WO2016165545A1 - Method and preparation for manipulating tumour microenvironment to remove tumour resistance by targeting mtor - Google Patents

Abstract

Provided in the present invention are a pharmaceutical compositions and kits containing rapamycin or an analogue thereof, and chemotherapy drugs for treating tumours or reversion of tumour resistance, and a use of rapamycin and an analogue thereof in manipulating the tumour microenvironment to reverse tumor resistance by targeting mTOR.

Description

Method and preparation for manipulating tumor microenvironment by targeting mTOR to eliminate tumor resistance Technical field

The present invention is in the field of biopharmaceuticals, and more particularly, the present invention relates to methods and formulations for manipulating tumor microenvironment by targeting mTOR to eliminate tumor resistance.

Background technique

The tolerance of tumors to chemotherapeutic drugs is a major obstacle to the treatment of cancer, and it is also the root cause of poor clinical outcomes and even eventually failure. With the advancement of basic research, new chemotherapy drugs and treatment programs have been continuously introduced, but the problem of tumor resistance has not yet been solved.

In clinical practice, the initial chemotherapy of many tumors can generally achieve better results, but then it is inevitable to relapse, and the treatment effect after relapse is often worse, the main reason is that the remaining tumor cells (including stem cells) have been treated with chemotherapy. The drug produces resistance, which is a decrease in sensitivity to treatment. It is estimated that more than 90% of cancer deaths are resistant. Therefore, the study of tumor resistance mechanisms and new treatment methods is extremely important to overcome the difficulties faced by anti-cancer treatment.

In the past clinical or experimental mouse-based preclinical studies including patients with advanced malignant solid tumors such as advanced prostate cancer and breast cancer, the anti-cancer treatment with genotoxic drugs as the main body has very limited effects. Most of the reasons are due to the fact that chemotherapy drugs remove massive tumors and kill a large number of cancer cells, and also cause extensive structural and functional damage to the microenvironment surrounding the cancer cells due to their inevitable off-target effects, and cause a large number of lesions. The stromal cells develop therapeutic activation, which forms multiple protective barriers for the survival, repair, and re-addition of cancer cells during the treatment interval. The non-autonomous change of this non-cancerous cell under clinical stress ultimately leads to widespread recurrence and metastasis of the disease in the post-treatment phase, which is manifested as a high mortality rate in clinical oncology.

Therefore, it is necessary in the field to continuously explore new ways to overcome the drug resistance of cancer to improve the clinical treatment effect of cancer.

Summary of the invention

It is an object of the present invention to provide methods and formulations for manipulating the tumor microenvironment by targeting mTOR to eliminate tumor resistance.

In a first aspect of the invention, there is provided a use of rapamycin or an analogue thereof for the preparation of a pharmaceutical composition for eliminating tumor resistance.

In a preferred embodiment, the rapamycin or analog thereof manipulates the tumor microenvironment by targeting mTOR, thereby eliminating tumor resistance.

In another preferred embodiment, the tumor resistance is the resistance of the tumor to chemotherapeutic drugs.

In another preferred embodiment, the rapamycin analogue comprises: RAD001.

In another preferred embodiment, the tumor includes, but is not limited to, prostate cancer, breast cancer, colorectal cancer, lung cancer, skin cancer.

In another aspect of the invention, there is provided a pharmaceutical composition for treating or eliminating tumor resistance, the pharmaceutical composition comprising: rapamycin or an analogue thereof; and a chemotherapeutic agent.

In a preferred embodiment, the chemotherapeutic drug comprises: mitoxantrone, doxorubicin, cyclophosphamide; or the rapamycin analogue comprises: RAD001.

In another preferred embodiment, the chemotherapeutic drug is other chemotherapeutic drug other than rapamycin; preferably, the chemotherapeutic drug is: a genotoxic drug, such as mitoxantrone, doxorubicin, cyclophosphonate Amide and the like.

In another preferred embodiment, the pharmaceutical composition comprises rapamycin and mitoxantrone, and the mass ratio of rapamycin to mitoxantrone is from 1 to 5:1; preferably from 1.5 to 4:1; more preferably 2 to 3:1.

In another preferred embodiment, the pharmaceutical composition comprises the rapamycin analogue RAD001, doxorubicin and cyclophosphamide, and the mass ratio of RAD001, doxorubicin and cyclophosphamide is: (0.8 to 1.2) : (0.8 to 1.2): (25 to 35); preferably (0.9 to 1.1): (0.9 to 1.1): (28 to 32); more preferably 1:1: 30

In another aspect of the invention, there is provided the use of the pharmaceutical composition for the preparation of a kit for treating a tumor or eliminating tumor resistance.

In another aspect of the present invention, a kit for treating a tumor or eliminating tumor resistance, the kit comprising: rapamycin or an analog thereof and a chemotherapeutic drug; or the kit Included are pharmaceutical compositions as described.

In a preferred embodiment, the kit further includes instructions for use.

In another preferred embodiment, the following chemotherapy regimen is described in the instructions for use:

(1) administering to the subject a chemotherapeutic drug, as well as rapamycin or an analogue thereof (dissolvable, mixed together);

(2) After 2 weeks, the step (1) is repeated; every 2 weeks as a administration cycle, 2 to 20 administration cycles (for example, 3 to 10 administration cycles) are performed.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

DRAWINGS

Figure 1. A series of factors in prostate stromal cells (PSC27) are activated after DNA damage occurs.

In Fig. 1A, phosphorylation was observed at the mTOR S2448 site, and phosphorylation was also observed in the T389 site of S6K1 and the S65 site of 4E-BP1, one of the substrates. The S235/236 site of the biochemical substrate S6 of S6K1 was subsequently phosphorylated. Total mTOR, S6K1, 4E-BP1 and S6 are controls for each of the phosphorylated protein forms; β-actin is the loading control.

Figure 1B, immunofluorescence staining of PSC27 after ¹³⁷ Cs gamma ray (IR) and/or rapamycin (Rapa) treatment. The antibody used in the experiment, anti-p-mTOR (S2448).

Figure 2. DNA replication, senescence and proliferation of stromal cells.

Figure 2A shows the results of BrdU detection of PSC27 after IR and/or Rapa processing.

Figure 2B shows a bright field photograph of cells stained with 12-hour galactosidase reagent.

Figure 2C is a graphical representation of the percentage of galactosidase reagent staining positive cells between groups.

Figure 3. Analysis of DNA damage after stromal cells were treated under various conditions.

Figure 3A, PSC27 stains the picture at the wound at the double-strand break after IR and/or Rapa treatment. Antibody, anti-γH2AX.

Figure 3B shows statistical analysis of DNA damage after PSC27 cells were treated with IR.

Figure 3C shows statistical analysis of DNA damage after PSC27 cells were treated with MIT.

Figure 3D shows statistical analysis of DNA damage after PSAT27 cells were treated with SAT.

Figure 4. Proliferation potential curves of PSC27 cells under various conditions. Control, untreated primitive stromal cells; RS, replicative senescent cells that have been passaged several times in vitro and reached the end of the division; Rapa/IR, matrix after gamma irradiation and simultaneous treatment with rapamycin Cells; DMSO/IR, stromal cells treated with gamma radiation and simultaneously treated with DMSO as a solvent control; IR only, stromal cells irradiated with gamma rays only; control/DMSO, stromal cells treated only with DMSO .

Figure 5. Interaction between signal-regulated pathway-associated protein IKK and mTORC1 and functional activation of NF-κB after stromal cell DNA damage.

