WO2023088358A1

WO2023088358A1 - Treatment of cancer with a glucocorticoid receptor agonist and an hiv protease inhibitor

Info

Publication number: WO2023088358A1
Application number: PCT/CN2022/132540
Authority: WO
Inventors: Dun Yang; Jing Zhang; Shenqiu ZHANG; Thaddeus ALLEN; Qiong SHI; Yan LONG; Ting Zhang; Hongmei Li; Chenglu YANG
Original assignee: Chengdu Anticancer Bioscience, Ltd.; J. Michael Bishop Institute Of Cancer Research
Priority date: 2021-11-17
Filing date: 2022-11-17
Publication date: 2023-05-25

Abstract

Disclosed herein are methods of treating cancer in a subject, comprising administration to the subject of (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitors. In some embodiments, the administration of the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitors results in restoration of contact inhibition, increased formation of intercellular junctions, reduced cell proliferation and/or increased vacuolization of cells growing in multilayer zones.

Description

[Title established by the ISA under Rule 37.2] TREATMENT OF CANCER WITH A GLUCOCORTICOID RECEPTOR AGONIST AND AN HIV PROTEASE INHIBITOR

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims priority to International Application PCT/CN2021/131182, filed November 17, 2021, which is hereby incorporated by reference in its entirety.

BACKGROUND

In normal cells, cell-cell and cell-matrix contacts mediate contact inhibition, which is the inhibition of cellular proliferation when cells reach confluence and make connections and they stop proliferating. In contrast, loss of contact inhibition of proliferation (CIP) is a hallmark of cancer cells, which tend to grow past confluence and become multilayered.

Contact inhibition of proliferation acts as a checkpoint that prevents uncontrolled cellular proliferation. The defect in the checkpoint might confer a unique vulnerability that becomes apparent only when the cells are overgrowing. However, how cancer cells evade contact inhibition remains unknown. There is no report in the literature of any chemicals that can re-establish contact inhibition in cancer cells. Therefore, compounds that can restore contact inhibition of cancer cells, and methods for identifying such compounds that can restore contact inhibition in cancer cells are needed.

SUMMARY

In certain aspects, disclosed herein are methods of treating cancer in a subject, comprising administration to the subject of (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) . In certain aspects, disclosed herein are methods of reducing cancer cell proliferation, comprising administration to a subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the cell proliferation is reduced compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor (s) .

In certain aspects, disclosed herein are methods of reducing cancer cell proliferation in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor (s) .

In certain aspects, disclosed herein are methods of inducing vacuolization of cancer cells, comprising administration to a subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the vacuolization is characterized by an increase in the presence of vacuoles compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor (s) .

In certain aspects, disclosed herein are method of inducing vacuolization of cancer cells in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor (s) .

In certain aspects, disclosed herein are methods of inducing cell death of cancer cells, comprising administration to a subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the number of cells undergoing cell death is increased compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor (s) .

In certain aspects, disclosed herein are methods of inducing cell death of one or more cancer cells in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor (s) .

In some embodiments, the one or more glucocorticoid receptor agonists are selected from the group consisting of: a natural glucocorticoid receptor agonist, a synthetic glucocorticoid receptor agonist, a hydrocortisone-type glucocorticoid receptor agonist, a methasone-type glucocorticoid receptor agonist and an acetonide related glucocorticoid receptor agonist. In some embodiments, the glucocorticoid receptor agonist comprises hydrocortisone. In some embodiments, the glucocorticoid receptor agonist comprises dexamethasone.

In some embodiments, a hydroxyl group of the HIV protease inhibitor interacts with the carboxyl group of the protease active site amino acid Asp25 by a hydrogen bond. In some embodiments, the HIV protease inhibitor contacts one or more amino acids of HIV protease selected from the group consisting of Gly27, Asp29, Asp30, and Gly48. In some embodiments, the HIV protease inhibitor inhibits activation of SREBP-1. In some embodiments, the HIV protease inhibitor inhibits proteasome activity.

In some embodiments, wherein the HIV protease inhibitor inhibits the proteasomal 20S subunit. In some embodiments, the HIV protease inhibitor inhibits AKT/PKB. In some embodiments, the HIV protease inhibitor comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.

In some embodiments, the one or more glucocorticoid receptor agonist (s) comprises hydrocortisone and the one or more HIV protease inhibitor comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.

