WO2014210411A1

WO2014210411A1 - Histone deacetylase inhibitors and methods of use

Info

Publication number: WO2014210411A1
Application number: PCT/US2014/044487
Authority: WO
Inventors: Sanjay K. Srivastava; Srinivas BOREDDY
Original assignee: Texas Tech University System
Priority date: 2013-06-28
Filing date: 2014-06-27
Publication date: 2014-12-31

Abstract

The present invention provides methods of making and using pharmaceutical compositions of Histone deacetylase inhibitors with other inhibitors for the treatment of solid malignancies by identifying a solid tumor; and providing a therapeutic amount of a pharmaceutical composition to the solid tumor, wherein the pharmaceutical composition includes a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the therapeutic amount of a pharmaceutical composition suppress the growth of the solid tumor.

Description

HISTONE DEACETYLASE INHIBITORS AND METHODS OF USE

Technical Field of the Invention

The present invention relates generally to the treatment of solid tumors and specifically to the use of histone deacetylase inhibitors (HDACi) in combination with other inhibitors for the treatment of solid tumors. Background Art

Without limiting the scope of the invention, its background is described in connection with the treatment of solid tumors. Cancer is one of the leading causes of death and typical treatments include surgery, chemotherapy, and radiotherapy. However, all of these approaches pose significant drawbacks for the subject. For example, surgery may not successfully remove all neoplastic tissue and can be contraindicated due to the health of the subject. Chemotherapy involves the administration of cytotoxic chemical agents which are associated with undesirable side effects, including alopecia, nausea and vomiting, hematoxicity, neurotoxicity, nephrotoxicity, cardiotoxicity and hepatotoxicity. In addition, cancer cells commonly develop resistance to most anticancer agents, thus rendering chemotherapy ineffective over time. Radiation therapy involves the treatment of both cancer cells and normal cells using ionizing radiation which deposits energy that injures or destroys cells in targeted tissues by damaging their genetic material and subsequently interfering with a cell's ability to grow and/or replicate. Radiotherapy can be used to treat localized solid tumors (e.g., skin, tongue, larynx, brain, breast, prostate, colon, uterus, lung, kidney, head and neck, and/or cervix) and systemic forms of cancer (e.g., leukemias and lymphomas). Signal transduction pathways have been implicated to play important roles in cellular responses to ionizing radiation and their disruption in cancer cells results in enhanced cytotoxic effects following radiation exposure. Acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcriptional factors is also mediated through acetylation.

A growing number of histone deacetylases (HDACs) have been identified and have been shown to play an important role in cell proliferation and differentiation. HDACs are believed to be associated with a variety of different disease states including, but not limited to cell proliferative diseases and conditions such as leukemia, melanomas/squamous cell carcinomas, breast cancer, prostate cancer, bladder cancer, lung cancer, ovarian cancer and colon cancer. Histone deacetylase inhibitors are potent inducers of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells in culture and in tumor bearing animals. However, Histone deacetylase inhibitors failed to exert any significant effect against solid tumors such as pancreatic cancer. As a result, histone deacetylase inhibitors are approved for the treatment of hematological malignancies but had limited efficacy against solid tumors.

U.S. Patent Application Publication No. 2013/0065963, entitled, "Histone Deacetylase Inhibitors and Methods of Use Thereof," discloses methods of sensitizing a cancer cell to the cytotoxic effects of radiotherapy are also provided as well as methods for treating cancer and methods for treating neurological diseases. Histone deacetylase inhibitors have been used to prevents and inhibits metastasis, e.g., U.S. Patent No. 8,299, 126, entitled, "Treatment of Canine Hemangiosarcoma with a Histone Deacetylase Inhibitor," discloses a method of treating cancer, particularly canine hemangiosarcoma that includes the continuous and regular administration of a formulation including a histone deacetylase inhibitor as part of the standard canine diet. Disclosure of the Invention

The present invention provides compositions and combinations of compositions of Histone deacetylase inhibitors with other inhibitors for the treatment of solid malignancies.