Figure 5A, Immunoprecipitation analysis of protein interactions in stromal cell lysates under control and radiation. IgG, control immunoglobulin; mTOR, antibody used in the experimental group for immunoprecipitation. Cell lysates, total cell lysate. Reverse IP, reverse immunosuppression; IgG, control immunoglobulin; IKKα, antibody used in the experimental group for immunoprecipitation.

Figure 5B, IKKα phosphorylation, IKBα protein degradation, and NF-κB functional activation, including nuclear transport of p65 and p50. --tubulin, cytoplasmic protein loading control; Histone H3, nuclear protein loading control.

Figure 6. Effect of NF-κB reporter system and mTORC1 major component proteins on functional activation of NF-κB.

Figure 6A is a reporting system for detecting functional activation of NF-κB under experimental conditions. A reporter vector encoding firefly luciferase was used to transfect PSC27 cells, and rapamycin and cell lysate after irradiation treatment were used for signal generation reactions.

Figure 6B shows the phosphorylation status of the S2448 site under mDNA damage conditions after mTOR is silenced by shRNA.

Figure 6C, raptor was silenced by shRNA and tested for expression under DNA damage conditions.

Figure 6D shows the functional activation of NF-κB before and after irradiation of mTOR and raptor by shRNA. Rapa, rapamycin treatment group, experimental positive control.

Figure 7. Effect of drug treatment or transcript silencing on the DDSP secretory phenotype in stromal cells.

Figure 7A shows the transcription of multiple DDSP effectors in PSC27 after treatment with 25 nM rapamycin.

Figure 7B shows the effect of shRNA knockdown of S6K1 on T38 phosphorylation of S6K1 in cells.

Figure 7C shows S65 phosphorylation of 4E-BP1 before and after DNA damage after transfection of stromal cells with an overexpression vector encoding 4E-BP1.

Figure 7D shows the expression changes of PSC27 secreting marker proteins including CXCL1, CCL8, WNT16B, IL8, MMP12, SPINK1, AREG, etc. after S6K1 knockdown or 4E-BP1 overexpression, respectively.

Figure 7E. ELISA-based biochemical assays for specific protein factors such as IL8.

Figure 8. Whole genome-wide microarray analysis of the expression of secreted proteins after DNA damage and the development of secretory phenotypes under rapamycin treatment conditions. C, uninjured primitive PSC27 cells; XRT, irradiated cells; X+R, cells treated with radiation and rapamycin; C vs XRT, irradiated cells compared to primitive cells C vs X+R, cells treated with radiation and rapamycin were compared to naive cells.

Figure 9. Functional effects of different IKK subunits on NF-κB activation under DNA damage conditions.

Figure 9A, after knocking out the IKKα subunit by shRNA, immunofluorescence was used to detect changes in phosphorylation and expression levels of IKKα after gamma irradiation.

Figure 9B, after knocking out the IKKβ subunit by shRNA, immunofluorescence was used to detect changes in phosphorylation and expression levels of IKKβ after IR.

Figure 9C shows the effect of IKKα subunit and IKKβ subunit on NF-κB activity after stromal cell DNA damage, respectively, after knockdown, and rapamycin treatment was combined with subunit knockout as a control.

Figure 10. Biological effects between mTOR and IKKα subunits under chemical stress and gene toxic stress.

Figure 10A, HeLa cells in TNFα (20 ng/ml, 60 min), PSC27 cells after 10 Gy gamma ray irradiation, cell lysates were immunoprecipitated (IgG as control, anti-p-IKK for the experimental group), and then Immunoblot analysis was performed for the presence of p-mTOR and p-IKK.

Figure 10B is a time plot of interaction between mTOR and IKK after gamma ray irradiation in PSC27 cells. The results of the cell lysate analysis at the segmental time node between 7 days and 20 days are shown.

Figure 11. Immunoprecipitation determines the mode of action between mTOR and IKKα under DNA damage conditions. The PSC27 cells were separately or co-transfected with the tag expression vectors Flag-IKKα and GST-mTOR, and subjected to gamma ray irradiation. The cells were pretreated with drug PP242 to avoid phosphorylation of Flag-IKKα by the GST-mTOR to be activated. Flag-IKKα in the cell lysate was then immunoprecipitated by anti-Flag, and the sediment was suspended in ATP-containing kinase buffer. Since no exogenous mTOR enters the reaction system during the process of immunization, the phosphorylation of IKKα in all subsequent results should be caused by mTOR under coprecipitation. Calf intestinal phosphatase (CIP) treated samples were obtained from a parallel experiment in a negative control. Immunoblots were detected using p-IKKα, IKKα and mTOR antibodies, and actin was a loading control.

Figure 12. Activation of NF-κB signaling pathway in stromal cells under exogenous IL-1α treatment or endogenous IL-1α knockout conditions.

Fig. 12A, PSC27 cells were pretreated with recombinant human IL-1α, and then the expression levels or retention amounts of p-IKKβ, IKKβ, IKBα, IRAK1 in the cell lysates of the original cells and IL-1α experimental group were compared and analyzed. The p65 and p50 antibodies were used to determine nuclear translocation of the NF-κB subunits p65 and p50.

Figure 12B shows the expression of IL-1α in stromal cells before and after IL-1α knockdown by shRNA, and GAPDH is a loading control.

Figure 12C shows the expression or retention of NF-κB signaling-related proteins in stromal cells after knockdown of IL-1α by shRNA, β-actin as a cytoplasmic protein loading control, and Histone H1 as a nuclear protein loading control.

Figure 13. Changes in intracellular p38, Akt and mTOR under DNA damage conditions after PSC27 stromal cell line drug-treated Akt, or transcript silenced PI3K.

Fig. 13A shows the modification of intracellular related proteins in the original state and gamma ray after treatment of PSC27 cells with pan-Akt chemical reagent MK-2206.

Figure 13B shows the expression of PIK3CA in stromal cells before and after DNA damage by shRNA knocking out the PI3K catalytic subunit p110.

Figure 13C shows the modification of intracellular related proteins in the original state and gamma ray irradiation after shRNA knockdown of the PI3K catalytic subunit p110.

Figure 14. Systematic analysis of p38, IKK subunits in stromal cells under regulation of DNA damage stress in DDSP signaling pathway. After shRNA RNA knocked out IKKα and IKKβ subunits separately or simultaneously, or SB203580 inhibited p38 kinase activity, stromal cells were modified and expressed by a series of signal transduction-related proteins before and after gamma irradiation. --actin, intracellular protein loading control; Histone H1, nuclear protein loading control.

Figure 15. Functional activation of NF-κB under conditions of drug inhibition of Akt and/or PI3K in stromal cells. Take After pretreatment of PSC27 cells with the specific drug LY294002 and/or MK-2206, the biological activity status of NF-κB was compared under the condition of DNA damage.

Figure 16. Inhibition of stromal cell mTOR activity mediated by rapamycin, a functional effect of stromal cells altering various phenotypes of cancer cells under conditions of DNA damage.

Figure 16A, Changes in proliferative properties of prostate cancer.

Figure 16B shows changes in two-dimensional migration characteristics of prostate cancer.

Figure 16C, Changes in two-dimensional invasive properties of prostate cancer.

Figure 16D, Changes in drug resistance characteristics of prostate cancer. In the above experiments, except for the Hela cell line, which is the source of cervical cancer, the others are human prostate sources (BPH1 is a benign control; M12, PC3, DU145, and LNCaP are experimental malignant cell lines).

