroRNA-221 is an important regulator, whose up regulation has been described in several types of cancers and several reports suggest that miR-221 can be used as a therapeutic target for cancer. Many tumor suppressors have been reported to be targets of miR-221. miR-221 regulates ceil cycle through p27 (19) and apoptosis through PUMA (20).
  • miR-221 By targeting the estrogen receptor (ER) it blocks the action of tamoxifen and hence targeting miR-221 can promote susceptibility to tamoxifen-mediated ceil death in ER positive breast cancers (21).
  • Other tumor suppressor targets of miR-221 include PTEN (22), p57 (23), FOX03A (24), and TIMP3 (22).
  • PTEN 22
  • p57 23
  • FOX03A 24
  • TIMP3 TIMP3
  • Beclin-1 the mammalian homologue of Atg6 of yeast is a promoter of autophagy.
  • beclin-1 is altered in different disease states including cancer. In several types of cancer aberrant mRNA/protein expression of beclin-1 has been observed (26). The underlying mechanism of this altered expression of beclin-1 is largely unknown.
  • oncogenes e.g., mda-7/IL-24, miR-221, and beclin-1.
  • Ad.mda-7 infection down regulates miR-221 , which in turn up regulates beclin-1 and promotes toxic autophagy that switches to apoptosis.
  • miR-221 is a downstream participant in mda-7/TL ⁇ 24-mediated cell death and cells overexpressing miR-221 are resistant to mda-7/IL-24-mediated cell death.
  • ROS plays a key role in this pathway and a novel mda-7/IL-24-miR-221 -beclin-1 axis is critical in mda ⁇ 7/IL-24 ⁇ mediated cell death.
  • the miR-221 and anti-imR-221 constructs were from GeneCopoeia (Rockville, MD).
  • Beclin-1 3’UTR construct was from Origene (Rockville, MD).
  • Beclin-1 construct was from Addgene (Cambridge, MA).
  • the Beclin-1 -UTR mutant was cloned from the wild type Beclin-1 -UTR by standard site-directed mutagenesis (27).
  • Cell lines used in this study included DU-145, MCF-7, T-47D, MDA-MB-231, ZR-751, SK-BR-3, RPMI- 7951, NB-1691, SK-N-SH, IM-PHFA, RWPE-1, HMEC, and A549. These cells were obtained from the American type culture collection (ATCC) (Manassas, VA), with the exception of IM- PHFA, which was established in our laboratory (28), and were maintained as described by the ATCC. ATCC authenticates these cell lines using short tandem repeat (STR) analysis. All the cell lines were expanded and frozen immediately after receipt. The cumulative culture length of the cells w'as less than 6 months after recovery. Early passage cells were used for all experiments.
  • ATCC American type culture collection
  • STR short tandem repeat
  • HMEC Human mammary' epithelial cells
  • NB- 1691 cells were a kind gift from Dr. Alan Houghton from St Jude children’s research hospital (Memphis, TINT). All the cell lines were frequently tested for mycoplasma contamination using a mycoplasma detection kit from Sigma. Stable clones expressing miR-221 and beclin-1 were established in MDA-MB-231 cells as described previously (28).
  • RNA and microRNA-enriched fractions of RNA were isolated from cells using the RNA and microRNA isolation kits, respectively, fro Qiagen (Hilden, Germany ) Real time PCR was performed with the taqman master mix and probe were from Applied Biosystems, Foster City, CA. Data were analyzed by Graphpad prism software
  • Transient transfection and reporter gene assay were used the lipofectamine reagent from Invitrogen, Carlsbad, CA.
  • luciferase assay cells were transfected with the 3’UTR construct of beciin-1 with or without miR-221 with the pRLTK luc construct encoding reni!la luciferase control. Cells were incubated for 24 hours and then luciferase assays were done using the dual -luciferase assay kit from Promega, Madison, WI.
  • Cell proliferation assay Cell proliferation was measured by standard MTT (3 -(4, 5-di methyl thiazol-2-yl)-2, 5 diphenyl tetrazolium bromide) assay as described earlier (1 1). Colony formation assays were done as described previously (29).
  • Tumor xenograft studies were establi shed subcutaneously in both flanks of 6-week old female athymic mice (Charles River Laboratories, Wilmington, MA) by injecting 0.5 x 106 MDA-MB-231 or MDA-MB-231 cells overexpressing miR-221 or beclin-1 mixed with Matrigel in a 1 : 1 ratio. Once tumors reached a measurable size of approximately 100 mm3, the mice were divided into different groups and treated as described in the figure and figure legend. When the tumors in the control group reached the maximum allowable limit, mice were sacrificed and tumor weight was measured. Tumor size was also measured and plotted. Animals were maintained under the guidelines of the National Institute of Health and under evaluation and approval of the Institutional Animal Care and Use Committee (Virginia Commonwealth
  • ROS Reactive oxygen species
  • Live-dead assay The number of live and dead cells was observed by confocal laser microscope (Zeiss, Germany) after staining with live/dead staining reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol. The images were analyzed by Zeiss software.
  • Apoptosis assay MDA-MB-231 cells were treated as indicated in the figure. After 72 hours, cells were analyzed for apoptosis using the Annexin-V-FITC/propidium iodide apoptosis detection kit (BD Biosciences, San Jose, CA) and subjected to flow cytometry analysis using BDFACS CantoII and BDFACS DIVA software (BD Biosciences, San Jose, CA).
  • MDA-7 regulates mill-221.
  • mda-7/IL-24 is recognized for its specific and selective tumor cell-killing effects without harming normal ceils.
  • the microRNA-enriched fraction was isolated and real time PCR was done for a series of microRNAs related to cell death/apoptosis.
  • microRNAs including miR-200c, let7c, and miR-320 were found to be deregulated after treatment with mda-7/IL-24 (Fig. 8).
  • miR-200c which regulates tumor metastasis and epithelial-mesenchymal transition, was found to be down regulated by mda-7/IL- 24.
  • microRNA- 185 a tumor suppressor, reported in many cancers was found to be up regulated in mda ⁇ 7/IL-24 ⁇ infec ⁇ ed cells.
  • miR-221 is one of the microRNAs reported in a number of cancers and it exhibits an expansive role in different pathways deregulated in cancer. miR-221 targets p27, a key modulator of cell cycle (19) and PUMA, a proapoptotic gene that is degraded by miR-221 (20) PTEN, a potent tumor suppressor is also down regulated by miR-221 (22) As shown in Fig.
  • miR-221 was down regulated in mda-7/[L-24-treated MDA-MB-231 cells, while no alteration was observed in the level of miR-222.
  • mda-7/IL-24-mediated down regulation of miR-221 occurred in a dose- dependent manner, which correlated with exogenous protein expression (MDA-7/IL-24) and inhibition of cell growth (FIG. IB).
  • the down regulation of miR-221 by mda-7/IL-24 also occurred in a temporal manner in a time point kinetic study (FIG. 1C).
  • mda-7/IL-24 down regulates miR-221 in diverse cancer cell lines.
  • Breast cancer is classified on the basis of hormone receptor expression [estrogen receptor (ER) and progesterone receptor (PR)] and also HER2/Neu status.
  • ER estrogen receptor
  • PR progesterone receptor
  • Triple negative breast cancers express higher levels of miR-221 than ER/PR/HER2 positive breast cancers (31).
  • miR-221 was down regulated by rnda-7/TL-24 in MDA-MB-231 cells, a triple negative breast cancer cell line.
  • MDA-7/IL-24 signals through receptor dimers consisting of an R1 type receptor and an R2 type receptor (IL-2QR1 and IL-20R2; IL-22R1 and IL-20R2; or a unique receptor pair IL-20R1 and IL-22R1) in order to activate downstream signaling events (5, 6).
  • R1 type receptor and an R2 type receptor IL-2QR1 and IL-20R2; IL-22R1 and IL-20R2; or a unique receptor pair IL-20R1 and IL-22R1
  • A549 cells lung cancer cells which lack a full set ofRl and R2, IL-20/IL-22, receptors
  • DU-145 cells prostate cancer cells containing both receptor types
  • miR-221 expression decreased in DU-145 cells following treatment with His-MDA-7, while the level remained unchanged in A549 cells, which lacks the cognate receptor pairs (FIG. 2C).