The present invention provides a method of treating one or more pancreatic tumor cells by identifying one or more pancreatic tumor cells and providing a therapeutic amount of a pharmaceutical composition to the one or more pancreatic tumor cells, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the histone deacetylase inhibitor is Vorinostat (SAHA), Panabinostat, Trichostatin A, or depsipeptide and the therapeutic amount of a pharmaceutical composition suppress the growth of the one or more pancreatic tumor cells. For example, the one or more transcription factor inhibitors may be a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The one or more transcription factor inhibitors may be a Bcl2 inhibitor and the composition further include a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The composition may further include a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof. The present invention also provides a deacetylase inhibitor composition for use in the treatment of pancreatic tumor cells comprising: providing a therapeutic amount of a pharmaceutical composition to the one or more pancreatic tumor cells, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the histone deacetylase inhibitor is Vorinostat (SAHA), Panabinostat, Trichostatin A, or depsipeptide and the therapeutic amount of a pharmaceutical composition suppress the growth of the one or more pancreatic tumor cells. The one or more transcription factor inhibitors may be a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The one or more transcription factor inhibitors may be a Bcl2 inhibitor and the composition further comprises a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The deacetylase inhibitor composition may further comprising a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

The present invention provides a deacetylase inhibitor composition for use in the treatment of a solid tumor comprising: providing a therapeutic amount of a pharmaceutical composition to the solid tumor, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the therapeutic amount of a pharmaceutical composition suppress the growth of the solid tumor. The solid tumor may be a pancreatic cancer solid tumor. The histone deacetylase inhibitor may be selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof. The one or more transcription factor inhibitors may be a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a Sp 1 inhibitor or a combination thereof. The one or more transcription factor inhibitors may be a Bcl2 inhibitor and the composition may further comprises a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The pharmaceutical composition may further comprises a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

The present invention provides a method of inhibiting a epithelial-to-mesenchymal transition comprising the steps of: identifying one or more cells capable of undergoing a epithelial-to- mesenchymal transition; and providing a therapeutic amount of a pharmaceutical composition to the one or cells, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the liistone deacetylase inhibitor is Vorinostat (SAHA), Panabinostat, Trichostatin A, or depsipeptide and the therapeutic amount of a pharmaceutical composition suppress the epithelial-to-mesenchymal transition.

The present invention provides a method of treating a solid tumor by identifying a solid tumor; and providing a therapeutic amount of a pharmaceutical composition to the solid tumor, wherein the pharmaceutical composition comprises a liistone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the therapeutic amount of a pharmaceutical composition suppress the growth of the solid tumor.

The solid tumor may be a pancreatic cancer solid tumor. The liistone deacetylase inhibitor may be selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof. The one or more transcription factor inhibitors may be a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The composition may further include a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof. The composition may further include a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

The present invention provides a method of inhibiting a epithelial-to-mesenchymal transition by identifying one or more cells capable of undergoing a epithelial-to-mesenchymal transition; and providing a therapeutic amount of a pharmaceutical composition to the one or cells, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the histone deacetylase inhibitor is Vorinostat (SAHA), Panabinostat, Trichostatin A, or depsipeptide and the therapeutic amount of a pharmaceutical composition suppress the epithelial-to-mesenchymal transition.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier; a therapeutic amount of a istone deacetylase inhibitor disposed in the pharmaceutically acceptable carrier; and a therapeutic amount of one or more transcription factor inhibitors disposed in the pharmaceutically acceptable carrier, wherein the therapeutic amount of a pharmaceutical composition suppress the growth of the solid tumor.

The solid tumor may be one or more pancreatic tumor cells. The istone deacetylase inhibitor may be selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof. The one or more transcription factor inhibitors may be a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a S l inhibitor or a combination thereof. The composition may further include a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof disposed in the pharmaceutically acceptable carrier. The composition may further include a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof disposed in the pharmaceutically acceptable carrier.

Description of the Drawings

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

FIGURES 1A-1C are images showing histone deacetylase inhibitors induce E-Cadherin expression in pancreatic cancer cells.

FIGURE 2 is an image showing histone deacetylase inhibitors induce E-Cadherin expression transcriptionally. FIGURES 3 A and 3B are images showing immunoflorescence of histone deacetylase inhibitor induced E-Cadherin expression.