Figure 17. In vitro, stromal cells were pretreated with rapamycin for 8 days, and then reconstituted with prostate cancer cells PC3 and inoculated into immunodeficient mice (intraperitoneal injection). After an 8-week in vivo growth period, tumors of tumor-bearing mice were measured and used for statistical analysis. 8 animals per group.

Figure 18. Growth and volume determination of tumors in experimental mice under chemotherapeutic conditions under pre-clinical conditions.

Figure 18A, Working diagram, the distribution of experimental time points for an 8-week course of treatment, including recombinant tissue inoculation, drug supply, tumor measurement, cycle number design, and the like.

Fig. 18B is a comparative analysis of the imaging images of tumor-bearing mice in different tumor conditions under the conditions of bioluminescence.

Figure 18C is a statistical analysis of the results of measuring tumor terminal volume at the end of a pre-clinical experiment for immunodeficient mice under the conditions of Figure 18B. The traditional chemotherapy drug used throughout the process is mitoxantrone. 10 animals per group.

Figure 19. Effect of rapamycin analogue RAD001 on post-translational modification, transcriptional molecule activation, and expression of DDSP phenotypic marker effector in breast stromal cells (HBF1203)-related signaling pathways following DNA damage.

Figure 19A, after ionizing radiation, the mTOR S2448 site rapidly phosphorylates, and its substrate S6K1 (and its substrate) and 4E-BP1 corresponding sites also subsequently phosphorylation. RAD forms a biochemical blockade after post-translational modification of the above signaling pathway regulators.

Figure 19B, IKKα phosphorylation, IKBα protein degradation, and NF-κB functional activation, including nuclear transport of p65 and p50. The presence of RAD001 caused significant inhibition of these changes.

Figure 19C shows the expression changes of HBF1203 secretory marker proteins including MMP1, MMP12, SFRP2, SPINK1, WNT16B, IL6, EREG, CXCL1, etc. after S6K1 knockout or 4E-BP1 overexpression, respectively.

Figure 20. Effect of rapamycin analogue RAD001 on activation of IKKβ and IKKα subunits in cytoplasm of breast stromal cells HBF1203, and NF-κB in stromal cells treated with exogenous IL-1α or endogenous IL-1α knockout. The activation state of the signal pathway.

Fig. 20A, recombinant human IL-1α was pretreated with HBF1203 cells, and then the expression levels or retention amounts of p-IKKβ, IKKβ, IKBα, IRAK1 in the cell lysates of the original cells and IL-1α experimental group were compared and analyzed. The p65 and p50 antibodies were used to determine nuclear translocation of the NF-κB subunits p65 and p50.

Figure 20B shows the expression or retention of NF-κB signaling-related proteins in stromal cells after knockdown of IL-1α by shRNA, β-actin as a cytoplasmic protein loading control, and Histone H1 as a nuclear protein loading control.

Figure 21. Therapeutic regimen of rapamycin analogue RAD001 in combination with traditional chemotherapeutic drugs and the anticancer effect of preclinical trials.

Figure 21A, Time distribution, dosing sequence and test design of SCID of immunodeficient mice subjected to conventional chemotherapy and RAD001 adjuvant therapy.

Figure 21B is a statistical analysis of the results of measuring the tumor terminal volume at the end of the pre-clinical experiment for the experimental mice in the above conditions. The traditional chemotherapeutic drug used throughout the process was doxorubicin/cyclophosphamide. 10 animals per group.

detailed description

The inventors have intensively studied for the first time to reveal a new mechanism of rapamycin or its analogs in reversing tumor resistance, and on the basis of this, provide drugs or drugs useful for treating tumors or reversing tumor resistance. A new use of a kit, as well as a combination formulation of rapamycin or an analog thereof with a chemotherapeutic drug.

As used herein, a "tumor" can be an in situ tumor or a metastatic tumor, including a refractory tumor in which resistance is present. Preferably, the tumor is a solid tumor. For example, the tumor includes: breast cancer, colorectal cancer, lung cancer, skin cancer, and the like.

The present inventors have found in the study that rapamycin or its analog can manipulate the tumor microenvironment by targeting mTOR, thereby abolishing the malignant phenotype of cancer cells and making the tumor sensitive to classical chemotherapy.

Accordingly, the present invention provides a novel medical use of rapamycin or an analogue thereof for the preparation of a medicament for eliminating tumor resistance.

The present inventors have also found that rapamycin or an analog thereof can significantly enhance the tumor suppressing effect in combination with a chemotherapeutic drug such as mitoxantrone. The synergistic effect of rapamycin or its analogs with chemotherapeutic drugs is through the following mode of action: rapamycin or its analogs affect the tumor microenvironment by targeting mTOR, making the tumor sensitive to chemotherapeutic drugs, thereby chemotherapeutic drugs The effect of medication is more ideal.

The rapamycin or its analog and the chemotherapeutic drug can be administered in the form of a pharmaceutical composition, or both can be separately present in one kit.

The present invention also provides a composition (drug) comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of said rapamycin or An analog thereof with an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the chemotherapeutic agent (e.g., mitoxantrone, doxorubicin, cyclophosphamide) ), as well as a pharmaceutically acceptable carrier.

As used herein, the term "contains" means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the term "contains."

As used herein, the term "effective amount" or "effective amount" refers to a composition or activity that is capable of functioning or active on a human and/or animal and is acceptable for use by humans and/or animals.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

Typically, the rapamycin or analog thereof can be formulated with a chemotherapeutic drug (eg, mitoxantrone, doxorubicin, cyclophosphamide) in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. Wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8.

The present invention provides a kit for treating a tumor, which comprises rapamycin or an analog thereof and a chemotherapeutic drug (such as mitoxantrone, doxorubicin, cyclophosphamide). More preferably, the kit further includes instructions for use to guide the clinician to administer the medicine in a correct and reasonable manner.

For ease of administration, the rapamycin or an analog thereof is combined with a chemotherapeutic drug (such as mitoxantrone, doxorubicin, cyclophosphamide) or isolated rapamycin or an analog thereof or chemotherapy The drug (such as mitoxantrone, doxorubicin, cyclophosphamide) can be placed in a unit dosage form and placed in a kit. "Unit dosage form" refers to a dosage form required for preparing a drug for single administration for convenience of administration, including but not limited to various solid agents (such as tablets), liquid agents, capsules, and sustained release agents.

In order to obtain a good therapeutic effect, the inventors have made a treatment plan through repeated research and comparison, including: (1) administration of a chemotherapy drug to a subject, and rapamycin or an analog thereof; (2) two weeks later, Step (1) is repeated; every two weeks as a dosing cycle, 2 to 20 dosing cycles are performed.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

Materials and Method

1. Cell culture

(1) Cell line and culture

The normal human prostate primary stromal cell line PSC27 and the normal human primary mammary stromal cell line HBF1203 were propagated and passaged in PSCC complete medium. Prostate benign epithelial cell line BPH1, prostate cancer epithelial cell line M12, DU145, PC3, LNCaP and VCaP (purchased from ATCC), both in 5% FBS in RPMI-1640 complete medium, at 37 ° C, 5% CO ₂ conditions Cultivate in an incubator. The breast cancer epithelial cell line MDA-MB-231 (purchased from ATCC) was cultured in DMEM medium containing 5% FBS.