  • Overexpression of the 1L-20R2 or IL-22R1 receptors in A549 cells rendered these cells sensitive to miR-221 down regulation after treatment with MDA-7/IL-24 recombinant protein
  • miR-221 Over expression of miR-221 rescues cells from mda-7/IL-24-mediated cell death.
  • MDA-MB-231 cells were transfected with a pCDNA3.I or miR-221 vector and infected with Ad. null or Ad.mda-7
  • MDA-7/IL-24 in glioblastoma multiforme cells increases thioredoxin and manganese super oxide dismutase (SOD2) levels, without altering SOD1 protein levels (32).
  • SOD2 manganese super oxide dismutase
  • MDA-7/IL-24-mediated cell killing relies on reactive oxygen species (ROS) generation, which is one of the key mediators of MDA-7/IL-24 toxicity in cancer cells (33)
  • ROS reactive oxygen species
  • Beclin-1 is a direct target of miR-221.
  • miR-221 inhibits autophagy induction, which leads to heart failure by deregulating the p27/CDK2/mTOR pathway (34).
  • a regulatory link between miR-221 and Beclin-1 has been suggested, since Beciin-1 is regulated by HDAC6 (35) and HDAC6 is regulated by miR-221 (36).
  • HDAC6 HDAC6
  • miR-221 a regulatory role of mda ⁇ 7/IL-24 in toxic autophagy has been described (37).
  • MDA-MB-231 cells were transfected with a pCDNA3.1 vector or a miR-221 expressing construct.
  • Overexpression of miR-221 resulted in beclin-1 down regulation (FIG. 5A) and increasing doses of miR-221 significantly down regulated beciin-1 at the transcript and protein level in a dose-dependent manner in MDA-MB-231 cells (Figs. 5B, 5C and FIG. 1 1).
  • FIG. 5A beclin-1 down regulation
  • Figs. 5B, 5C and FIG. 1 To validate the role of miR-221 on the transcriptional regulation of beclin-1, we performed a luciferase reporter gene assay using a 3’ UTR beclin-1 construct that covers 600-bp downstream of the beclin-1 stop codon.
  • the miR-221 transfected HeLa cells showed a significantly lower luciferase activity than the vector-transfected cells (FIG. 5D) suggesting that beclin-1 is a potential target of miR-221.
  • the assay was validated with a mutated 3’ UTR construct of beclin-1.
  • miR-221 failed to down regulate the mutant construct which has no binding site for miR-221 (FIG. 5D). It was reported earlier that nida ⁇ 7/IL-24 over expression led to enhanced beclin-1 expression (35). These data confirm that beclin-1 is a potential target of miR- 221 and suggest a mechanism of mda-7/IL-24-mediated autophagy regulation through a miR-221 and beclin-1 pathway.
  • Rapamycin another autophagy inducer, up regulates beclin-1 (38).
  • beclin-1 38
  • different cancer cell lines were transfected with miR- 221 and treated with Rapamycin.
  • Rapamycin up regulated beclin-1 protein levels
  • ectopic expression of miR-221 decreased this upregulation (FIG. 13).
  • This result confirms miR-221 - mediated beclin-1 regulation and also explains yet another mechanism showing miR-221 can deregulate Rapamycin-induced autophagy.
  • mda-7/IL-24 regulates miR-221 expression in vivo.
  • MDA-MB-231 cells over expressing miR-221 or miR-221 plus beclin-1 were injected subcutaneously to establish tumor xenografts in female athymic nude mice. After a palpable tumor (100 mm3) developed in approximately 10 days, the tumors were injected with 8 intramural injections over a 3 -week period with 1 X 108 viral particles of Ad. null or Ad.nida-7 In control vector-transfected cells a significant growth inhibitor ⁇ ' effect was evident, but in miR-221 over expressing cells the effect of Ad.
  • m da-7 was less apparent both in the injected left tumor, and in the uninjected right tumor, as previously observed when infecting these cells in vitro (FIG. 6 A and B).
  • overexpressing beclin-1 in miR-221 -transfected cells sensitized these cells to mda-7/IL-24-induced cell death.
  • the expression of miR-221 was confirmed by RQ-PCR (FIG.
  • miR-221 an oncogenic miRNA
  • mda-7/IL-24 an oncogenic miRNA
  • Using a panel of breast, lung, prostate, and neuroblastoma cell lines we document a significant decrease in the level of miR-221 following adenoviral -mediated delivery of mda-7/IL ⁇ 24
  • This down regulation of miR-221 correlates with mda-7/FL-24-mediated ceil death and over expression of miR-221 blocks cell death induced by mda-7/IL-24.
  • MDA-7/IL-24 Production and secretion of MDA-7/IL-24 following treatment with purified recombinant cytokine or infection with Ad.mda-7 decreases cell growth and induces apoptosis in cancer cells, but not in normal cells. Additionally, secreted MDA-7/IL-24 also induces apoptosis in surrounding cells as well as distant tumor cells through“bystander” antitumor effects (28) Furthermore, MDA-7/IL-24 protein induces production of endogenous MDA-7/TL-24 through an autocrine/paracrine loop (1 1). Using recombinant His-MDA-7 we found that MDA-7/IL-24 also down regulates miR-221, uniquely in IL-20/IL-22 receptor positive cancer ceils.
  • miR-221 represents a novel downstream target of mda-7/IL ⁇ 24 specific to cancer cells that mediates its biological anti -cancer functions both in vitro and in vivo.
  • mda-7/IL-24-mediated down regulation of miR-221 is ROS-dependent and treatment with anti-oxidants can reverse this process. These results accentuate a path for the development of rational combinatorial approaches for the treatment of aggressive tumors by combining mda-7/IL-24 with other in-clinic RGS- inducing chemotherapeutic agents.
  • MicroRNAs play a central role in regulating different normal and
  • miR-221 is significantly upregulated in different cancers (17).
  • To identify potential new targets of miR-221 we investigated the expression of some of the major proteins involved in autophagy, apoptosis and cell cycle. The regulation of beclin-1 expression by miR-221 was demonstrated by transfection of tumor cells with a miR-221 mimic, which resulted in a decrease in beclin-1 expression at both the mRNA and protein level. This relationship was confirmed further by transfection with anti -miR-221, which resulted in up regulation of beclin-1 expression.
  • Beclin-1 -mediated protective/toxic autophagy plays a decisive role in cell survival /death and aberrant expression of beclin-1 has been reported in different diseases including cancer (26, 46-49). Rapamycin is a well described autophagy inducer (50) and it also up regulates beclin-1 (36). Our results confirm that miR-221 not only degrades basal but also Rapamycin- and mda-7/TL-24-induced beclin-1 expression.
  • Example 2 Novel Mechanism of mda-7/IL-24- induction of Cancer Cell Specific Death
  • mda-7/IL-24 Melanoma differentiation associated gene-7/Interleukin-24 displays broad spectrum anti-cancer activity in vitro, in vivo in preelinical animal models and in a phase I/II clinical trial in patients with advanced cancers, without harming normal cells or tissues.
  • mda-7/IL-24 regulates a specific subset of miRNAs, including cancer associated miR-221.
  • Both ectopic expression of mda-7/IL-24 or treatment with recombinant His- MDA-7 protein down regulate miR-221, while up regulating p27 and PUMA, in a panel of cancer ceils culminating in cell death.
  • Mda-7/lL-24-induced cancer ceil death was dependent on reactive oxygen species induction and was rescued by over expressing miR-221.
  • Beclin-1 was identified as a new transcriptional target of miR-221 and mda-7/IL ⁇ 24 regulated autophagy through a miR- 221 /beclin-1 feedback loop.
  • miR-221 overexpressing MDA-MB-231 clones were more aggressive and resistant to mda-7/IL ⁇ 24-mediated cell death than MDA-MB-231 parental clones.
  • ROS inducers regulate miR-221 expression remains to be deciphered. We are working on this pathway to have a detailed knowledge of the system. This could lead to new combinatorial approaches to selectively target cancers for cell death.
  • MDA-7/TL-24 is a protein that is known to act as a tumor suppressor in several cancers, and its role in negatively regulating cancer cell survival is experimentally suggested and our results show that MDA-7/IL-24 can be used as a therapy for multiple diverse cancers.
  • Subtraction hybridization identified genes displaying differential expression as metastatic human melanoma cells terminally differentiated and lost tumorigenic properties by treatment with recombinant fibroblast interferon and mezerein. This approach permitted cloning of multiple genes displaying enhanced expression when melanoma ceils terminally differentiated, called melanoma differentiation associated (mda) genes.