In some embodiments, the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) are administered to the subject or contacted with the cancer cells concurrently. In some embodiments, the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) are administered to the subject or contacted with the cancer cells sequentially. In some embodiments, the one or more glucocorticoid receptor agonist (s) are administered to the subject or contacted with the cancer cells prior to the administration of or contact with the one or more HIV protease inhibitor (s) . In some embodiments, the one or more glucocorticoid receptor agonist (s) are administered to the subject or contacted with the cancer cells after administration of or contact with the one or more HIV protease inhibitor (s) .

In some embodiments, the cancer cells overexpress MYC compared to an otherwise identical non-cancer cell. In some embodiments, the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell. In some embodiments, the cancer cells express BRAF ^V600E. In some embodiments, the cancer cells exhibit loss of TP53 expression. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is lung cancer.

In some embodiments, the administration of or contacting with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) results in a reduction in cancer proliferation, increased cancer cell death, or combinations thereof, compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) . In some embodiments, the administration of or contacting with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) results in a 2-fold or greater reduction in cancer cell proliferation as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .

In some embodiments, the administration of or contacting with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) results in a 2-fold or greater increase in cancer cell death as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) . In some embodiments, the cancer cell death is non-apoptotic cell death. In some embodiments, the non-apoptotic cell death is characterized by an increase in the presence of vacuoles compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .

In some embodiments, the increase in the presence of vacuoles is at least 2 fold or more greater than the presence of vacuoles in cancer cells treated with the one or more HIV protease inhibitor (s) alone. In some embodiments, the increase in the presence of vacuoles is at least 5-fold or more greater than the presence of vacuoles in cancer cells treated with the one or more glucocorticoid receptor agonist (s) alone. In some embodiments, the vacuoles have a maximum diameter of about 50 μm. the vacuoles comprise regions of high acidity in the periphery of the vacuoles. In some embodiments, the vacuoles comprise condensed and minimized nuclei at inner periphery of vacuoles. In some embodiments, one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick TFEB or ERM1 are localized to the vacuoles of the cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor (s) .

In some embodiments, cancer cells exhibit increased intercellular junctions as compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) . In some embodiments, the cancer cells exhibit increased plasma membrane localization of B-catenin. In some embodiments, the cancer cells exhibit increased plasma membrane localization of ZO-1.

In some embodiments of the methods described herein, the cancer cells are grown in vitro. In some embodiments, the cancer cells contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) when grown to confluence, exhibit a greater area of cells growing in a single monolayer compared to an area of cells growing in a multi-layer, as compared to otherwise identical cancer cells grown to confluence and not treated with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) . In some embodiments, greater than 50%of the area of the cancer cells contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) and grown to confluence, exhibit growth in a monolayer.

In certain aspects, described herein is a method of inducing vacuolation of one or more cancer cells, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells, following contact with the one or more glucocorticoid receptor agonist (s) and one or more HIV protease inhibitor (s) , are characterized by the presence of vacuoles; wherein the vacuoles: (i) have a maximum diameter of about 50 μm; and (ii) one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick, TFEB or ERM1 are localized to the vacuoles.

In certain aspects, described herein is a method of inducing non-apoptotic cell death of one or more cancer cells, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells, following contact with the one or more glucocorticoid receptor agonist (s) and one or more HIV protease inhibitor (s) , are characterized by the presence of vacuoles; wherein the vacuoles: (i) have a maximum diameter of about 50 μm; (ii) one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick, TFEB or ERM1 are localized to the vacuoles; and (iii) comprise condensed and minimized nuclei at inner periphery of vacuoles.

In some embodiments, a hydroxyl group of the HIV protease inhibitor interacts with the carboxyl group of the protease active site amino acid Asp25 by a hydrogen bond. In some embodiments, the HIV protease inhibitor contacts one or more amino acids of HIV protease selected from the group consisting of Gly27, Asp29, Asp30, and Gly48. In some embodiments, the HIV protease inhibitor inhibits activation of SREBP-1. In some embodiments, the HIV protease inhibitor inhibits proteasome activity. In some embodiments, the HIV protease inhibitor inhibits the proteasomal 20S subunit. In some embodiments, the HIV protease inhibitor inhibits AKT/PKB. In some embodiments, the one or HIV protease inhibitor (s) comprises an inhibitor selected from the group consisting of: Saquinavir, Lopinavir, and Nelfinavir Mesylate.