(2) In vitro experimental treatment

To cause DNA damage, PSC27 cells were grown to 80% using ¹³⁷ Cs gamma rays (dosage 10 Gy, abbreviated as IR) for ionizing radiation. In addition, 1 μM mitoxantrone (PSC27-MIT), 10 μM satraplatin (Satraplatin/JM216, PSC27-SAT) was also used in parallel experiments to cause DNA damage. Six hours after the drug treatment, the cells were briefly washed three times with PBS, left in the culture solution for 7 to 10 days, and then subjected to subsequent experiments.

2. Gene knockout vector and immunological test antibody

The lentiviral vectors encoding shRNAs used in the experiments were purchased from Open Biosystems (Lafayette, CO), USA.

The sources of antibodies against the following antigens are: γH2AX (Upstate); mTOR (BioLegend; Invitrogen); phospho-mTOR (Ser2448) (R&D); GST, Raptor (Millipore); Rictor, 4EBP1, phosphor-4EBP1, S6K1, phospho -S6K1 (Cell Signaling); IKKα, phospho-IKKα, phospho-Akt (S473) (Cell Signaling); IKKβ, IKKγ (Abeam); IKBα (MyBioSource); phospho-IKKβ (US Biological); p65, Histone H1, Histone H3, β-actin, β-tubulin (Santa Cruz); NF-κB1 (p50) (eBioscience); p38, phosphor-p38 (Enzo Life Sciences); PI3K-p110 (Abgent); PI3K-p85 (Epitomics); Akt , IL-1α, IL-8 (R&D); CXCL1 (GeneTex); CCL8 (Abcam); WNT16B (BD Pharmingen); MMP12 (OriGene) and AREG (LifeSpan), used for immunoblotting and/or immunofluorescence precipitation assay . Recombinant human IL-1α (HPLC-purification (159aa)) was supplied by US Biological.

3. BrdU embedded labeling and β-galactosidase staining analysis

The cells were seeded in a 35 ml culture dish at a volume of 1.5 × 10 ⁵ /ml (a cover glass was placed inside), cultured for 1 day, and synchronized with a 0.5% FBS medium for 3 days to allow most cells to be in the G0 phase. . Before termination of cell culture, BrdU (final concentration 30 μg/L) was added, and incubation was carried out for 4-6 h at 37 °C. The culture was discarded and the slides were washed 3 times with PBS. Methanol / acetic acid was fixed for 10 min. The fixed slides were air dried and 0.3% H ² O ² -methanol was used to inactivate the endogenous oxidase for 30 min. Block with 5% normal rabbit serum. Formaldehyde amine denatured nucleic acid at 100 ° C for 5 min. After cooling in an ice bath, it was washed with PBS, and the primary antibody was added with anti-mouse BrdU monoclonal antibody (working concentration 1:50), and the negative control was PBS or serum. The ABC method was used to detect the hematoxylin or eosin. The total number of cells and BrdU-positive cells in 10 high power fields were randomly counted under the microscope, and the marker index (LI) was calculated.

The β-galactosidase staining was performed by simply washing the cells to be stained with PBS and fixing them at room temperature for 5 minutes using 4% formaldehyde. After washing with PBS, it was incubated at 37 ° C for 12-18 h without CO ₂ and then observed under a microscope.

4, immunofluorescence analysis

Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) and rabbit polyclonal antibody anti-SFRP2 (Santa Cruz), and secondary antibody Alexa

488 (or 594)-conjugated F(ab') _{2 was} added to the slides covered with fixed cells in sequence. The nuclei were counterstained with 2 μg/ml of 4',6-diamidino-2-phenylindole (DAPI). The most representative image was selected from the three observation fields for data analysis and results display. The FV1000 laser scanning confocal microscope (Olympus) was used to obtain confocal fluorescence images of cells.

5. Matrix-epithelial co-culture and in vitro experiments

The PSC27 cells were cultured for 3 days with DMEM + 0.5% FBS medium, and then the abundance of the cell population was washed with 1 time PBS. After simple centrifugation, the supernatant was collected and stored as a conditional medium at -80 ° C or used directly. Prostate epithelial cells were cultured in vitro in this conditional medium for 3 days. For chemotherapy resistance, epithelial cell lines are cultured in low serum DMEM (0.5% FBS) ("DMEM"), or in conditioned medium, while mitoxantrone is used to treat cells for 1 to 3 days at concentrations close to each IC ₅₀ values of cell lines, followed by a bright field microscope.

6. Whole genome-wide expression microarray analysis (Agilent expression microarray)

The procedure and method for genome-wide expression chip (4 x 44k) analysis of the normal human prostate primary stromal cell line PSC27 is identical to that previously published (Sun, Y., Campisi, J., Higano, C., Beer) , TM, Porter, P., Coleman, I., True, L. and Nelson, PS 2012. Treatment-Induced Damage to the Tumor Microenvironment Promotes Prostate Cancer Therapy Resistance through WNT16B. Nat. Med. 18: 1359-1368).

7. Quantitative PCR (RT-PCR) for gene expression

The total RNA in the growth phase was extracted with Trizol reagent and reverse transcribed into cDNA. The reverse transcription reaction product cDNA was diluted 50-fold as a template.

AceQ SYBR Green Master Mix 10ul

According to the above standard, the reaction conditions were: pre-denaturation at 95 ° C for 15 s, then 95 ° C for 5 s, 60 ° C for 31 s, 40 cycles; the melting curve conditions were 95 ° C for 15 s, 60 ° C for 30 s, and 95 ° C for 15 s. The samples were reacted on an ABI ViiA7 (ABI) instrument. The expression of β-actin was used as an internal reference. After the reaction was completed, the amplification of each gene was analyzed by software analysis, and the corresponding number of domain value cycles were derived. The relative expression of each gene was calculated by the 2-ΔΔCt method. The peaks and waveforms of the melting surve are analyzed to determine if the resulting amplification product is a specific single-purpose fragment.

8. Western blot analysis

(1) Total cell protein extraction

After simple washing of the cells in ice-cold PBS buffer, RIPA cell lysis buffer (Invitrogen) containing 1 mM PMSF (protease inhibitor) was added, and the cells were lysed on ice for 30 min, and the cell lysate was collected with a cell scraper at 1 ° C for 12,000 °C. Centrifuge at rpm for 15 min, take the supernatant, and store at -80 °C.

(2) BCA protein quantification

The BCA protein quantification kit (Pierce) was prepared by mixing reagent A and reagent B in a ratio of 1:50 to prepare a working solution for use. The standard protein was diluted to a concentration of 0 μg/μl, 25 μg/μl, 50 μg/μl, 100 μg/μl, 250 μg/μl, 500 μg/μl, 750 μg/μl, 1000 μg/μl, 2000 μg/μl. Add 5 μl of standard protein or 5 μl of sample to the plate, add 100 μl of BCA working solution, mix well and then bath at 37 ° C for 30 min, and read the absorbance at 570 nm with a microplate reader. A standard curve is drawn by taking the absorbance value as the ordinate and the standard protein concentration as the abscissa. The concentration of the sample was calculated from the standard curve.