FIGURES 4A and 4B are images showing the effect of histone deacetylase inhibitor on E- Cadherin transcriptional repressors.

FIGURES 5 A and 5B are images showing SNAIL 1 and SNAIL2 which are not involved in histone deacetylase inhibitor-induced E-Cadherin expression.

FIGURE 6 is an image showing ZEB1 overexpression abrogates histone deacetylase inhibitor- induced E-Cadherin up regulation.

FIGURES 7A-7C are images showing histone deacetylase inhibitors suppress tumor growth and induce E-Cadherin expression in vivo. FIGURES 8A-8C are images showing histone deacetylase inhibitors inhibiting epithelial to mesenchymal transition in pancreatic cancer by inhibiting the expression of ZEB 1.

Description of Embodiments While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

As used herein, the term "tumorigenesis" denotes the neoplastic process leading to the appearance of a tumor. In the present invention, the inhibition of tumor growth is related not to toxicity but to differentiation of the tumor cells toward a normal phenotype. As used herein, the term "metastasis" denotes movement via the circulatory system of a disease- producing agent (such as cancer cells) from an original site of disease to another part of the body with development of a similar lesion in the new location.

As used herein, the term "about" or "approximately" denotes within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

As used herein, the term "pharmaceutically acceptable" denotes to compositions that are "generally regarded as safe," e.g., that are physiologically tolerable and can also mean approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals. As used herein, the term "Pharmaceutically acceptable salts" denotes salts of inhibitors of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartatic acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, madelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2 -hydroxy ethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p- toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like. As used herein, the term "carrier" or "pharmaceutically acceptable carrier" denotes to a diluent, adjuvant, excipient, or vehicle with which the compound is administered and can be sterile liquids, due to its high insolubility in water, oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The histone deacetylase inhibitor can be suspended in oil. Carriers such as micelles or dextrans can be used to deliver the agent in an aqueous solution or suspension.

As used herein, the term "therapeutically effective amount" denotes the amount which, when administered to is sufficient to effect such treatment for the disease.

As used herein, the term "treatment" or "treating" denotes any administration of a compound of the present invention and includes: preventing the disease from occurring in an animal which may be predisposed to the disease but does not yet experience or display the pathology or symptomatology of the disease, inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased, or ameliorating one or more symptom of the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased. As used herein the term "unit dosage form" as used herein denotes to physically discrete units suitable as unitary dosages for subjects (e.g., animals, usually humans), each unit containing a predetermined quantity of agent(s) in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention will depend on a variety of factors including, but not necessarily limited to, the particular agent employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The present invention provides a method of treating one or more tumor cells by identifying one or more tumor cells and providing a therapeutic amount of a pharmaceutical composition to the one or more tumor cells. At a minimum the pharmaceutical composition comprises a histone deacetylase inhibitor. The pharmaceutical composition may be delivered in conjunction with one or more transcription factor inhibitors. This may be as a pharmaceutical composition comprising a histone deacetylase inhibitor and one or more transcription factor inhibitors. However, the pharmaceutical composition may be separate pharmaceutical composition, i.e., a first pharmaceutical composition comprises a histone deacetylase inhibitor and a second pharmaceutical composition comprising one or more transcription factor inhibitors. Alternatively the pharmaceutical composition may be a single pharmaceutical composition having multiple layers or zones of one or more transcription factor inhibitors and histone deacetylase inhibitors. These compositions can be immediately released, time released, sustained released or a combination or mixture of these releases to accomplish the desired release. Although the composition is particularly effective against solid tumors it is effective against any cancer cell, precancerous cell, and the like and the skilled artisan will readily understand that the pharmaceutical compositions disclosed herein may be used to treat any cancerous cell.

The present invention may be administered by various methods including but not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes or any other standard routes of administration.