In some embodiments, the one or more glucocorticoid receptor agonist (s) comprises hydrocortisone. In some embodiments, the one or more glucocorticoid receptor agonist (s) comprises dexamethasone. In some embodiments , the one or more glucocorticoid receptor agonist (s) comprises hydrocortisone and the one or more HIV protease inhibitor (s) comprises an inhibitor selected from the group consisting of: Saquinavir, Lopinavir, and Nelfinavir Mesylate. In some embodiments, the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell.

In some embodiments, the cancer cells overexpress MYC-Nick compared to otherwise identical non-cancer cells. In some embodiments, the cancer cells express BRAF ^V600E. In some embodiments, the cancer cells exhibit loss of TP53 expression. In some embodiments, the cancer is metastatic. the cancer is lung cancer. In some embodiments, the cancer is metastatic. the cancer is liver cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

Figure 1 (left) are micrograph images of murine liver cancer cells stably expressing MycNick that have been incubated with Hydrocortisone (HC) and stained with propidium iodide. Also depicted is a graph (right) showing the percentage of area of cell growth where cells are grown in monolayers ( “monolayer zone” ) and the percentage of area of cell growth where cells are grown in multilayers ( “multilayer zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO or the indicated concentrations of HC.

Figure 2 (right) are images of HCC cells incubated with DMSO, the glucocorticoid receptor (GR) antagonist RU486, Hydrocortisone (HC) or both RU486 and HC for 12 days and stained with propidium iodide. Also depicted is a graph (right) showing the relative number of cells after incubation with DMSO, the glucocorticoid receptor (GR) antagonist RU486, Hydrocortisone (HC) or both RU486 and HC.

Figure 3 (left) are micrograph images of murine liver cancer cells stably expressing MycNick that have been incubated with Hydrocortisone (HC) . Also depicted is a graph (right) showing the percentage of area of cell growth with vacuoles ( “the vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO or the indicated concentrations of HC.

Figure 4 (left) are micrograph images of hepatocellular carcinoma (HCC) cells over-expressing MycNick that have been incubated with DMSO, the glucocorticoid receptor (GR) antagonist RU486, Hydrocortisone (HC) or both RU486 and HC. Also depicted is a graph showing the percentage of area of cell growth with vacuoles ( “the vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) .

Figure 5 (left) are micrograph images of hepatocellular carcinoma (HCC) cells over-expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) , saquinavir, or both saquinavir and HC. Also depicted is a graph showing the percentage of area of cell growth with vacuoles ( “the vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) .

Figure 6 are micrograph images of cells stably expressing MycNick that have been stained with Lysosensor ^TM Green DNA-189 (left) . Cells were incubated with Saquinavir or both Saquinavir and Hydrocortisone.

Figure 7 (left) are micrograph images of cells stably expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) , saquinavir, or both saquinavir and HC, and stained with propidium iodide. Also depicted is a graph (right) showing the percentage of area of cell growth where cells are grown in monolayers ( “monolayer zone” ) and the percentage of area of cell growth where cells are grown in multilayers ( “multilayer zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, Hydrocortisone (HC) , saquinavir, or both saquinavir and HC.

Figure 8 (left) are micrograph images of murine liver cancer cells stably expressing MycNick that have been incubated with Hydrocortisone (HC) , lopinavir at a concentration of 0.625 uM, or both Lopinavir and HC. Also depicted is a graph (right) showing the percentage of area of cell growth with vacuoles ( “vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, lopinavir, or both lopinavir and HC.

Figure 9 (left) are micrograph images of murine liver cancer cells stably expressing MycNick that have been incubated with Hydrocortisone (HC) , lopinavir at a concentration of 2.5 uM, or both lopinavir and HC. Also depicted is a graph (right) showing the percentage of area of cell growth with vacuoles ( “the vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, lopinavir, or both lopinavir and HC.

Figure 10 (left) are micrograph images of HCC cells stably expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) or lopinavir ( “CI024” ) , and stained with propidium iodide. Also depicted is a graph (right) showing the relative number of cells 11 days after exposure to DMSO, Hydrocortisone (HC) or CI024.

Figure 11 (left) are micrograph images of cells stably expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) , lopinavir, or both lopinavir and HC, and stained with propidium iodide. Also depicted is a graph (right) showing the percentage of area of cell growth where cells are grown in monolayers ( “monolayer zone” ) and the percentage of area of cell growth where cells are grown in multilayers ( “multilayer zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, Hydrocortisone (HC) , Lopinavir, or both Lopinavir and HC.