  • mda-7 One mda gene, mda-7, has risen to the top of the list based on its relevance to cancer and now inflammation and other pathological states, which based on presence of a secretory sequence, chromosomal location and an EL- 10 signature motif has been named interleukin-24 (MDA-7/IL-24).
  • MDA-7/IL- 24 has proven to be a potent, near ubiquitous cancer suppressor gene capable of inducing cancer ceil death through apoptosis and toxic autophagy in cancer cells in vitro and in pre-clinical animal models in vivo.
  • MD A-7/IL-24 embodied profound anti-cancer activity in a Phase I/II clinical trial following direct injection with an adenovirus (Ad. mda-7, INGN-241) in tumors in patients with advanced cancers.
  • MDA-7/IL-24 has been implicated in many pathological states involving inflammation and may play a role in inflammatory bowel disease, psoriasis, cardiovascular disease, rheumatoid arthritis, tuberculosis and viral infection. This review provides an up-to-date review on the multifunctional gene mda- 7/IL-24, which may hold potential for the therapy of not only cancer, but also other pathological states.
  • MDA-7 Melanoma differentiation associated gene-7
  • IL- 24 interleukin-24
  • MDA-7/IL-24 was discovered several decades ago, new discoveries of the role that MDA-7/EL-24 plays in normal physiology as well as in multiple human pathologies are still unfolding. So far, researchers have confirmed that MDA-7/IL-24 is not only involved in normal immune function and wound healing, but it also has several additional beneficial effects in a variety of human diseases.
  • MDA-7/IL-24 functions as an anti -cancer gene in multiple diverse cancers including melanoma (Sarkar et ah, 2008), prostate cancer (Greco et ah, 2010; Lebedeva et al.
  • MDA-7/EL-24 provides protection against autoimmune diseases and bacterial infections (Leng et al, 2011; Ma et al, 2009). MDA- 7/IL-24 is also relevant in inflammation (Pasparakis et al, 2014), rheumatoid arthritis (Kragstrup et al, 2008) and cardiovascular diseases (Vargas-Alarcon et al, 2014).
  • MDA-7/IL-24 we discuss in detail the roles of MDA-7/IL-24 in both normal physiology as well as the various disease states mentioned above. We begin with a discussion of the characteristic features of MDA-7/IL-24 that allows this molecule to play a key role in normal cellular function as well as contributing to a variety of disease states.
  • MDA-7 was initially identified and cloned from terminally differentiating human melanoma cells in the Fisher laboratory' by Jiang in 1993 and reported in detail in 1995 (Jiang and Fisher, 1993; Jiang et al., 1995) HO-1 human metastatic melanoma cells were treated with a combination of recombinant human fibroblast interferon (EFN-beta) and mezerein (MEZ) to induce terminal differentiation and suppression of growth and turnorigenic abilities. Next subtraction hybridization of cDNA libraries was performed to assess genes that were differentially expressed in melanoma cells before and after terminal differentiation (Jiang and Fisher, 1993).
  • EFN-beta recombinant human fibroblast interferon
  • MEZ mezerein
  • MDA-7 was identified as one of the transcripts that was induced in terminally differentiating melanoma cells (Jiang and Fisher, 1993; Jiang et al., 1995). In subsequent years, MDA-7 was found to have tumor suppressive abilities against several different cancer indications, while leaving normal counterparts unharmed (Jiang et al., 1996; Su et al., 1998). In 2001, Huang and colleagues in the Fisher laboratory' identified the genomic structure and chromosomal localization of MDA-7 (Huang et al., 2001) They determined that MDA-7 was located in a region of the chromosome that contained a cluster of genes associated with the IL-10 cytokine family (Huang et al., 2001).
  • MDA-7 also had an IL-10 signature sequence and w'as specifically expressed in tissues associated with the immune system including the spleen, thymus and peripheral blood leukocytes (Huang et al., 2001). Given the conserved chromosomal location, presence of a putative secretory motif, an IL-10 signature sequence and the expression profile of MDA-7, the Human Gene Organisation (HUGO) designated this gene as interleukin-24 (II, -24) (Sarkar et al., 2002a). Additionally in 2002, Caudell and colleagues provided evidence that MDA-7/IL-24 had functional immunostimulatory attributes justifying its designation as an interleukin (Caudell et al., 2002).
  • MDA-7/IL-24 Located on chromosome lq32-33 in humans, MDA- 7/IL-24 is a secreted cytokine that belongs to the IL-10 gene family (Caudell et al., 2002; Huang et al., 2001). MDA-7/EL-24 contains seven exons and six introns. The cDNA of MDA-7/IL-24 is 1,718 base pairs and the protein encodes 206-amino acids (Huang et al., 2001). Being a secreted cytokine, MDA-7/IL-24 has a 49-amino acid N terminal hydrophobic signal peptide that allows for protein secretion (FIG. 16).
  • MDA-7/IL-24 The predicted tertiary structure of MDA-7/IL-24 is that of a compact globular molecule comprised of four strongly helical regions interspersed by loops of unpredicted structure (Sauane et al, 2003b). MDA-7/IL-24 can also form N-linked glycosylated dimers though intermolecular disulfide bonds and these dimers are functionally active (Mumm et al., 2006).
  • U20S human osteosarcoma cells treated with siRNA to SRp55 were assessed using a splice-specific microarray analysis to identify the relevance of SRp55 on the splicing patterns of genes involved in apoptosis. At least 4 isoforms of AIDA-7 were identified, out of which one isoform (that lacks exons 2 and 3) was sensitive to splicing by SRp55 and silencing SRp55 splicing activity caused an increase in this isoform. In a follow-up study, Whitaker and colleagues identified and
  • the splice variants identified were mda ⁇ 7/IL ⁇ 2453,5 - lacking exons 3 and 5 (described and characterized previously by (Allen et al., 2004)); mda-7/IL-2455 - lacking exon 5; mda-7/IL-2462,3 - lacking exons 2 and 3; mda-7/IL-2452,5 - lacking exons 2 and 5; rnda-7/IL- 2452,3,5 - lacking exons 2, 3 and 5, and mda-7/IL-2452 - lacking exon 2. All 7 exons were present in the full-length transcript.
  • mda-7/IL-24 isoforms might vary based on different cell-types.
  • Full length MDA-7/IL-24 as well as spliced isoforms 65, 62,3,5, 62,5 and 62 were capable of reducing U20S cell viability with no effect on the viability of non-cancerous immortalized NOK cells (Whitaker et al., 2011).
  • deletion of the signal peptide (Ml) did not disrupt the tumor inhibitory effects of MDA-7/IL-24.
  • all the other deletions, except M4 caused a loss of tumor inhibitory effects.
  • M4 showed tumor suppressive effects in Hela and DU-145 cells but did not affect normal prostate epithelial P69 cells and was capable of inducing cancer cell -specific apoptosis (Gupta et al., 2006b).
  • MDA-7/IL-24 protein gets ubiquitinated and degraded via the 26S proteasome.
  • Tian and colleagues mutated each of the 10 lysine sites within the MDA-7/IL-24 protein and converted them to arginine (Tian et al., 2012). They identified lysine 123 as the critical internal lysine involved in MDA-7/IL-24 ubiquitination.
  • EL- 10 cytokine family members signal through receptor dimers that consist of an R1 type receptor (with a long cytoplasmic domain) and an R2 type receptor (with a short cytoplasmic domain).
  • the IL-10 cytokine family of receptors has three R1 and two R2 subunits.
  • the R1 subunits are IL-lORi, IL-20R1 and IL-22R1 and the R2 subunits are IL-20R2 and IL-10R2.
  • MDA-7/IL-24 can also signal and induce growth suppression and apoptosis in a cancer-selective manner using the IL- 20R1/1L22-R1 heterodimeric receptors (Dash et ah, 2014; Pradhan et ah, 2017).
  • the mechanism through which these two R1 receptor dimers promote signaling after interacting with MDA-7/IL-24 remains to be determined.
  • MDA-7/IL-24 can be produced by immune cells (myeloid cells and lymphoid cells and monocytes) in response to treatment with lipopolysaccharides or specific cytokines (Buzas et ah, 201 1). Physiological levels of MDA-7/IL-24 are induced in Th2 lymphocytes by stimulation with phorbol my ri state acetate (PMA) and Ionomycin and in T cells, especially CD4+ naive and memory cells activated by anti-CD3 monoclonal antibody (Sahoo et ah, 2011; Schaefer et ah, 2001).