As used herein, the term "histone deacetylase inhibitor" and "HDACi" denotes but is not limited to various classes of anticancer compounds, e.g., hydroxamic acids such as SAHA (suberoylanilide hydroxamic acid); trichostatins (e.g., trichostatin A, oxamflatin, ABHA, scriptaid, pyroxamide, and propenamide); short-chain fatty acids (e.g., butyrate, valproate, and phenylbutyrate); epoxyketone-containing cyclic tetrapeptides (e.g., trapoxins, HC-toxin, chlamydocin, diheteropeptin, WF-3161, Cy-1 and Cy-2); non-epoxyketone-containing cyclic tetrapeptides (e.g., FR901228, apicidin, and cyclic-hydroxamic-acid-containing peptides); benzamides (e.g., MS-27-275, CI-994, and benzamide analogs); depudecin; and organosulfur compounds. For example, the histone deacetylase inhibitor may be compositions disclosed in U.S. Patent No. 7, 154,002 which is incorporated in its entirety by reference herein.

The present inventors discovered that unlike in any other cancer type, histone deacetylase inhibitors were specifically up regulating Bcl2 protein in pancreatic cancer and hence were not effective in suppressing the growth of these tumor cells. In the clinic studies histone deacetylase inhibitors failed to exert any significant effect against solid tumors such as pancreatic cancer. The present invention provides the use histone deacetylase inhibitors along with Bcl2 inhibitor or inhibitors of transcription factor such as NF-kB, STAT3 or Spl for the treatment of solid tumors. Although individual inhibitors have been tried in clinical trials without much success, the combination of the present invention has never has been tried against solid tumors.

The individual inhibitors have been tried in clinical trials against solid tumors but have been found to be ineffective. Specifically, Bcl2 inhibitor and inhibitors of transcription factor such as NF-kB, STAT3 or Spl have been tried in the treatment of solid tumors but have been found to have little or no effect on solid tumors. As a result, the combination of histone deacetylase inhibitors along with a Bcl2 inhibitor and/or inhibitors of transcription factor as seen in the present invention provide surprising and unexpected results in the synergistic effect between the combination of histone deacetylase inhibitors and Bcl2 inhibitors and/or one or more transcription factor inhibitors. Histone deacetylase inhibitors such as Vorinostat (SAHA), Panabinostat and Trichostatin A epigenetically modifies histones by deacetylation and decrease the access of transcription factors to the promoter region of the responsive genes. Emerging evidences suggest that histone deacetylase inhibitors play a major role in cancer treatment. For example, SAHA and depsipeptide were approved by FDA for the treatment of refractory cutaneous T-cell lymphoma. However, histone deacetylase inhibitors have nothing or modest activity against solid tumors such as pancreatic cancer.

In the present invention, it was found that histone deacetylase inhibitors (SAHA, Panabinostat, Trichostatin A) drastically up regulate Bcl2 expression (>100 fold) in the pancreatic tumor cells. Bcl2 is an anti-apoptotic protein, overexpression of which lead to resistance to chemotherapy in almost every tumor model. It was found that the transcription factors such as NF-kB, STAT3 and Spl play significant role in the up regulation of Be 12. The present invention shows that histone deacetylase inhibitor treatment cause increased expression of Spl, phosphorylation of NF-kB and acetylation of STAT3. Acetylated STAT3 translocate to the nucleus by CD44 2/4 variants and cause up regulation of Bcl2. The present invention shows that combining Bcl2, STAT3, NF-kB, Spl or CD44 inhibitors along with histone deacetylase inhibitors significantly suppress the growth of pancreatic tumor cells, which was not possible by HDACi alone or any other inhibitors. So this combination of specific inhibitors with histone deacetylase inhibitors has significant advantages over histone deacetylase inhibitors alone for the treatment of solid tumors.

Combining Bcl2 inhibitors along with histone deacetylase inhibitors will have better outcome as compared to either inhibitor alone for the treatment of solid tumors. Histone deacetylase inhibitors were proven to be effective against hematological malignancies but were not effective against solid tumors. The present invention shows the reason for the ineffectiveness of histone deacetylase inhibitors against solid tumors. Based on the evidence of the present invention shows, that combination of histone deacetylase inhibitors along with Bcl2 or other inhibitors mentioned above would be effective for solid malignancies.