Figure 12 (left) are micrograph images of cells stably expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) , lopinavir (at a concentration of 2.5 uM) , or both Lopinavir and HC, and stained with propidium iodide. Also depicted is a graph (right) showing the percentage of area of cell growth where cells are grown in monolayers ( “monolayer zone” ) and the percentage of area of cell growth where cells are grown in multilayers ( “multilayer zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, Hydrocortisone (HC) , lopinavir, or both lopinavir and HC.

Figure 13 (left) are micrograph images of HCC cells stably expressing MycNick that have been incubated with DMSO or lopinavir ( “CI024” ) for 7 days, and stained with LysoSensor ^TM Green.

Figure 14 are micrograph images of cells stably expressing MycNick that have been stained with Lysosensor ^TM Green DNA-189 (left) . Cells were incubated with lopinavir (at a concentration of 2.5 uM) or both lopinavir and Hydrocortisone.

Figure 15 (left) left) are micrograph images of murine liver cancer cells stably expressing MycNick that have been incubated with Hydrocortisone (HC) , nelfinavir mesylate, or both nelfinavir mesylate and HC. Also depicted is a graph (right) showing the percentage of area of cell growth with vacuoles ( “vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, Nelfinavir Mesylate, or both nelfinavir mesylate and HC.

Figure 16 are micrograph images of cells stably expressing MycNick that have been stained with Lysosensor ^TM Green DNA-189 (left) . Cells were incubated with nelfinavir nesylate or both nelfinavir mesylate and Hydrocortisone.

Figure 17 (left) are micrograph images of cells stably expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) , nelfinavir mesylate, or both nelfinavir mesylate and HC, and stained with propidium iodide. Also depicted is a graph (right) showing the percentage of area of cell growth where cells are grown in monolayers ( “monolayer zone” ) and the percentage of area of cell growth where cells are grown in multilayers ( “multilayer zone” ) with murine liver cancer cells expressing MycNick incubated with DMSO, Hydrocortisone (HC) , nelfinavir mesylate, or both nelfinavir mesylate.

Figure 18 is a graph depicting tumor volume of mice treated with vehicle, hydrocortisone (HC001) , saquinavir, or saquinavir and Hydrocortisone in a BRAF ^V600E lung cancer mouse model.

Figure 19 is a graph depicting body weight of mice treated with vehicle, hydrocortisone (HC001) , saquinavir, or saquinavir and Hydrocortisone in a BRAF ^V600E lung cancer mouse model.

Figure 20 is a graph depicting tumor volume of mice treated with vehicle, hydrocortisone (HC001) , nelfinavir mesylate, or nelfinavir mesylate and Hydrocortisone in a BRAF ^V600E lung cancer mouse model.

Figure 21 is a graph depicting body weight of mice treated with vehicle, hydrocortisone (HC001) , nelfinavir mesylate, or nelfinavir mesylate and Hydrocortisone in a BRAF ^V600E lung cancer mouse model.

Figure 22 are micrograph images of HCC cell lines stably expressing MycNick incubated with DMSO, Hydrocortisone (HC001) and lopinavir ( “CI024” ) .

Figure 23 are micrograph images of HCC cell lines stably expressing MycNick incubated with DMSO, Hydrocortisone (HC001) and lopinavir ( “CI024” ) .

Figure 24 are micrograph images of HCC cell lines stably expressing MycNick incubated with Hydrocortisone (HC001) or lopinavir ( “CI024” ) for 5 days, HCC cells after 4 days or 7 days following withdrawal of Hydrocortisone (HC001) or CI024, and HCC cells 3 days after re-exposure to Hydrocortisone (HC001) or CI024.

Figure 25 are micrograph images of HCC cells stably expressing MycNick that have been incubated with DMSO, Hydrocortisone (HC) or lopinavir ( “CI024” ) for 11 days, and stained with propidium iodide. Stars indicate condensed and minimized nuclei.

Figure 26 (left) are micrograph images of HCC cells stably expressing MycNick that have been incubated with DMSO or lopinavir ( “CI024” ) for 15 days, and stained with anti-phosphorylated Histone 3 Serine 10 (H3 Ser10P) antibody or DAPI to visualize nuclei. Also depicted is a graph (right) showing quantitation of number of phosphorylated Histone 3 Serine 10 stained cells per field in multilayer zones and monolayer zones.