  • PMA phorbol my ri state acetate
  • Ionomycin Ionomycin
  • MDA-7/IL-24 is induced by antigenic stimulation with iipopoiysaccharide, concanavalin A and cytokines (Caudell et ah, 2002; Wolk et ah, 2004).
  • B cell receptor signaling also triggers MDA- 7/IL-24 expression in B-lymphocytes (Maarof et ah, 2010).
  • Non-lymphoid cells can also produce physiological levels of MDA-7/IL-24 in response to cytokines secreted by immune cells (Persaud et ah, 2016).
  • MDA-7/IL-24 FUNCTIONS UNDER PHYSIOLOGICAL CONDITIONS .
  • MDA-7/IL-24 is produced by various immune cells and exerts a range of immune functions (Persaud et ah, 2016). At lower physiological concentrations, MDA-7/IL-24 mainly functions as a cytokine. MDA-7/IL-24, when secreted, interacts with distinct sets of receptors including IL-20R1/IL-20R2, IL-22R1/IL-20R2 or IL-22R1/IL-20R1 receptor complexes (Dash et al., 2014; Dumoutier et al., 2001; Wang and Liang, 2005, Wang et al, 2002) Most immune cells lack the cognate pairs of receptors and chiefly express IL-20R2.
  • MDA-7/IL-24 assessed the secretion profile of peripheral blood mononuclear cells treated with MDA-7/IL-24 protein, which showed increased secretion of immune modulatory cytokines such as IL-6, IL-Ib, IFN-g, TNF-a, IL-12 and GM-CSF (Caudell et al, 2002).
  • IFN-g in turn upregulates IL ⁇ 22R1 expression in keratinocytes, which facilitates formation of IL-22R 1 /IL-2GR2 receptor pairs and induces innate immunity responses (Wolk et al, 2004)
  • MDA-7/IL-24 also induces several additional changes in normal skin cells.
  • MDA-7/TL-24 also impedes B cell maturation to plasma ceils by regulating several transcription factors, which are important for plasma cell differentiation
  • MDA-7/IL-24 plays a diverse role in pro-inflammatory, infectious and autoimmune skin diseases, which is discussed in further detail below (Persaud et al., 2016).
  • MDA-7/IL-24 Apart from these immune and dermatologic functions, several studies have also reported other biological functions of MDA-7/IL-24 in vascular diseases and inflammatory' bowel disease (Persaud et al., 2016). MDA-7/TL-24 is also expressed in normal cultured fetal membranes, suggesting a potential role in normal pregnancy (Nace et al., 2010).
  • STEM CELLS AND DIFFERENTIATION.
  • Tumors are comprised of heterogeneous cell populations with diverse biological properties.
  • Cancer stem cells are immortal cells within tumors which display the property of self-renewal. They can divide and differentiate to give rise to a heterogeneous cell population, in which subsets of cells can form distant tumors (Talukdar et a!., 2016). Stem cells detach from the primary tumor, migrate and generate tumors at distant sites. Cancer stem cells can relapse and metastasize making the need for specific therapies against them essential (Talukdar et al., 2016). They are also resistant to conventional therapies and divide more rapidly (Morrison et al., 2013).
  • mda-7/IL ⁇ 24 inhibits the growth of breast cancer stem cells. Specifically, infection of
  • Ad.mda-7 decreased proliferation of breast cancer initiating ceils without harming normal stem cells (Bhutia et al., 2013).
  • Over expression of mda-7/IL-24 induces apoptosis and ER stress in sorted stem cell populations of breast cancer cells, which is similar to what is observed in unsorted breast cancer cells (Bhutia et al., 2013).
  • Over expression of mda-7/IL-24 also decreases the self renewal capabilities of cancer stem cells.
  • mda-7/IL-24 suppresses b-catenin/Wnt signaling (Chada et al , 2005; Sieger et al., 2004) and regulates the proliferation of stem cells.
  • the Wnt/b catenin pathway is one of the key signaling pathways that promotes self-renewal of stem cells (Xu et al., 2016). Wnt proteins interact with Frizzled and LRP receptors to signal b-catenin to activate Wnt target genes (MacDonald and He, 2012) It can also signal through ROR/RYK receptors as an alternative pathway (Green et al., 2014). In cancer, these are dynamically expressed and this causes an imbalance in the proliferation and differentiation of cancer stem cells. Alteration of the b-catenin signaling pathway increases the survival of stem cells. This suggests that mda-7/IL-24- mediated blockage in proliferation of ste cells is facilitated through the b-catenin pathway.
  • mda-7/IL-24 Over expression of mda-7/IL-24 by an adenoviral system increased the expression of tumor suppressors including PTEN, E-cadherin, GSK-3p, and APC and down regulated proto- oncogenes involved in b-catenin and PI3K signaling (Gupta et al, 2006a).
  • b-catenin translocates to the plasma membrane from the nucleus upon mda-7/TL-24 treatment, which reduces the transcriptional activity of TCF/LEF (Mhashilkar et al., 2003) This up-regulates the expression of E-cadherin- -catenin adhesion in a cancer-selective manner.
  • mda-7/TL- 24 regulates cell-cell adhesion by modulating these signaling cascades (Mhashilkar et al , 2003). These effects are not common in normal cells and are specific for cancer cells.
  • Ad.mda-7 down regulates the tendency of breast cancer cells to form mammospheres and also inhibits the formation of distant tumors (Bhutia et al, 2013). A small proportion of stem cells are the progenitors of metastatic tumors, even after surgery, and they tend to be resistant to radiotherapy (Eyler and Rich, 2008).
  • mda ⁇ 7/IL-24 can induce apoptosis by down regulating Akt, Bel2, and Bcl-xL as described earlier (FIG. 18).
  • APOPTOSIS Programmed cell death or apoptosis plays a pivotal therapeutic role in cancer drug sensitivity (Naik et al., 1996).
  • apoptosis (Hanahan and Weinberg, 2000). It involves a series of signaling events that are disrupted in cancer. Cancer cells bypass the apoptotic signaling pathway and evade this mechanism of cell death (Femald and Kurokawa, 2013)
  • Much of the research focusing on cancer therapeutics involves the ability of the therapy to induce apoptosis, specifically in cancer cells (Lebedeva et al., 2003a). Side effects of chemotherapy are due to non-selective toxicity toward normal cells. Understanding the mechanism by which cancer cells evade the general apoptotic pathways is critical to develop new therapies against cancer.
  • mda-7/IL-24 regulates ER (endoplasmic reticulum) stress and the mitochondrial apoptotic pathway (Fisher, 2005; Gopalkrishnan et al., 2004; Lebedeva et al., 2003c; Lebedeva et al., 2005a, Lebedeva et ah, 2005b, Sauane et al., 2008; Sieger et ah, 2004).
  • Over expression of mda-7/IL-24 has been shown to induce apoptosis in different cancer cells without any harmful effect to normal cells (reviewed in Fisher, 2005). This cancer cell-specific death is both time- and dose-dependent.
  • SB203580 an inhibitor of the p38MAPK pathway, inhibits Ad.mda-7-induced apoptosis.
  • the p38MAPK or mitogen protein kinase pathway is altered due to over expression of mda-7/IL-24 (Sarkar et al., 2002b). This induces GADD genes (growth arrest and DNA damage) leading to cell cycle arrest and cell death (Sarkar et al., 2002b).
  • GADD genes growth arrest and DNA damage leading to cell cycle arrest and cell death
  • AIF -mediated apoptosis by mda- 7/1L-24 has recently been demonstrated to occur uniquely in neuroblastoma (Bhoopathi et ah,
  • miR-221 targets PUMA, a proapoptotic gene, blocking apoptosis (Pradhan et al.,
  • mda-7/IL-24 down regulates miR-221, which in turn up regulates PUMA inducing cell death (Pradhan et al., 2017)
  • mda-7/IL-24 down regulates the expression of anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl2, while inducing pro-apoptotic proteins such as Bid, Bim, Bax, and Bak (Menez.es et al, 2014)
  • pro-apoptotic proteins such as Bid, Bim, Bax, and Bak
  • mda-7/!L-24 increases the Bax/Bc!2 ratio (Pei et a! , 2012).
  • Previous studies also demonstrated a role of PERK in mda-7/IL-24-mediated cell death (Park et al, 2008).