Histone deacetylase inhibition is known to inhibit the Epithelial-to-Mesenchymal Transition (EMT) in various cancer models by suppressing the acetylation of histone H3 and H4 of E- Cadherin promoter. However, the exact mechanism of epithelial to mesenchymal transition inhibition by histone deacetylase inhibition is not known. In the present study, using various histone deacetylase inhibitors such as Vorinostat (SAHA), Tricostatin A (TSA) and Panobinostat (LBH-589), the present invention examines the molecular mechanisms of Epithelial-to- Mesenchymal Transition inhibition in pancreatic tumor cells in vitro and in vivo. The expression of epithelial markers such as E-Cadherin and cytokeratin 18 were significantly up regulated in PanC-1, BxPC-3 and HPAC cells treated with histone deacetylase inhibitors, whereas the expression of mesenchymal marker vimentin decreased. Morphology of the cells also changed indicating that histone deacetylase inhibitors suppressed epithelial to mesenchymal transition in pancreatic cancer cells. To determine whether histone deacetylase inhibition correlates with the down-regulation of key transcriptional suppressors of E-Cadherin such as SNAIL 1, SNAIL2, TWIST and ZEB, cells were treated with panobinostat. The present invention revealed that the SNAIL1 expression was drastically increased, whereas expression of SNAIL2, TWIST and ZEB were decreased by histone deacetylase inhibition in all the cell lines. Since, it has been previously shown that histone deacetylase activity is required to suppress E- Cadherin expression by its repressors, HDACl/2 was co-immunoprecipitated with SNAIL 1 to see whether SNAIL 1 also requires histone deacetylase activity to repress E-Cadherin expression in the model. The present invention revealed that this interaction was more pronounced in control cells as compared to histone deacetylase inhibitors treated cells. These results suggest that although SNAIL1 expression was significantly increased by histone deacetylase inhibition, it remained ineffective in E-Cadherin promoter. To evaluate other repressors, PanC-1 cells were transfected with SNAIL2 and ZEB 1 and treated with histone deacetylase inhibitors. Interestingly, the results show that SNAIL2 overexpression was unable to abrogate increase in E-Cadherin by histone deacetylase inhibition. However, ZEB1 overexpression completely blocked the induction of histone deacetylase-mediated E-Cadherin expression. To validate in vitro observations in an in vivo model, BxPC-3 cells were subcutaneously implanted in athymic nude mice. When the tumors reached about 70mm³, 20mg/Kg panobinostat was administered intraperitonealy three times a week while control group received vehicle alone. After 4 weeks of treatment, the growth of pancreatic tumors was significantly suppressed in panabinostat treated mice. Moreover, the tumors from panabinostat treated mice showed an increase in E-Cadherin and decrease in ZEB expression. The expression of other E-Cadherin repressors SNAIL 1 and SNAIL2 were also down regulated. In conclusion, the in vitro and in vivo results clearly show that increase in E-Cadherin by histone deacetylase inhibition was due to the down regulation of ZEB1.

Cell culture: PanC-1 and HPAC cells were cultured in DMEM medium, whereas BxPC-3 cells were cultured in RPMI supplemented with 10% FBS and antibiotics. RT-PCR: Pancreatic cancer cells were treated with histone deacetylase inhibitors and total RNA was isolated by Trizol method. RNA was subjected to RT-PCR analysis using Verso 1-step PCR kit (Thermo scientific, IL) for E-Cadherin expression. Immunoflorescence: PanC-1 cells were treated with panobinostat for 72 hours and fixed with methanokacetone (50:50, -20 C). Cells were probed with E-Cadherin and actin primary antibody followed by Alexa Fluor-594/488 secondary antibody. Cells were photographed under florescence microscopy. Transfections: All the transfections were performed using FuGene 6 reagent (Roche, IN), according to manufacturer's instructions. Western blot analysis: Cells were exposed to varying concentrations of deguelin for the indicated time periods. Cell lysates were subjected to SDS-PAGE and the proteins were transferred onto PVDF membrane and incubated overnight with the desired primary antibody. Subsequently, the membrane was incubated with appropriate secondary antibody, and the immunoreactive bands were visualized using enhanced chemiluminescence kit (NEN Life Science Products, Boston, MA) according to the manufacturer's instructions. The same membrane was re-probed with the antibody against actin (1 :50,000 dilution) and used as an internal control for equal protein loading after stripping with stripping buffer. Pancreatic cancer cells (BxPC-3, Panc-1, HPAC) were treated with 10μΜ ABT for 1 hour followed by treatment with 25nM panobinostat for 72 hours and lysed on ice as described by us previously. Whole-cell extracts were prepared as mentioned above. The cell lysate was cleared by centrifugation at 14,000g for 30 minutes. Cell lysate containing 10-80 μg protein was resolved by 6-12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred onto polyvinylidene fluoride membrane. After blocking with 5% non-fat dry milk in Tris buffered saline, membrane was incubated with the desired primary antibody (1 : 1000 dilutions) overnight. Subsequently, the membrane was incubated with appropriate secondary antibody (1 :2000 dilutions) and the antibody binding was detected by using enhanced chemiluminescence kit according to the manufacturer's instructions. Each membrane was stripped and re-probed with antibody against actin (1 :20000 dilutions) to ensure equal protein loading.