(3) SDS-PAGE electrophoresis

Prepare 12% SDS-PAGE (5ml system containing 30% acrylamide 2ml, ddH2O 1.6ml, 1.5M pH8.8Tris-HCl 1.3ml, 10% SDS 50μl, 10% ammonium persulfate 50μl, TEMED 2μl), mix quickly Inject into a clean preset glass plate (Bio-Rad) gap, and add appropriate amount of deionized water on the top layer to promote gel polymerization. Allow to stand at room temperature for 30 minutes. After the separation gel is completely agglomerated, discard the upper layer of deionized water and filter it with filter paper. Net residual liquid. Prepare concentrated gel (2ml system contains 30% acrylamide 0.33ml, 1.0M pH6.8Tris-HCl 0.25ml, 10% SDS 20μl, 10% ammonium persulfate 20μl, TEMED 2μl), and mix immediately after adding to the upper layer of separation gel Insert a clean 10-tooth comb and let stand for 30 minutes at room temperature. After the gel is completely solidified, remove the comb and wash the sample tank several times with ddH2O. Place the gel in the electrophoresis tank (Bio-Rad) and add the running buffer. (containing 25 mM pH 8.0 Tris, 0.25 M Glycine, 0.1% SDS). The protein sample was mixed in a 5:1 ratio with 6× loading buffer (containing 300 mM pH 6.8 Tris-HCl, 12% SDS, 600 mM DTT, 60% glycerol, 0.6% bromophenol blue), boiled in water for 10 min, and cooled in an ice bath. 5 min, combined with protein quantification results, equal amounts of protein samples were added to each lane, and electrophoresed using a Bio-Rad electrophoresis apparatus, first with 80V. After the voltage was electrophoresed for about 20 minutes until the front of the bromophenol blue entered the separation gel, the voltage was raised to 120 V, and electrophoresis was continued for about 1 hour until the bromophenol blue band reached the bottom of the separation gel, and the electrophoresis was completed.

(4) Protein transfer film

After SDS-PAGE electrophoresis, the concentrated gel and the sample-free area were excised, and the nitrocellulose filter was briefly soaked in an electrophoretic transfer buffer. On the electrotransfer device (Bio-Rad), Bio-Rad 3 mm filter paper, nitrocellulose filter, gel, Bio-Rad 3 mm filter paper were sequentially placed from the anode to the cathode. Electrotransfer was performed at a voltage of 100 V for 1.5 h. After the completion of the transfer, the transfer effect was judged by pre-staining Marker and staining with 0.1% Ponceau Stain (Ponceau Stain), and decolorized with ddH ₂ O for 5 min.

(5) Antibody labeling and ECL detection

The nitrocellulose filter was blocked in a blocking solution (TBST (0.1% Tween-20in TBS) containing 5% skim milk powder) for 1 hour at room temperature. Incubate overnight in a primary anti-hybrid solution at 4 °C. Rinse 3 times with TBST at room temperature for 2 minutes each time. HRP-conjugated secondary antibody hybrids prepared in blocking solution were added and incubated for 0.5 hour at room temperature. The filter was rinsed 3 times with PBST at room temperature for 2 min each time.

SuperSignal West0Pico kit (Pierce) medium proportion of substrate and enhancer mixed, evenly added to the filter, incubated for 1 minute at room temperature, exposed X-ray film, developed, fixed, scanned X-ray film and saved for analysis .

9. Protein interaction immunoprecipitation analysis

Stromal cells (or epithelial cells) are stimulated by gamma rays, genotoxic drugs or chemical factors in vitro, and after a certain period of time, the cell lysates are collected and used for subsequent protein-protein interaction detection. Take 1.2 ml of cell lysate, place in a centrifuge tube, add protein beads A agarose 20 ul, and shake gently for 1 h on a 4° shaker to remove non-specific hybrid proteins and reduce background. After centrifugation, the supernatant was collected in another centrifuge tube, and 2 to 5 ug of the primary antibody and 25 ul of the protein A agarose beads were added to the supernatant, and the mixture was shaken slowly for 4 overnight on a 4° shaker. The A agarose beads were collected by centrifugation, the supernatant was discarded, and the pellet was resuspended in 2 x SDS loading buffer 25 ul, boiled for 2 to 5 min, and then used for polyacrylamide gel electrophoresis.

10. Detection of in vitro kinase activity (without cell conditions)

The stromal cells, PSC27, were treated under different conditions and treated with 1 ml of lysate containing 0.3% CHAPS. The tag expression vectors Flag-IKKα (purchased from Addgene) and GST-mTOR (purchased from Addgene) were used to co-transfect cells 48 hours prior to irradiation. Total cell lysates were collected after 72 hours of treatment, PP242 (Fisher-Scientific, 20 nM) and control were added to the medium 3 hours prior to irradiation to limit mTOR kinase activity. The lysate contained a protease inhibitor, a phosphatase inhibitor and 20 nM PP242. Lysates were incubated with anti-Flag for 12 hours and then incubated with 25 [mu]l Protein G Sepharose beads for 1 hour. Anti-Flag-mediated immunoprecipitate was washed 4 times with lysate to remove PP242 with ATP (20 mM Hepes at pH 7.7, 2 mM MgCl2, 2 mM MnCl2, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM p-Nitrophenyl Phosphate [PNPP] ], 300 μM orthovanadate, 1 mM Benzamidine, 2 mM PMSF, 1 mM DTT, 10 μg/mL aprotinin, 1 μg/mL Leupeptin, 1 μg/mL pepstatin, 1 mM DTT) were washed 3 times. Immunoprecipitates were also incubated with calf intestinal phosphatase (CIP) for 45 min at 37 °C to inhibit co-settled mTOR activity as a special control for this experiment. Kinase assays for GST-mTOR using sediment were performed at 30 ° C for 60 min using kinase buffer and 10 μM ATP and [γ- ³² P]ATP (0.5 μCi per kinase reaction). To terminate the experiment, 8 μL of 4×SDS sample buffer was added to each reaction and boiled for 10 min. The phosphorylation status of IKKα in the immunoprecipitate was subsequently determined by immunoblotting using a phospho-IKKα-specific antibody. In addition, antibodies against GST were used in red in a parallel experiment to detect the expression of mTOR in the lysate.

11. NF-κB transcriptional activity reporter expression vector

Covers multiple NF-κB binding sites and optimized IL-2 minimal promoter as a reporter vector for NF-κB activated transgenic system (NAT system) NAT11-Luc2CP-IRES-nEGFP (obtained from Hokkaido University, Japan) in experiments Used as a positive control for NF-κB activity.

12. Mouse xenograft test and pre-clinical chemotherapy procedure

ICF SCID mice (body weight about 25 g) of immunodeficient mice of about 6 weeks old were used in the animal experiments of the present invention. PSC27 stromal cells and epithelial cells in a 1: 4 ratio mixture, and each graft containing 1.25 × 10 ⁶ cells, for tissue reconstruction. The transplanted tumor was implanted into the mouse by intraperitoneal injection, and the animal was euthanized 8 weeks after the end of the transplant operation. The tumor volume was calculated according to the following formula: V = (π / 6) x ((l + w) / 2) ³ (V, volume; l, length; w, width).

In the pre-clinical chemotherapy trial, mice that were transplanted subperitoneally were fed a standard experimental diet, and two weeks later, the chemotherapy drug mitoxantrone (0.2 mg/kg dose) and/or rapamycin (0.5 mg/kg dose) was administered. ) intraperitoneal administration. The time point was the first day of the 3rd, 5th, and 7th week, and the entire course of treatment was administered in 3 cycles, each cycle being 2 weeks. At the end of the course of treatment, tumors in mice were collected for volume measurement and histological analysis. Each mouse cumulatively received mitoxantrone 0.6 mg/kg body weight and rapamycin 1.5 mg/kg body weight. The chemotherapy trial was completed by the end of the 8th week.