  • ROS reactive oxygen species
  • ROS inducers enhance cell death mediated by mda-7/IL-24 (Lebedeva et al, 2005b; Sauane et al, 2008). These results confirm the role of ROS and mitochondrial membrane potential as an important component in cell death promoted in cancer cells by the cytokine MDA-7/IL-24.
  • mda-7/IL-24 also up regulates SARI, a tumor suppressor, which is cancer-specific (Dash et al, 2014). Ectopic expression of mda-7/IL-24 induces SARI mRNA and protein in a broad panel of cancer cells (Dash et al, 2014). SARI expression is required for the anti-tumor effects of mda-7/TL-24. Recombinant MDA-7/TL-24 protein also induces SARI expression through binding to its cognate receptors, IL-20R1/IL-20R2/IL-22R1 (Dash et al, 2014).
  • the FasL signaling pathway is another pathway activated by Ad.mda-7, which results in cancer-cell selective apoptosis (Gopalan et al, 2005).
  • Ad.mda-7 induces activation of the transcription factors c-Jun and ATF2 (activating transcription factor 2) inducing FasL -Fas (Gopalan et al, 2005).
  • siRNA targeting Fas decreased mda-7/IL-24-induced cell death in ovarian cancer cells (Gopalan et al, 2005). This work reveals a role of mda-7/IL-24 in regulating the Fas- FasL signaling cascade to induce cancer cell death.
  • mda-7/IL-24 up regulates PKR (serine/threonine protein kinase) in non-small cell lung cancer, which is independent of p53 expression (Mhashilkar et al., 2003).
  • PKR serine/threonine protein kinase
  • the regulation of PKR by mda-7/TL-24 is post transcriptional (Gupta et al, 2006a).
  • Exogenous recombinant mda-7/IL-24 also induces PKR and mda ⁇ 7/IL-24 interacts with PKR in cancer cells (Pataer et al, 2005)
  • Apoptosis mediated by mda-7/IL-24 is independent of p53 mutations and functions (Gupta et al, 2006a; Su et al, 2003). It is established that mda-7/TL-24 induces apoptosis in diverse breast cancer cells, e.g., MCF7 (p53-wt), MDA-MB-231 (mutant p53), MDA-MB-453 (mutant p53), and T47D (mutant p53) (Chada et al., 2006). Based on different genetic
  • mda-7/IL-24-induced apoptosis is distinct from other identified tumor suppressors
  • MDA-7/IL-24 protein functions as an anti -angiogenic molecule (Chada et al, 2004a; Nishikawa et al., 2004). It binds to its cognate receptor pairs and induces phosphorylation and nuclear translocation of STAT3 (Chada et al, 2004a) This receptor interaction induces BAX protein leading to cell death (Gupta et al, 2006a). This process is ST AT3 -independent as other interleukins (IL ⁇ I0, IL-19, IL-20, and IL-22) also activate STAT3 without promoting cell death (Mosser and Zhang, 2008).
  • mda-7/TL-24 binds IL-20/IL-22 receptor complexes resulting in acti vation of the JAK/STAT cascade.
  • S tudies have shown that mda-7/IL-24 induces apoptosis of cancer cells independent of the JAK/STAT pathway (Sauane et al., 2003a). Specifically, inhibitors of JAK/STAT pathway do not inhibit apoptosis mediated by mda-7/IL-24 (Sauane et al., 2003a). These results demonstrate that mda-7/Il, ⁇ 24 is independent of tyrosine kinase activation.
  • Autophagy is the process of degradation of organelles located in the cytoplasm. This process is complex owing to its differential context dependent role.“Is autophagy good or bad for life and cancer?” is a difficult question to answer (Bhutia et al., 2013). Sometimes it is protective, helping cancer cells to survive adverse conditions but it can also be toxic towards cancer cells (Bhutia et al., 2013; Liu and Debnath, 2016) (FIG. 19) Small molecules that can control autophagy may in certain contexts provide therapeutic benefit. Autophagy is a conserved phenomenon and it promotes tumor growth in advanced cases of cancer.
  • mda ⁇ 7/IL-24 induces autophagy, which is mediated by PERK (Park et al., 2008) and Beclin-1 (Bhutia et al, 2010).
  • mda-7/IL-24 regulates a subset of microRNAs, including the oncogenic microRNA, miR-221 (Pradhan et al., 2017). Beclin-1 was identified as a new transcriptional target of miR-221 (Pradhan et al, 2017).
  • mda-7/IL-24 down regulates miR-221, which in turn induces beclin-1, leading to autophagy (Pradhan et al., 2017). Cleavage of LC3, a marker of autophagy, is also observed.
  • CD95 is an important regulatory' molecule in the induction of autophagy mediated by mda-7/IL-24 (Park et al., 2009).
  • ANGIOGENESIS Cancer and metastatic spread depends on an adequate supply of nutrients and oxygen to cells (Welch and Fisher, 2016) Additionally, removal of waste products also requires new blood and lymph vessels. The process of formation of new blood vessels is called angiogenesis, which represents another hallmark of cancer (Han ah an and Weinberg, 2011). Angiogenesis is regulated by a number of activator and inhibitor molecules. Although not as effective as anticipated when used as a single agent, angiogenesis inhibitors combined with other therapeutic agents are showing promise in the treatment of various cancers.
  • mda-7/IL-24 Over expression of mda-7/IL-24 in HUVEC cells or human umbilical vascular endothelial cells inhibits endothelial cell differentiation (Dash et ah, 2010, Wang et ah, 2016) Similarly, treatment of tumor xenografts with mda-7/IL-24 reduces expression of angiogenesis markers (Bhutia et ah, 2012).
  • VEGF Vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • the PBK/Akt pathway is another signaling cascade known to regulate angiogenesis (Karar and Maity, 2011) and mda-7/IL-24 down regulates phospho Akt and can therefore negatively modulate angiogenesis (Dash et ah, 2010).
  • mda-7/IL-24 has been shown to impede the migration of cancer cells (Ramesh et ah, 2004). Also, over expression of mda-7/IL-24 results in a decrease in the in vitro invasion of an array of different cancer cell types (Ramesh et ah, 2004). Lung cancer cells showed an inhibition in migration and invasion by modulating a number of signaling cascades (Panneerselvam et ah, 2015). Focal adhesion kinase (FAK) and matrix
  • MMPs metalloproteinases
  • mda- /IL- 24 regulates a number of molecules related to metastasis, e.g., cyclin-Bl, TGF-b, Survinin, Twist, ICAM-1, and CD44 (Huo et ah, 2013). Also, E-cadherin, NF-KB, and PERK are regulated by mda-7/IL-24 (Panneerselvam et ah, 2013). mda-7/IL ⁇ 24-mediated inhibition in invasion and metastasis is both receptor-dependent and receptor-independent (Menezes et ah, 2014).
  • SYNERGISTIC EFFECTS Cancer is a complex process that is mediated by multiple genetic and epigenetic changes that impact directly and indirectly on a number of pivotal signaling pathways involved in cell growth, survival, resistance to apoptosis, and additional physiologically relevant processes (Hanahan and Weinberg, 2000, 2011). Considering this complexity, it is not surprising that a single targeting molecule fails to provide complete therapy resulting in a cure in most cancers. Conversely, a combinatorial approach using multiple target-selective agents directed toward specific signaling abnormalities in defined cancers have shown promise in cancer therapy.
  • Table 1 Combinatorial enhancement of therapy by combining mda ⁇ 7/IL ⁇ 24 with other therapeutic modalities.
  • BYSTANDER ACTIVITY Evidence of bystander activity of mda-7/TL-24 (Su et ai 2005) was shown in vivo in animal studies, where tumor cells were injected in both flanks of nude mice (Pradhan et a!., 2017, Sarkar et a! , 2007, Sarkar et a! , 2008; Sarker et a! , 2005, Su et ah, 2005). A tumor on one flank was treated while the tumor on the other flank was left untreated. Tumor measurements showed a decrease in tumor size in the treated as well as the untreated tumor.
  • the inhibitory action on distant tumors can be explained by the anti-tumor“bystander” activity of the secreted mda ⁇ 7/IL ⁇ 24 cytokine and its ability to induce apoptosis and promote production of MDA-7/IL-24 through dimeric receptor pairs in the untreated tumor (Menezes et ah, 2014; Sauane et ah, 2008).