Figure 27 (left) are micrograph images of HCC cells stably expressing MycNick that have been incubated with DMSO or lopinavir ( “CI024” ) for 15 days, and stained with anti- CyclinA1 antibody or DAPI to visualize nuclei. Also depicted is a graph (right) showing quantitation of the relative number of cyclin A1 positively stained cells.

Figure 28 are micrograph images of HCC cells stably expressing MycNick that have been incubated with DMSO or lopinavir ( “CI024” ) for 12 days, and stained with anti-Beta Catenin antibody and DAPI to visualize nuclei. The florescence intensity (gray value) at each pixel (distance) is depicted. Also depicted is a graph (right) showing quantitation of overall gray value (peak –valley) of images of the cells incubated with DMSO or CI024, and stained with anti-Beta Catenin antibody.

Figure 29 depicts chemical structures of exemplary glucocorticoid receptor agonists, according to present disclosure.

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

The term “glucocorticoid receptor agonist” as used herein, refers to a compound that can bind the glucocorticoid receptor.

The term “CIR compound” as used herein, refers to a compound that arrests cell cycle progression only when cells are overgrowing passing confluence. In other words, a CIR compound arrest proliferation of cells cultured in high but not low density. Alternatively, the compound might elicit death in cells that are still actively proliferating when they have reached the confluence. However, the CIR compound is generally inert to the same cells when they are dividing at sub-confluence or have stopped proliferation.

The term “vacuolization” as used herein, means the increased presence of vacuoles in a cell. In certain embodiments, the vacuoles have a maximum diameter of about 50 μm.

The term “restoring contact inhibition” or “contact inhibition restoration” or “CIR” as used herein refers to the ability of cancer cells to re-establish intercellular junctions and stop proliferating when grown to confluence in vitro or when growing in vivo.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to restore contact inhibition in a cancer cell.

The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease.

As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

“About” a number, as used herein, refers to range including the number and ranging from 10%below that number to 10%above that number. "About" a range refers to 10%below the lower limit of the range, spanning to 10%above the upper limit of the range.

Abbreviations used in this application include the following: CIP, which refers to contact inhibition of proliferation; CIR, which refers to contact inhibition restoration; HC, which refers to hydrocortisone; GR, which refers to glucocorticoid receptor, DEX, which refers to dexamethasone; CI024, which refers to lopinavir; CI062, which refers to saquinavir; and CI063, which refers to nelfinavir mesylate.

It must be noted that, as used in the specification and the appended claims, the singular forms “a, ” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Briefly, and as described in more detail below, described herein are methods of treating cancer comprising administering to a subject in need thereof a sufficient amount of one or more glucocorticoid receptor agonists and one or more HIV inhibitors. In certain aspects, described herein are methods of restoring contact inhibition of cancer cells, reducing cancer cell proliferation, inducing vacuolization of cancer cells and/or inducing cancer cell death; comprising administering to a subject in need thereof a sufficient amount of one or more glucocorticoid receptor agonists and one or more calcineurin inhibitors. Additionally, described herein are methods of identifying compounds that restore contact inhibition, reduce cancer cell proliferation, induce vacuolization of cancer cells and/or induce cancer cell death.

Glucocorticoid Receptor Agonists

In certain aspects, described herein are methods comprising administration to the subject of one or more glucocorticoid receptor agonists. Glucocorticoid receptor agonists are selected from the group consisting of: a natural glucocorticoid receptor agonist, a synthetic glucocorticoid receptor agonist, a hydrocortisone-type glucocorticoid receptor agonist, a methasone-type glucocorticoid receptor agonist and an acetonide glucocorticoid receptor agonist. In some embodiments, the glucocorticoid is a selective glucocorticoid receptor agonist (SEGRAM) . In some embodiments, the methods comprise administering 1, 2, 3, 4 or 5 distinct glucocorticoid receptor agonists.