In the pre-clinical chemotherapy trial of breast cancer cell line MDA-MB-231 and breast stromal cell line HBF1203, the chemotherapy drug doxorubicin/cyclophosphamide was administered 2 weeks after tumor implantation (1.0 mg/kg and 30.0 mg/d, respectively). The kg dose) and/or RAD001 (1.0 mg/kg dose) were administered intraperitoneally. The time point, frequency of administration and frequency were all combined with the above treatment regimen of mitoxantrone/rapamycin. Each mouse cumulatively received doxorubicin/cyclophosphamide 3.0 mg/kg, 90.0 mg/kg body weight, and RAD001 was 3.0 mg/kg body weight. The entire duration of the chemotherapy trial was 8 weeks.

13. Biostatistical methods

In the present invention, all in vitro experiments involving cell proliferation rate, migration, invasiveness and viability, and in vivo experiments of mouse xenografts and chemotherapy treatment were repeated three times or more, and the data were presented in the form of mean ± standard error. Statistical analysis is based on raw data, by one-way analysis of variance or a two-tailed Student’s The t-test was calculated, and the result of P < 0.05 was considered to have a significant difference.

Example 1. One of the responses of stromal cells to genotoxic stress, mTOR is activated intracellularly

Anticancer drugs can cause significant cellular physiology fluctuations under clinical conditions, including DNA physical damage and autonomic repair reactions. The consequences mainly include apoptosis, autophagy and aging procedures. The cells enter the senescence phase shortly after DNA damage, but remain metabolically active and physiologically viable for several months, and exhibit a secretory state characterized by lysosomal expansion and positive galactosidase staining. This process is called the DNA Damage Secretion Program (DDSP). First, it is necessary to confirm whether mTOR, the main signaling molecule that responds to various external stimuli such as drug stress, nutrients and amino acid supply by mammalian cells, is activated in stromal cells.

The experimental data indicate that once the human prostate stromal cell line PSC27 is treated by ionizing radiation, mTOR is highly phosphorylated at Ser2448 (Fig. 1A), and this change is often involved in cell-to-external stimulation as a biological activity and function of the kinase. A biomarker that reacts. Accompanying mTOR activation is phosphorylation of its downstream target S6K1 and its biochemical substrates S6 and 4E-BP1. In general, phosphorylation of S6K1 allows cells to precisely regulate ribosome biosynthesis and protein translation. Phosphorylation of 4E-BP1 prevents its own binding to eIF-4E and thus increases the initiation of translation of capped proteins. In contrast, pretreatment with rapamycin at a final concentration of 25 nM substantially abolished post-translational modifications of mTOR, S6K1 and 4E-BP1 in stromal cells under culture conditions. Phosphorylated mTOR mainly occurs in the cytoplasm, and the appearance of DNA single-double strand breaks in the nucleus following the gene poisoning attack is inconsistent. Although rapamycin did not significantly alter the morphology of naive cells, it significantly inhibited the phosphorylation level of mTOR in injured stromal cells (Fig. 1B). Cell cycle termination and significant galactosidase staining were positive markers of genotoxicity-induced cellular senescence.

However, the addition of rapamycin to the culture medium did not alter these phenotypes, suggesting that both the senescence and metabolic activities of these cells were maintained (Fig. 2A, 2B and 2C). It is worth noting that the focus of the wound caused by radiation-induced DNA damage remains unchanged even after rapamycin is added to the medium (Figure 1B; Figures 3A and 3B), indicating that rapamycin does not alter DNA physical properties. The state of the wound. As supporting evidence, two drugs commonly used in prostate cancer patients in the clinic, mitoxantrone (MIT) and satraplatin (SAT), once used for administration in the same experiment, will produce Very similar effects (Figures 3C and 3D). Although for some transformed cells, mitoxantrone can attenuate DNA damage signals and transform irreversible senescence growth repression into reversible resting state repression, the inventors' research proves that it is triggered in stromal cells. A completely different reaction. The total population doubling is a response index of cell proliferation potential, but it is not altered by rapamycin. This trend becomes a reality once stromal cells enter the senescence stage under genotoxic stress (Fig. 4).

The vast majority of regulatory pathways involved in the secretion of synaptic stimuli have been reported in the past, including the transcription factors NF-κB and C/EBPβ, but those with mTOR functional involvement have been largely ignored. To fill this gap, the inventors chose to lyse cells on day 10 after DNA damage and then perform deep analysis by mTOR antibody-mediated immunoprecipitation. Interestingly, the binding between mTOR and IKKα and Raptor in irradiated cells is clear. Significantly enhanced, although the total protein amount of each protein in the lysate did not change before and after DNA damage (Fig. 5A). Although there is a physical bond between mTOR and IKKβ and IKKβγ, this effect is weak and does not show much change. To confirm this, the inventors conducted an experiment in which an IKKα antibody was used before collecting the sediment, and it was found that the amount of mTOR was increased instead of Raptor (Fig. 5A). The above data indicate that there is an interaction between the α subunit of IKK and mTOR; and the molecular aggregation between IKKα/β/γ and the mTOR complex confirms that some molecules involved in mTOR can bind to each other to cause transcriptional complex NF. - Activation of κB.

Example 2, as a key signal node in the progress of DDSP program, the mTOR/Raptor complex points to the IKK/NF-κB pathway once DNA damage occurs.

The inventors next evaluated the biological relevance of the mTOR pathway for the development of the DNA damage secretory program (DDSP) and attempted to elucidate its underlying mechanisms. First, the inventors examined the expression and activation of several components of the NF-κB pathway in the cytoplasm or nucleus. DNA damage caused phosphorylation of IKKα at the Ser176/180 site, whereas the presence of rapamycin caused a significant decrease in its signal (Fig. 5B). Consistent with the changes in the IKK subunit, even after stromal cells were irradiated, a large amount of IκBα remained in the cytoplasm, and the p65/p50 nuclear translocation as one of the downstream responses was treated with rapamycin. The next was quickly abolished (Figure 5B). Using a set of reporter systems (Fig. 6A) that the inventors created in the experiment to detect NF-κB transcriptional activity in the nucleus, it was found that transcriptional inhibition against mTOR or Raptor can significantly prevent functional activation of the NF-κB complex, Although mTOR or Raptor specific shRNA (Fig. 6B, 6C) had a synergistic effect under the conditions of the combined use and was roughly equivalent to the results produced by rapamycin at a working concentration of 25 nM (Fig. 6D).

In view of the fact that stromal cells respond to DNA damage by synthesizing and releasing a plurality of soluble factors, the inventors used rapamycin for cells and subsequently irradiated them. The upregulation of the DDSP marker factor was significantly attenuated or substantially abolished compared to changes caused by genotoxic treatment, and the magnitude of the change at the transcriptional level was limited to a minimum range (Fig. 7A). Once knocked out of S6K or 4E-BP1 high expression (Figures 7B and 7C), or treated with rapamycin, a portion of the DDSP factor associated with DNA damage is significantly reduced, including CXCL1, CCL8, WNT16B, IL-8, MMP12 , SPINK1 and AREG (Fig. 7D). In particular, an ELISA-based biochemical assay performed on a specific protein factor (such as IL-8) confirms this (Fig. 7E). However, there are always a large number of synthetic proteins encoded by DDSP in the case of genotoxicity, but not affected by rapamycin, suggesting the biological complexity of this stress-responsive program regulatory mechanism (Fig. 7A). The inventors also obtained supporting evidence from genome-wide chip expression data, which is more detailed and thorough information (Fig. 8).