In vivo: All the studies involving animals were approved by the Institutional Animal Care and Use Committee (IACUC). Twenty 4-6 week old female athymic nude mice (Charles River, Wilmington, MA) were injected with 1X10⁶ exponentially growing BxPC-3 cells on the both flanks of nude mice. After 7 days mice were randomized and treated group received 20mg/kg panobinostat every 72 hours for 42 days. Tumors were measured thrice a week using vernier calipers. At the end of the experiment animals were euthanized and tumors were excised. Control and treated tumors were lysed and subjected to western blot.

Statistical analysis: All the statistical analysis was performed using Prism 4.0 (GraphPad Software Inc., San Diego, CA). Results were expressed as mean ± SD of the at least three independent studies. Data was analyzed by Student's t-test or one way ANOVA followed by Bonferroni's post hoc analysis for multiple comparisons. Differences were considered statistically significant at p<0.05.

FIGURES 1A-1C are images of gels showing histone deacetylase inhibitors induce E-Cadherin expression in pancreatic cancer cells. To see whether histone deacetylase inhibitors inhibit epithelial to mesenchymal transition (EMT) by up regulating E-Cadherin expression, BxPC-3, HP AC and PanC- 1 cells were treated with different concentrations of panobinostat, trichostatin A and veronistat. Cells were collected and E-Cadherin expression was evaluated by western blot. As shown in FIGURES 1A-1C, epithelial markers, such as E-Cadherin and cytokeratin 18 were up regulated in histone deacetylase inhibitor treated cells, as compared to control cells. Pancreatic cancer cells were seeded in a 6-well plate and treated with histone deacetylase inhibitors for 72 hours. Cells were collected and whole cell lysates were analyzed for epithelial markers' expression by western blot. Actin was used as a loading control.

FIGURE 2 is an image of a gel showing histone deacetylase inhibitors induce E-Cadherin expression transcriptionally. To test these cells were treated with histone deacetylase inhibitors and RT-PCR analysis was performed whether histone deacetylase inhibitors transcriptionally up regulate E-Cadherin expression in pancreatic cancer cells. Interestingly, all the histone deacetylase inhibitors significantly increased mRNA levels of E-Cadherin, indicating that E- Cadherin was transcriptionally up regulated.

PanC-1 cells were treated with panobinostat and SAHA for 72 hours and total RNA was isolated by Trizol method. RNA was subjected RT-PCR using Verso 1-step PCR kit (Thermo Scientific) to analyze the levels of E-Cadherin RNA transcripts. GAPDH was used as a loading control.

FIGURES 3A and 3B are images showing immunoflorescence of histone deacetylase inhibitor induced E-Cadherin expression. E-Cadherin up regulation by histone deacetylase inhibitors was further confirmed by immunoflorescence. PanC-1 cells were treated with panobinostat for 72 hours and cells were immunostained for E-Cadherin expression. Similar to the western blot analysis, as compared to control cells, panobinostat treated cells showed intense red color staining indicating E-Cadherin. PanC-1 cells were treated with panobinostat (25nM) for 72h and cells were subjected to immunoflorescence to visualize E-Cadherin (red) and actin (green).