  • secreted MDA-7/IL-24 requires a complete set of dimeric cell surface receptors (Dash et al, 2014; Dumoutier et al, 2001; Wang et al, 2002).
  • Secreted MDA-7/IL-24 binds to the dimeric receptor pair and induces cancer cell death (Dash et al, 2014; Menezes et al, 2014).
  • IL-20R1/IL-20R2 antibodies it has been demonstrated that mda-7/IL-24-mediated cell death is receptor-dependent (Chada et al, 2004a).
  • Normal cells also promote‘"bystander” activity after exposure to mda-7/IL-24, which results in production and secretion of MDA-7/IL-24 without inducing toxicity or cell death.
  • MDA-7/IL-24 induces IL-6, TNF-a, IFN-g, IL-Ib, and IL- 12, which are potent immunoregulatory molecules (Caudell et al., 2002; Deng et al., 2011;
  • MDA-7/IL-24 has been extensively studied in cancer. In addition to its function as a tumor suppressor and apoptosis-toxic autophagy inducing cytokine in cancer, MDA-7/IL-24 has also been reported to play a significant role in inflammation, cardiovascular disease, autoimmune diseases and viral replication.
  • Inflammation The skin is the largest organ in the body and plays an essential role in promoting immunity and defense against pathogenic microorganisms. However, dysregu!ated immune reactions can cause chronic inflammatory skin diseases. Extensive crosstalk between the different cellular and microbial components of the skin regulates local immune responses to ensure efficient host defense, to maintain and restore homeostasis, and to prevent chronic disease. In this section, we briefly discuss recent findings that highlight a role of MD A-7/IL-24 in inflammation. IL-19 and MDA-7/IL-24 belong to the IL-20 subfamily and are known to be involved in host defense against bacteria and fungi, tissue remodeling and wound healing
  • MDA-7/IL- 24 may be a member of a complex cascade of cytokines involved in inflammation as MDA-7/IL- 24 can induce expression of many cytokines, including TNF-a, IL-6 and interferon-g (IFN-g) (Wang and Liang, 2005).
  • MDA-7/IL-24 and its receptor expression pattern supports a major physiological function related to epidermal functions, such as wound healing, and abnormalities may be part of the cause of pathological skin conditions such as psoriasis.
  • IBD inflammatory bowel disease
  • Ultrichos disease chronic inflammation of all parts of the digestive tract may bring about inflammatory bowel disease (IBD). This includes primarily ulcerative colitis and Crohn’s disease. The symptoms for both of these conditions include severe diarrhea, pain, fatigue and weight loss. Genomic abnormalities and environmental factors can trigger IBD. Andoh and colleagues assessed the expression ofMDA-7/IL-24 in inflamed mucosa of IBD patients and determined the molecular mechanism that resulted in MDA-7/IL-24 expression in coionic subepithehal myofibroblasts (Andoh et ah, 2009). They demonstrated that MDA-7/IL-24 expression is enhanced in the inflamed mucosa of active IBD patients.
  • MUC genes are the primary component of the mucin barrier that divides the intestinal microbiota and the intestinal epithelium. MUC genes also play an important role in the pathogenesis of IBD. This study showed that IL-24 expression is elevated in inflamed mucosa of IBD patients compared to control patients. Work done by other researchers show that the IL-10 subfamily of cytokines is involved in immune regulation and inflammatory responses.
  • MDA-7/IL-24 is synthesized by peripheral B cells, CD4+ T cells, CD8+ T cells and monocytes. Overall, MDA-7/IL-24 can promote a suppressive inflammatory effect on colonic epithelial cells and mucosal inflammation in IBD.
  • Psoriasis is a common chronic inflammatory skin disease resulting from a complex interplay among the immune system, keratinocytes, susceptibility genes, and
  • MDA-7/IL-24 was elevated in psoriatic skin compared to normal skin. It is also reported that MDA-7/TL-24 can induce different psoriasis-associated factors, which can promote inflammation and epidermal hyperplasia (Kumari et al., 2013).
  • MDA-7/IL-24 The interleukin 10 family of cytokines including MDA-7/IL-24 has been implicated in the pathogenesis of psoriasis (Kunz et al., 2006; Leng et al., 201 1 ; Romer et al, 2003; Weiss et al., 2004, Wolk et a!., 2009) These reports also showed an increased expression of MDA-7/IL-24 in psoriatic skin compared to normal skin. MDA-7/IL-24 was mainly produced by keratinocytes, myeloid cells, and T cells (Conti et a! , 2003; Kunz et ah, 2006, Zheng et aL, 2007b).
  • MDA-7/TL-24 receptors are also found in keratinocytes and they signal by activating STAT3 (Dumoutier et al., 2001 ; Kunz et al., 2006; Parrish-Novak et al., 2002).
  • STAT3 over expression is also observed in psoriatic skin conditions and the expression of constitutively active STAT3 in epidermal keratinocytes also caused psoriasis-like skin inflammation in mice (Sano et al., 2005), which suggests an important role for epidermal STAT3 signaling in psoriasis (Kumari et al., 2013).
  • Cardiovascular Disease vascular calcification is a symptom of cardiovascular disease. Wang and colleagues showed that low concentration of ! 1202 treatment induced abnormal proliferation of vascular endothelial cells and MDA-7/TL-24 inhibited this proliferation (Wang et al., 2016). They also showed that MDA-7/IL-24 could inhibit apoptosis by inhibiting RQS production in vascular endothelial cells. MDA-7/IL-24 is also involved in the down regulation of several genes that regulate cardiovascular disease. The authors concluded that MDA-7/TL-24 can provide a basic therapeutic strategy for treating vascular disease and cancer by inhibiting ROS production in vascular cells.
  • MDA-7/IL-24 Lower levels of MDA-7/IL-24 were observed in hypertensive rats compared to controls, and anti-hypertensive therapy increased MDA-7/IL-24 levels. Hypertension is also a hallmark of cardiovascular disease. MDA-7/TL-24 was identified as one of the 16 differentially regulated genes in spontaneously hypertensive rats. MDA-7/TL-24 also regulates the expression of inflammation- and hypertension-related genes in a H202-treated mouse vascular smooth muscle cell line, MOYAS. This study also showed that MDA-7/IL-24 attenuates H202- induced activation of PBK/Akt and Erk.
  • MDA-7/IL-24 can inhibit ROS production by regulating mitochondrial ROS release mediated by PBK/Akt and Erk pathway in H202 -treated vascular smooth muscle cells, VSMG’s (Lee et al., 2012). This inhibition of ROS in VSMC leads to reduced cell growth and migration.
  • Another study by Chen and colleagues also indicated that adenovirus-mediated expression of MDA-7/EL- 24 could inhibit pulmonary arterial smooth muscle cell line (PAC1-SMC) migration and proliferation, leading to reduced intimal hyperplasia (Chen et al., 2003).
  • PAC1-SMC pulmonary arterial smooth muscle cell line
  • MDA-7/EL-24 may be a novel therapeutic target for cardiovascular disease and/or hypertension.
  • MDA-7/TL-24 inhibits b-GP -induced vascular smooth muscle cell calcification.
  • Activation of the Wnt/p-catenin pathway by b-GP is inhibited by MDA-7/IL-24, which indicates that the inhibitor ⁇ ' effect of MDA-7/IL-24 on VSMC calcification correlates with the inactivation of the Wnt/p-catenin pathway.
  • the authors did not explore the role of Jak/Stat pathway mediated b-GP -induced VSMC calcification by MDA-7/EL-24 in this study, they showed an effect of MDA-7/TL-24 on inhibition of the Wnt/p-catenin pathway using a neutralizing antibody to MDA-7/IL-24.
  • MDA-7/EL-24 This inhibition by MDA-7/EL-24 correlates with suppression of apoptosis, and the expression of osteoblast markers and calcification by down regulation of BMP- 2 and the Wnt/p-catenin pathway. They also showed that p-GP increased the expression of calcification and osteoblastic markers in VSMCs (Persaud et al., 2016). This effect is specifically inhibited by MDA-7/IL-24 suggesting that MDA-7/IL-24 suppresses downstream molecules by inhibiting BMP-2 expression. The inhibitory effect MDA-7/IL-24 on VSMC calcification is mediated at least in part through anti-apoptotie activity.
  • statins which are hydroxy-3-niethylglutaryl coenzyme A reductase inhibitors.