Example 3, NF-κB signaling pathway is linked to the kinase activity of the mTORC1 complex

To elucidate the differences in mTOR signaling mediated by different IKK subunits at DNA damage, the inventors removed the IKKα and IKKβ subunits prior to gamma ray irradiation with gene-specific shRNA, respectively (Figs. 9A and 9B). Elimination of the alpha subunit substantially abolishes the nuclear activity of NF-κB, although the effect of the removal of the beta subunit is higher. When both subunits were absent, only minimal levels of activation of the NF-κB pathway were detected in the presence of DNA damage (Fig. 9C).

Although IKKα is associated with mTOR and appears to be functionally involved in the signalling of the activated mTORC1 complex (Figure 5A; Figures 10A and 10B), is there a direct interaction or two between the two molecules? The relationship is indirectly mediated by other factors and it is still unclear. To clarify this, the inventors conducted further experiments using the drug PP242, a novel ATP competitive, selective mTOR kinase inhibitor. First, in vitro experiments were performed to determine whether mTOR directly phosphorylates IKKα, ie, co-transfected PSC27 cells with GST-mTOR and Flag-IKKα expression vectors (Fig. 11). Prior to irradiation, cells were treated with PP242 to prevent IKKα from being phosphorylated by the oncoming activated mTOR. After the radiation treatment, the cells were lysed, Flag-IKKα was immunoprecipitated, and then suspended in the kinase buffer together with the ATP added to the mixture; since no exogenous mTOR was added to the system after the immunoprecipitation, this detection process All observed IKKα phosphorylation events are necessarily caused by mTOR that has settled together. Western blot results demonstrated that IKKα phosphorylation at the S176 site in the context of genotoxicity, whereas phosphorylation of mTOR against IKKα was confirmed by another treatment involving calf intestinal phosphatase (Figure 11). Thus, IKKα phosphorylation actually occurs downstream of mTOR activated after DNA damage, and this reaction can be reversed by dephosphorylation. The inventors' data demonstrate that there is a physical bond between mTOR and the IKK complex, which is a response to external stress, in which IKKα is the main interaction directly with mTOR by assembling the total IKK complex. Protein component. Interestingly, when rapamycin was used to replace PP242 in a parallel experiment, the phenomenon of IKKα phosphorylation (S176) did not appear, suggesting a potential difference in function between the two mTOR inhibitors.

Example 4: The downstream and upstream signaling pathways of mTOR are active in injured stromal cells.

As a stress-inducing factor, IL-1α is important for the establishment and maintenance of secretory factor-related stromal cell phenotypes including IL-6 and IL-8, and such factors are essential for enhanced growth termination. Less pro-inflammatory factors. Stromal cells damaged by gene virulence can express high levels of IL-1α transcripts, intracellular proteins, cell surface binding proteins and very limited exocrine proteins. Even with these traits, the functional association between IL-1α on the cell surface-bound state and mTOR-activated mTOR remains absent. It is important to understand whether rapamycin inhibits and how IL-1α-mediated signaling changes in the progression of DDSP in the background. Interestingly, IKKβ was phosphorylated immediately after exposure to stromal cells (Fig. 12A). As a key component of the IL-1α/IL-1R signaling pathway, IL-1α receptor-associated kinases IRAK1 and IκBα were both degraded. This trend was significantly inhibited by rapamycin treatment, indicating that this led to a blockade of IL-1R signaling. In addition, exogenous IL-1α was able to reverse this repression after addition to the medium, and nuclear transfer of the NF-κB complex including p65 and p50 was also substantially simultaneously restored (Fig. 12A).

In another reverse experiment, the elimination of IL-1α (Fig. 12B) resulted in a decrease in the level of IKKβ phosphorylation, while the IKKα phosphorylation status did not change (Fig. 12C). Although the level of IRAK1 protein did not change, the total amount of IκBα was significantly decreased, suggesting that the single line-mediated NF-κB pathway of IKKα is still active. The most further support, p65 The p50 nuclear signal is still present, indicating a sustained activation of the NF-κB complex (Fig. 12C).

Since mTOR activation is mostly due to the response of cells to external stimuli, it is to be confirmed whether PI3K/Akt, which is an upstream regulator of mTOR in mammalian cells, is directly responsible for DDSP-related mTOR activation. After using MK-2206, a highly selective, non-ATP competitive isomeric, broad-spectrum Akt inhibitor, the inventors found that mTOR phosphorylation is disappearing, and this process is associated with a decrease in phosphorylation of Akt at the S473 site. Simultaneously (Fig. 13A). However, regardless of how Akt inhibition is carried out, p38 activation is silent. In the absence of such evidence, it is not surprising that the removal of the catalytic subunit p110 of PI3K kinase (Fig. 13B) prevented Akt and mTOR phosphorylation, although the p85α regulatory subunit remained unchanged. State (Figure 13C). In summary, under each of the above assays, p38 phosphorylation, as an indicator of activation of the MAPK pathway, maintained a sustained activation state regardless of changes in the PI3K/Akt axis (Figure 14).

As a universal imprint of the DDSP event, the activity of the NF-kB complex is significantly increased after DNA damage, but can be ablated by the inhibition of PI3K or Akt, although some of the nuclear activity remains in the cell under the attack of the gene poison (Fig. 15). . Therefore, the regulatory pathway parallel to p38 from the upstream is working at the same time, or p38 mediates the DDSP signaling pathway through PI3K/Akt but ultimately involves functional activation of the NF-kB complex in the injured cells; more important The reason is that both possibilities exist in fact (Figure 14).

Example 5. Manipulating the microenvironment by targeting mTOR can abolish the malignant phenotype of cancer cells and make the tumor sensitive to classical chemotherapy

Next, the inventors examined various effects on the behavior of cancer cells once mTOR was used to inhibit the stromal cells' DDSP program. First, the inventors collected a conditional medium produced by stromal cell lines for stimulation of various epithelial cell lines in vitro. PSC27 was subjected to radiation in the presence of rapamycin for 10 days to fully develop the DDSP phenotype prior to collection of the conditioned medium, which was subsequently applied to epithelial cells. Interestingly, growth potential was generally minimized in several cell lines tested (Figure 16A). It has been reported in the past that conditional media produced by damaged stromal cells can promote the planar mobility of cancer epithelial cells and the invasiveness of penetrating gelatinous membranes; however, these properties of mTOR-deficient stromal cells are derived from conditional media. , but was significantly reduced (Figures 16B and 16C). At the same time, in an experiment to measure the cytotoxicity of DDSP broad-spectrum factors to protect epithelial cells from MIT presentation, the data obtained indicate that rapamycin-mediated mTOR functional stromal cells confer survival to cancer cells. The advantage no longer exists (Figure 16D).

On the basis of in vitro experiments, the inventors conducted further studies on mTOR as a pivotal mediation of DDSP progression by manipulating tissue reconstitution by stromal cells. After ionizing radiation, PSC27 cells were incubated for 10 days in the presence of rapamycin, and then mixed with PC3-Luc (a subtype of prostate cancer PC3 cell line, which can express firefly luciferase), and transplanted together with immunization. Deficient mice in vivo. After an 8-week course of treatment, the experimental mice were assayed for bioluminescence by the IVIS xenogen system and the instrument determined its tumor size. The present inventors have found that the growth potential of PC3 tumors is inhibited by 50% in vitro using rapamycin pretreatment in stromal cells (Fig. 17). Therefore, a single dose of rapamycin can avoid the tumor growth that DDSP improves.