FIGURES 4A and 4B are images of gels showing the effect of histone deacetylase inhibitor on E-Cadherin transcriptional repressors. Since, E-Cadherin expression is tightly regulated by its transcriptional repressors, such as SNAILl, SNAIL2, TWIST and ZEBl Cells were treated with various histone deacetylase inhibitors and whole lysates were subjected to western blotting to see the effect of histone deacetylase inhibitors on these repressors. Surprisingly, SNAILl was up regulated and SNAIL2 and ZEBl were down regulated, whereas TWIST was moderately altered in HDACi-treated pancreatic cancer cells. PanC-1 and HP AC cells were treated with HDCi for 72 hours and whole cell lysates were collected. Equal protein from control and treated was subjected to SDS-PAGE followed by western blot to analyze the expression of E-Cadherin repressors. Actin was used as a loading control.

FIGURES 5 A and 5B are images of gels showing SNAIL 1 and SNAIL2 which are not involved in HDACi-induced E-Cadherin expression. Since it was observed that down regulation of all E- Cadherin repressors, except SNAIL 1 by histone deacetylase inhibitor treatment, the next step was to see which one of these repressors was critically involved in HDACi-induced E-Cadherin up regulation and is E-Cadherin induced in spite of SNAIL 1 up regulation by histone deacetylase inhibitor treatment. Although SNAIL 1 was up regulated by HDACi its co-activator HDACl/2 was down regulated by HDACi (see FIGURE 5A). Hence SNAIL 1 was ineffective on E-Cadherin promoter. Furthermore, SNAIL2 overexpression could not abrogate HDACi- induced E-Cadherin induction (see FIGURE 5B). Taken together, the results indicate that SNAIL 1 and SNAIL2 were not involved in HDACi-induced E-Cadherin induction. PanC-1 cells were treated with panobinostat for 72 hours and whole cell lysates were isolated. FIGURE 5A shows equal amount of SNAIL 1 protein was immunoprecipitated in control and treated cells and immunoblotted for HDACI. FIGURE 5B indicates SNAIL2 was overexpressed in PanC-1 cells using FuGene 6, according to manufacturer's instructions, and treated with panobinostat for 72 hours. Equal amount of protein from control and treated cells was subjected to western blot.

FIGURE 6 is an image of a gel showing ZEB 1 overexpression abrogates HDACi-induced E- Cadherin up regulation. To evaluate whether ZEB 1 is involved in HDACi-induced E-Cadherin expression PanC-1 cells were pre-treated with TGF l for lh and followed by treatment with panobinostat for additional 72 hours. The results show that TGF l significantly induced ZEB 1 expression. However when these cells were treated with panobinostat, E-Cadherin induction was abrogated, indicating the involvement of ZEB 1 in HDACi-induced induction of E-Cadherin. PanC-1 cells were pre-treated with TGF l for 1 hour and co-treated with panobinostat for additional 72 hours. Cells were collected and subjected to western blot. Actin was used a loading control.

FIGURES 7A-7C are images showing histone deacetylase inhibitors suppressing tumor growth and inducing E-Cadherin expression in vivo. Around IX 10⁶ exponentially growing BxPC-3 cells were injected on the both flanks of the nude mice. After 7 days animals were randomized and the treated group received 20mg/kg panobinostat every 72 hours. FIGURE 7A is a plot of the tumor volume as a function of time. Tumors were measured twice a week using vernier calipers and presented as line diagram. FIGURE 7B is a bar plot of the tumor growth suppression by panobinostat by the end of the study. FIGURE 7C is an image of a gel showing the proteins present at the end of the studies when the animals were euthanized, tumors were carefully excised and snap frozen in liquid nitrogen. Tumors were lysed and equal amount of protein was subjected to western blot. *=P<0.05 statistically significant compared to control animals.

Panobinostat reverses epithelial to mesenchymal transition in vivo. BxPC-3 cells were injected into the flanks of nude mice and treated with 20mg/kg panobinostat over the period of 42 days. Tumor volume was measured by vernier calipers thrice a week. The results show that panobinostat suppressed the growth of BxPC-3 xenografts by 36%, as compared to control animals. At the end of the study tumors were excised and subjected to western blot. The results show that E-Cadherin expression was significantly increased, whereas ZEB1 expression was decreased in the tumors of panobinostat treated mice, as compared to control mice.