  • MDA-7/IL-24 appears to play a distinct role in cardiovascular disease. MDA-7/IL-24 can promote the growth of vascular smooth muscle cells by suppressing calcification and osteoblast marker expression, which is associated with
  • MDA-7/IL-24 also may provide benefit in the treatment of vascular disorders since it selectively inhibits rat pulmonary arterial smooth muscle cell growth and migration. Polymorphisms in the MDA-7/IL-24 gene also correlate with cardiovascular and metabolic risk factors, further supporting a relationship between MDA-7/IL-24 and cardiovascular diseases.
  • Rheumatoid arthritis is an inflammatory auto-immune disease that can lead to progressive joint damage and disability.
  • Cytokines including IL-1, IL-6, IL-8, IL-1Q, monocyte chemo-attractant protein I (CCL2/MCP-1), and tumor necrosis factor (TNFa) play an important role in RA.
  • SpA spondyloarthropathy
  • OA osteoarthritis
  • Tuberculosis Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis in humans. Although the lungs are the primary organs altered by TB infection, other parts of the body can also be affected. Wu and colleagues reported that active TB patients had decreased expression of MDA-7/IL-24 compared to individuals with latent TB infection. This observation led them to investigate the role of MD A-7/IL-24 in pulmonary TB patients. Since IFN-g plays an important role in TB infection, and the levels of IFN-g were similar to MDA-7/1L- 24 levels in these patients, they investigated the role of MDA-7/TL-24 on IFN-g expression.
  • PBMCs isolated from these individuals were stimulated with Mycobacterium tuberculosis early secreted Ag of 6 kDa (EAST-6) to determine the levels of gene expression .
  • EAST-6 Mycobacterium tuberculosis early secreted Ag of 6 kDa
  • IFN-g IFN-g levels
  • neutralizing MDA-7/TL-24 decreased IFN-g.
  • This upregulation of IFN-g with exogenous MDA-7/IL-24 w3 ⁇ 4s due to increased levels of IL-12a, IE-12b, IL-23a and IL-27.
  • MDA-7/IL-24 regulates EFN-g in TB patients and targeting MDA-7/IL-24 might be a treatment option for these patients.
  • Influenza virus replication Influenza infection also known as flu, is associated with mild to severe symptoms including fever, headaches, runny nose and fatigue.
  • MDA-7/EL-24 was known to influence TLR3-mediated apoptosis and influenza virus can stimulate the TLR3 receptor.
  • the reduction of viral replication by MDA-7/IL-24 could be independent of type I interferon.
  • MDA-7/TL-24 could inhibit Mel I and induce caspase 3 cleavage due to initiation of TLR3-mediated apoptosis. This was further demonstrated by TLR3 knockdown or by treating cells with a Pan-Caspase inhibitor. Inhibition of anti-apoptotic proteins Bcl2, Bax, and Bcl-xL was also observed following MDA- 7/IL-24 expression. They established that Mcll is the key factor in MDA-7/IL-24-mediated inhibition of influenza A virus replicati on. They also showed that MDA-7/EL-24 expressed by influenza A virus vector does not have any toxicity in mice. Another study by Seong and colleagues also showed that MDA-7/IL-24 expression decreased influenza viral replication (Seong et al., 2016).
  • MDA-7/IL-24 decreased the transcript level of the viral nucleoprotein (NP) gene following influenza vims infection as compared to viral infection alone, confirming an inhibitor ) ⁇ role of MDA-7/IL-24 in viral replication. Furthermore, an MDA-7/IL-24 expressing recombinant adenovirus did not induce toxicity as compared to a wild type adenovirus, suggesting that MDA-7/IL-24 can specifically target virus infected cells. Taken together, these studies suggest that MDA-7/IL-24 exerts potent inhibitor)' activity of influenza viral replication and can be used as a promising novel approach to suppress viral infections (Seong et al, 2016; Weiss et al., 2015).
  • MDA-7/IL-24 IMMUNOLOGICAL EFFECTS OF MDA-7/IL-24.
  • the role of MDA-7/IL-24 in normal physiology and disease pathology is quite diverse and depends principally on the source of production/secretion, and the target tissue.
  • MDA-7/IL-24 exerts immune- modulatory' functions in diverse autoimmune, infectious and immuno-patho!ogical diseases including Rheumatoid arthritis, Psoriasis, Inflammatory bowel diseases and others, as discussed in detail above (also reviewed in Persaud, De Jesus et al 2016).
  • MDA-7/IL-24 also plays a prominent role in host defense by inducing innate immune response in epithelial tissue during infection and inflammation by induction of chemokines and recruitment/activation of leukocytes (Jin et al., 2014; Tamai et al, 2012).
  • MDA-7/IL-24 Apart from these immune-modulatory roles in diverse biological diseases, MDA-7/IL-24 also exerts a profound immune stimulatory effect in the context of cancer. Forced expression of MDA-7/IL-24 induces IFN-g and IL-6 secretion from melanoma cells and displays potent anti tumor functions (Caudell et al., 2002; Chada et al., 2004b). Transduction of AID A-7/IL-24 via an adenoviral vector resulted in a significant increase in the CD3+ and CD8+ population, thereby facilitating immune activation and antitumor immunity. In one recent study, Ma et al.
  • MDA-7/IL-24 evaluated the efficacy of MDA-7/IL-24 in inhibiting colon cancer progression in murine models with an intact immune system and explored the immune-modulatory role of MDA-7/IL-24 in colon cancer progression (Ma et al., 2016b).
  • Menezes and colleagues in the Fisher laboratory' assessed the relevance of immune response in MDA-7/TL, ⁇ 24-mediated tumor suppression in a transgenic murine mouse model of breast cancer with an intact immune system (Menezes et al., 2015).
  • Ad.5-CTV replication competent cancer-selective adenovirus expressing MDA-7/IL- 24; a Cancer Terminator Vims
  • a significant increase in infiltrating CD8+T cells, along with increased IFN-g and granzyme B expression was also observed in non-treated tumors derived from MMTV-PyMT transgenic mice that received Ad5-CTV suggesting that MDA-7/IL-24 is capable of inducing a systemic immune response in an intact immune microenvironment (Menezes et ah, 2015).
  • Ad.mda-7 (INGN-241) showed a marked increase in CD3+ and CD8+ T cells at day 15 following injection as well as transient increases in circulating cytokines, such as EL-6, IL-10 and TNF-cx (Cunningham et ah, 2005; Tong et al., 2005). A few patients showed elevated levels of GM-CSF and IL-2 as well.
  • cytokines such as EL-6, IL-10 and TNF-cx
  • MDA- 7/EL-24 plays significant roles in a number of different human diseases.
  • MDA-7/IL-24 was primarily recognized for its role as a tumor suppressor in cancer.
  • MDA-7/IL-24 became available our understanding of its relevance in other diseases has also increased.
  • a detailed understanding of the molecular mechanisms defining the function of MDA-7/IL-24 have helped develop several predinical therapeutic options as well as therapeutic targets against cancer.
  • MDA- 7/IL-24 has already been tested in clinical trials for cancer and a Phase I clinical trial with MDA- 7/1L-24 (INGN 241) showed promising results (Cunningham et ah, 2005, Tong et ah, 2005).
  • the search for new molecules and compounds that can enhance or stabilize MDA-7/IL-24 protein are also ongoing.
  • combination therapies that would enhance MDA ⁇ 7/IL ⁇ 24-mediated tumor cell killing and prevent tumor growth and metastasis are also being identified and tested preciinically (Menezes et ah, 2014).
  • MDA-7/IL-24 therapeutic options are developed in one disease indication, they will also be valuable against other human diseases with MDA-7/IL-24 involvement. Further information gained regarding the role of MDA-7/TL-24 in diseases where MDA-7/IL-24 is over expressed will allow researchers and clinicans to develop new'er approaches to manage these conditions. Given the currently known functions of MDA-7/IL-24, it is likely that MDA-7/IL-24 will also be implicated in other disease indications. Such information will be critical for understanding the multifaceted role of MDA- 7/IL-24 in human physiology.
  • Example 4 MDA-7/IL-24 regulates the miRNA processing enzyme DICER through downregulation of MIT F
  • DICER, DICER promoter, and MITF plasmids were obtained from Addgene, MA, USA.
  • the MDA-7/IL-24 CAS9 construct and its vector control were from Genecopoeia, MD, USA.