Furthermore, a pre-clinical study simulating physiological reality in a clinical setting confirmed the effect of manipulating the tumor microenvironment in a pattern that targets mTOR. From the beginning of the third week, mice received an intraperitoneal injection of mitoxantrone (MIT) at a dose of 0.2 mg/kg on the first day of the third, fifth and seventh weeks (Fig. 18A). Therefore, there are a total of three two-week cycles during this chemotherapy period. During this period, rapamycin was administered as a drug targeting the tumor microenvironment in the form of a unit dosage form with MIT. At the end of the 8-week course of treatment, the group that rapamycin was involved in formed a tumor that was much smaller than the control group (Fig. 18B).

To confirm the pre-clinical effect, the inventors analyzed the data statistically and found that in addition to the 36.8% tumor shrinkage directly caused by MIT, rapamycin administration further caused a 55.7% reduction in tumor burden (Fig. 18C). Objectively, this has confirmed the in vivo effect of this drug on abolishing the drug resistance conferred by DDSP.

In summary, the combination of mitoxantrone and rapamycin has significantly improved the anti-tumor efficacy.

Example 6, treatment and effect of rapamycin analogue RAD001 combined with traditional chemotherapy drugs

The present invention verified the effect of rapamycin analogue RAD001 on post-translational modification, transcriptional molecule activation and expression of DDSP phenotypic effector factors of breast stromal cells (HBF1203)-related signaling pathways following DNA damage. Once HBF1203 was treated by ionizing radiation, the mTOR S2448 site rapidly phosphorylated, and its substrates S6K1 (and its substrate) and 4E-BP1 corresponding sites also subsequently phosphorylated, as shown in Figure 19A. RAD forms a biochemical blockade after post-translational modification of the above signaling pathway regulators. IKKα phosphorylation, IKBα protein degradation, and NF-κB functional activation, including nuclear translocation of p65 and p50. The presence of RAD001 caused significant suppression of these changes, as shown in Figure 19B. After S6K1 knockdown or 4E-BP1 overexpression, HBF1203 secreted marker proteins include expression changes of MMP1, MMP12, SFRP2, SPINK1, WNT16B, IL6, EREG, CXCL1, etc., as shown in Fig. 19C.

The present inventors also verified the effect of rapamycin analogue RAD001 on the activation of IKKβ and IKKα subunits in the cytoplasm of breast stromal cells HBF1203, and in stromal cells under exogenous IL-1α treatment or endogenous IL-1α knockout conditions. Activation status of the NF-κB signaling pathway. Recombinant human IL-1α was pretreated with HBF1203 cells, and then the expression levels or retention of p-IKKβ, IKKβ, IKBα, IRAK1 in the cell lysates of the original cells and IL-1α experimental group were compared and analyzed. The p65 and p50 antibodies were used to determine nuclear translocation of the NF-κB subunits p65 and p50, and the results are shown in Figure 20A. The expression or retention of NF-κB signal-related protein in stromal cells after knockdown of IL-1α by shRNA, β-actin was a cytoplasmic protein loading control, and Histone H1 was a nuclear protein loading control. The results are shown in Figure 20B.

The present inventors also verified the therapeutic regimen of the rapamycin analogue RAD001 in combination with a conventional chemotherapeutic drug and the anticancer effect of the pre-clinical trial. The time distribution of the SCID of immunodeficient mice after conventional chemotherapy and RAD001 adjuvant therapy, the order of administration and the design of the test are shown in Figure 21A. The traditional chemotherapeutic drug used in the whole process is doxorubicin/cyclophosphamide. Results of the measurement of tumor terminal volume at the end of the pre-clinical experiment for the experimental mice in the above conditions Analysis is shown in Figure 21B. The results showed that the administration of the rapamycin analogue RAD001 further caused a 52.9% reduction in tumor burden in addition to the 38.7% tumor shrinkage directly caused by the chemotherapeutic drug.

In summary, the combination of doxorubicin/cyclophosphamide and rapamycin analogue RAD001 significantly improved the anti-tumor efficacy.

In the present invention, "mitoxantrone" is administered directly at the same time as "rapamycin", and there is no such an old way of intermittent administration, which is time-consuming and labor-intensive, increases trauma, and ultimately affects the therapeutic effect. In addition, the present inventors provided combined therapy data for breast cancer, namely, the use of "doxorubicin/cyclophosphamide" in combination with the rapamycin analogue RAD001, which has changed only previously targeted cancer. The treatment idea of the cell itself incorporates the pathological effects of the microenvironment into the intervention category, thereby overcoming the serious limitations of the traditional anti-cancer treatment and has outstanding innovation significance.

All documents mentioned in the present invention are incorporated herein by reference in their entirety as if they are individually incorporated by reference. In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the invention.

Claims (13)

1. A use of rapamycin or an analogue thereof for the preparation of a pharmaceutical composition for eliminating tumor resistance.
2. The use according to claim 1, wherein said tumor resistance is resistance of a tumor to a chemotherapeutic drug.
3. The use according to claim 1, wherein the rapamycin or analog thereof manipulates the tumor microenvironment by targeting mTOR, thereby eliminating tumor resistance.
4. The use according to claim 1, wherein said rapamycin analogue comprises: RAD001.
5. The use according to any one of claims 1 to 4, wherein the tumor comprises: prostate cancer, breast cancer, colorectal cancer, lung cancer, skin cancer.
6. A pharmaceutical composition for treating a tumor or eliminating tumor resistance, characterized in that the pharmaceutical composition comprises:

   Rapamycin or an analogue thereof;

   Chemotherapy drugs.
7. The pharmaceutical composition according to claim 6, wherein said chemotherapeutic drug is a genotoxic drug; more preferably, said chemotherapeutic drug comprises: mitoxantrone, doxorubicin, cyclophosphamide;

   The rapamycin analogue comprises: RAD001.
8. The pharmaceutical composition according to claim 7, which comprises rapamycin and mitoxantrone, and a mass ratio of rapamycin to mitoxantrone is from 1 to 5:1; preferably It is 1.5 to 4:1; more preferably 2 to 3:1.
9. The pharmaceutical composition according to claim 7, which comprises a rapamycin analogue RAD001, doxorubicin and cyclophosphamide, and the mass ratio of RAD001, doxorubicin and cyclophosphamide is: (0.8) ～1.2): (0.8 to 1.2): (25 to 35); preferably (0.9 to 1.1): (0.9 to 1.1): (28 to 32); more preferably 1:1: 30
10. Use of the pharmaceutical composition according to any one of claims 6 to 9 for the preparation of a kit for treating a tumor or eliminating tumor resistance.
11. A kit for treating tumors or eliminating tumor resistance, characterized in that the kit comprises: Papamycin or its analogs and chemotherapeutic drugs; or

    The pharmaceutical kit according to any one of claims 6 to 9 is included in the kit.
12. The kit according to claim 11, wherein said kit further includes instructions for use.
13. The kit according to claim 12, wherein said instruction manual describes the following chemotherapy regimen:

    (1) administering to the subject a chemotherapeutic drug, as well as rapamycin or an analogue thereof;

    (2) After 2 weeks, the step (1) is repeated; every two weeks as one administration cycle, 2 to 20 administration cycles are performed.

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