FIGURES 8A-8C are images showing histone deacetylase inhibitors inhibit epithelial to mesenchymal transition in pancreatic cancer by inhibiting the expression of ZEB1. CI Casp-3: Cleaved Caspase -3 play role in inducing cell death (apoptosis).

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier; a therapeutic amount of a histone deacetylase inhibitor disposed in the pharmaceutically acceptable carrier; and a therapeutic amount of one or more transcription factor inhibitors disposed in the pharmaceutically acceptable carrier, wherein the therapeutic amount of a pharmaceutical composition suppress the growth of the solid tumor.

2. The composition of claim 1, wherein the solid tumor comprises one or more pancreatic tumor cells.

3. The composition of claim 1, wherein the histone deacetylase inhibitor is selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

4. The composition of claims 1 or 2, wherein the one or more transcription factor inhibitors is a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof.

5. The composition of claim 1, wherein the one or more transcription factor inhibitors is a Bcl2 inhibitor and the composition further comprises a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof disposed in the pharmaceutically acceptable carrier.

6. The composition of claim 1, further comprising a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof disposed in the pharmaceutically acceptable carrier.

7. A deacetylase inhibitor composition for use in the treatment of pancreatic tumor cells comprising: identifying one or more pancreatic tumor cells; and

providing a therapeutic amount of a pharmaceutical composition to the one or more pancreatic tumor cells, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the histone deacetylase inhibitor is Vorinostat (SAHA), Panabinostat, Trichostatin A, or depsipeptide and the therapeutic amount of a pharmaceutical composition suppress the growth of the one or more pancreatic tumor cells.

8. The deacetylase inhibitor composition of claim 7, wherein the one or more transcription factor inhibitors is a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof.

9. The deacetylase inhibitor composition of claim 7, wherein the one or more transcription factor inhibitors is a Bcl2 inhibitor and the composition further comprises a NF-kB inhibitor, a STAT3 inhibitor, a Sp 1 inhibitor or a combination thereof.

10. The deacetylase inhibitor composition of claim 7, further comprising a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

11. A deacetylase inhibitor composition for use in the treatment of a solid tumor comprising: identifying a solid tumor; and

providing a therapeutic amount of a pharmaceutical composition to the solid tumor, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the therapeutic amount of a pharmaceutical composition suppress the growth of the solid tumor.

12. The deacetylase inhibitor composition for use according to claim 11, wherein the solid tumor is a pancreatic cancer solid tumor.

13. The deacetylase inhibitor composition for use according to claim 11, wherein the histone deacetylase inhibitor is selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

14. The deacetylase inhibitor composition for use according to claim 11, wherein the one or more transcription factor inhibitors is a Bcl2 inhibitor, a NF-kB inhibitor, a STAT3 inhibitor, a

Spl inhibitor or a combination thereof.

15. The deacetylase inhibitor composition for use according to claim 11, wherein the one or more transcription factor inhibitors is a Bcl2 inhibitor and the composition further comprises a NF-kB inhibitor, a STAT3 inhibitor, a Spl inhibitor or a combination thereof.

16. The deacetylase inhibitor composition for use according to claim 11, wherein the pharmaceutical composition further comprises a second histone deacetylase inhibitor selected from Vorinostat (SAHA), Panabinostat, Trichostatin A, depsipeptide and a combination thereof.

17. A method of inhibiting a epithelial-to-mesenchymal transition comprising the steps of: identifying one or more cells capable of undergoing a epithelial-to-mesenchymal transition; and providing a therapeutic amount of a pharmaceutical composition to the one or cells, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor and one or more transcription factor inhibitors disposed in a pharmaceutically acceptable carrier, wherein the histone deacetylase inhibitor is Vorinostat (SAHA), Panabinostat, Trichostatin A, or depsipeptide and the therapeutic amount of a pharmaceutical composition suppress the epithelial-to-mesenchymal transition.