  • Ad.mda- 7, a replication incompetent Ad 5 expressing mda-7/IL-24, and Ad. null, a replication incompetent Ad5 without a gene insert, were constructed and amplified as described (25-27).
  • IM-PHFA cells were infected with Ad.5-His-mda-7 using a standard protocol.
  • DU-145, MDA-MB-231, A549, and RWPE-1 cells were obtained from the American Type Culture Collection (ATCC, VA, USA). They were cultured as per ATCC instructions and regularly tested for mycoplasma contaminations using mycoplasma detection kit (Sigma Aldrich, USA). GBM6 ceils were described (52) and provided by C. David James, University of California, San Franc sco. The 1M-PHFA cell line was established and maintained as described (19). DICER stable cell lines were generated using a standard protocol (19).
  • RNA and miRNA-enriched fractions were isolated from cell lines using RNA and miRNA isolation kits from Qiagen, Germany.
  • cDNA synthesis was done using the cDNA synthesis kit from Applied Biosystems, CA, USA.
  • RQ-PCR was performed using the TaqMan master mix from Applied Biosystems, CA, USA. Data were analyzed using GraphPad Prism software
  • MTT 3 -4, 5 -dim ethyl thiazol-2-yl-2, 5-diphenyl tetrazoliurn bromide
  • Standard protocol was used (18).
  • ROS Reactive Oxygen Species
  • Prostate cancer cell lines including DU-145 and DICER overexpressing stable clones of DU-145, were implanted in both flanks of 6-7 week-old athymic nude mice. After the tumor reached palpable size, they were injected with the adenoviruses 108 vp for 8 doses. Once the control tumors reached a maximum allowable limit, the animals were sacrificed, tumors were isolated, and tumor weight was measured. Also, tumor sizes were measured periodically using calipers and graphical representation was done. After completion of the experiment, tumors were fixed and the sections were evaluated immunohistochemicalJy.
  • MDA-7/IL-24 Regulates Mature miR-221 and Not pri-miR-221.
  • miR-221 downregulated the oncogenic microRNA, miR-221, in a broad- spectrum of cancer ceil lines (21).
  • Overexpression of miR-221 partially rescued cells from mda- 7/IL-24-mediated cell death.
  • MDA-7/IL-24 Downregulates the miRNA-Processing Enzyme DICER.
  • miRNA processing is complex: mature miRNAs develop through a series of posttranscriptional steps including production of pre-miRNA by Drosha/DGCR8, export into the cytoplasm by exportin-5, and digestion by DICER, an RNase III endonuclease. There are also reported miRNAs, which follow DICER-independent pathways, e.g., miR-451 (4).
  • mirtrons which use the splicing machinery and bypass Drosha-mediated cleavage.
  • DU- 145 prostate
  • MDA-MB-231 breast
  • lung A549 carcinomas
  • RWPE-1 normal prostate epithelial cells
  • Fig. 21, Fig. 27 Cell lysates were collected 72 hrs after treatment and Western blotting was done with the indicated antibodies.
  • DICER expression decreased with mda-7/IL-24 overexpression, whereas DROSHA and DGCR8
  • MDA-7/IL-24 is a secreted protein that requires a complete set of dimeric cell surface receptors (IL-2QR1/TL-20R2, IL-20R1/TL-22R1, or IL-20R2/IL ⁇ 22R1) for internalization and signaling (24).
  • IL-2QR1/TL-20R2, IL-20R1/TL-22R1, or IL-20R2/IL ⁇ 22R1 dimeric cell surface receptors
  • DICER Overexpression Rescues miRs Downregulated by MDA-7/IL-24. Since DICER expression decreased with mda-7/IL-24 treatment, we examined the expression of rniRNAs that were d eregulated by mda-7/IL-24 following overexpression of DICER. While mda-7/IL-24 downregulated miR-221 and miR-320 in control (non-transfected) cells, there was a significant rescue in the levels of miR-221 and miR-320 in DICER overexpressing cells (Fig. 22). In contrast, miR-451, a DICER-independent rniRNA (4), showed no significant change following
  • DICER Overexpression Rescues MDA-7/IL ⁇ 24-Mediated Cell Death. Since DICER is a downstream target of mda-7/IL-24, we determined if DICER overexpression could mitigate rnda- 7/!L-24-mediated effects on cell proliferation and death. Cells were transfected with a vector control or DICER and infected with either Ad. null or Ad.mda-7.
  • DICER-transfected ceils were resistant to mda-7/IL-24-mediated inhibitor ⁇ , effects on cell proliferation, as monitored by MET assays (Fig. 24). These antiproliferative effects were also evident in long-term colony-forming assays (Fig. 24). A strong inhibition in colony formation was seen in mda-7/IL-24-infected cells, which was partially but significantly rescued in DICER overexpressing cells.
  • overexpressing vector or DICER were injected subcutaneously to establish tumor xenografts in male athymic nude mice. After a palpable tumor developed in approximately 2 weeks, the tumors wore injected with 8 intratumorai injections over a 3 -week period with 1 X 108 viral particles of Ad null or Ad.mda-7. In control vector-transfected cells, significant growth inhibition was evident, but in DICER overexpressing cells the effect of Ad.mda-7 was diminished in both the injected left tumor, and in the uninjected right tumor, as previously observed in vitro (Figs. 25A, 25B, Fig. 32) The expression of MDA-7/IL-24 and DICER were confirmed by immunohistochemistry (Figs. 25C, Fig. 33). Taken together, these experiments demonstrate the signifi cance of DICER in mda- 7/IL-24-mediated cancer ceil death both in vitro and in vivo.
  • MDA-7/TL-24-Mediated DICER Regulation is Mediated by the Transcription Factor MITF.
  • DICER plays a central regulator ⁇ ' role in miRNA processing and biogenesis pathways.
  • SOX4 (28) and MITF (29) can transcriptionally activate DICER.
  • Infection with Ad.mda-7 dramatically inhibited MITF expression in all three cancer types (Figs. 26A, 26B, Fig. 34A).
  • the transcript levels ofMITF decreased with mda-7/IL-24 expression (Fig.
  • adenovector expressing the melanoma-differentiation associated gene-7 (mda-7/TL24): biologic outcome in advanced cancer patients. Mol Ther 11 : 160-172.
  • MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer.
  • RNA 16 1087-1095.

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Abstract

La présente invention concerne, entre autres, des procédés de niveaux de détection de DICER chez des patients soumis à un traitement pour des maladies associées à DICER (par exemple, un cancer, une maladie inflammatoire, une maladie infectieuse, une maladie auto-immune, une maladie cardiovasculaire). Les procédés selon l'invention sont utiles, entre autres, pour surveiller et déterminer l'efficacité de traitement en déterminant (détection) les niveaux de DICER ou de molécules en aval des voies de signalisation DICER (par exemple un miARN), chez des patients recevant, ayant reçu ou devant recevoir une thérapie à base de MDA-7.
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WO2023273422A1 (fr) * 2021-07-01 2023-01-05 中国医学科学院肿瘤医院 Isomère d'épissage de kinase d'adhésion focale et son application

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US20160024503A1 (en) * 2013-03-15 2016-01-28 Board Of Regents, The University Of Texas System miRNA BIOGENESIS IN EXOSOMES FOR DIAGNOSIS AND THERAPY
WO2018089995A1 (fr) * 2016-11-14 2018-05-17 Virginia Commonwealth University Thérapies anticancéreuses à base de mda-7 et procédés de détection de biomolécules

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Publication number Priority date Publication date Assignee Title
US20160024503A1 (en) * 2013-03-15 2016-01-28 Board Of Regents, The University Of Texas System miRNA BIOGENESIS IN EXOSOMES FOR DIAGNOSIS AND THERAPY
WO2018089995A1 (fr) * 2016-11-14 2018-05-17 Virginia Commonwealth University Thérapies anticancéreuses à base de mda-7 et procédés de détection de biomolécules

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UEDA ET AL.: "Dicer-regulated microRNAs 222 and 339 promote resistance of cancer cells to cytotoxic T-lymphocytes by down-regulation of ICAM-1", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 106, no. 26, 30 June 2009 (2009-06-30), pages 10746 - 10751, XP055363409, DOI: 10.1073/pnas.0811817106 *

Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2023273422A1 (fr) * 2021-07-01 2023-01-05 中国医学科学院肿瘤医院 Isomère d'épissage de kinase d'adhésion focale et son application

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