Examples of natural glucocorticoid receptor agonists include, but are not limited to: Cortisol (hydrocortisone) ; 11-Dehydrocorticosterone (11-oxocorticosterone, 17-deoxycortisone) = 21-hydroxypregn-4-ene-3, 11, 20-trione; 11-Deoxycorticosterone (deoxycortone, desoxycortone; 21-hydroxyprogesterone) = 21-hydroxypregn-4-ene-3, 20-dione; 11-Deoxycortisol (cortodoxone, cortexolone) = 17α, 21-dihydroxypregn-4-ene-3, 20-dione; 11-Ketoprogesterone (11-oxoprogesterone; Ketogestin) = pregn-4-ene-3, 11, 20-trione; 11β-Hydroxypregnenolone = 3β, 11β-dihydroxypregn-5-en-20-one [1] ; 11β-Hydroxyprogesterone (21-deoxycorticosterone) = 11β-hydroxypregn-4-ene-3, 20-dione; 11β, 17α, 21-Trihydroxypregnenolone = 3β, 11β, 17α, 21-tetrahydroxypregn-5-en-20-one [2] ; 17α, 21-Dihydroxypregnenolone = 3β, 17α, 21-trihydroxypregn-5-en-20-one [3] ; 17α-Hydroxypregnenolone = 3β, 17α-dihydroxypregn-5-en-20-one; 17α-Hydroxyprogesterone =17α-hydroxypregn-4-ene-3, 11, 20-trione; 18-Hydroxy-11-deoxycorticosterone = 18, 21-dihydroxypregn-4-ene-3, 20-dione [4] ; 18-Hydroxycorticosterone = 11β, 18, 21-trihydroxypregn-4-ene-3, 20-dione; 18-Hydroxyprogesterone = 18-hydroxypregn-4-ene-3, 20-dione [5] ; 21-Deoxycortisol = 11β, 17α-dihydroxypregn-4-ene-3, 20-dione, ; 21-Deoxycortisone = 17α-hydroxypregn-4-ene-3, 11, 20-trione; 21-Hydroxypregnenolone (prebediolone) = 3β, 21-dihydroxypregn-5-en-20-one; Aldosterone = 11β, 21-dihydroxypregn-4-ene-3, 18, 20-trione; Corticosterone (17-deoxycortisol) = 11β, 21-dihydroxypregn-4-ene-3, 20-dione; Cortisol (hydrocortisone) = 11β, 17α, 21-trihydroxypregn-4-ene-3, 20-dione; Cortisone = 17α, 21-dihydroxypregn-4-ene-3, 11, 20-trione; Pregnenolone = pregn-5-en-3β-ol-20-one; and Progesterone = pregn-4-ene-3, 20-dione.

Examples of synthetic glucocorticoid receptor agonists, include but are not limited to: Progesterone-type; Flugestone (flurogestone) = 9α-fluoro-11β, 17α-dihydroxypregn-4-ene-3, 20-dione; Fluorometholone = 6α-methyl-9α-fluoro-11β, 17α-dihydroxypregna-1, 4-diene-3, 20-dione; Medrysone (hydroxymethylprogesterone) = 6α-methyl-11β-hydroxypregn-4-ene-3, 20-dione; and Prebediolone acetate (21-acetoxypregnenolone) = 3β, 21-dihydroxypregn-5-en-20-one 21-acetate. Additional examples of synthetic glucocorticoid receptor agonists include, but are not limited to, progesterone derivative progestins such as chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, and segesterone acetate which possess glucocorticoid activity that can manifest clinically at high dosages.

Examples of hydrocortisone-type glucocorticoid receptor agonists, include but are not limited to: Chloroprednisone = 6α-chloro-17α, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione Cloprednol = 6-chloro-11β, 17α, 21-trihydroxypregna-1, 4, 6-triene-3, 20-dione; Difluprednate =6α,9α-difluoro-11β, 17α, 21-trihydroxypregna-1, 4-diene-3, 20-dione 17α-butyrate 21-acetate; Fludrocortisone = 9α-fluoro-11β, 17α, 21-trihydroxypregn-4-ene-3, 20-dione; Fluocinolone =6α,9α-difluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione; Fluperolone = 9α-fluoro-11β, 17α, 21-trihydroxy-21-methylpregna-1, 4-diene-3, 20-dione; Fluprednisolone = 6α-fluoro-11β, 17α, 21-trihydroxypregna-1, 4-diene-3, 20-dione; Loteprednol =11β, 17α, dihydroxy-21-oxa-21-chloromethylpregna-1, 4-diene-3, 20-dione; Methylprednisolone = 6α-methyl-11β, 17α, 21-trihydroxypregna-1, 4-diene-3, 20-dione; Prednicarbate = 11β, 17α, 21-trihydroxypregna-1, 4-diene-3, 20-dione 17α-ethylcarbonate 21-propionate; Prednisolone = 11β, 17α, 21-trihydroxypregna-1, 4-diene-3, 20-dione; Prednisone =17α, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione; Tixocortol = 11β, 17α-dihydroxy-21-sulfanylpregn-4-ene-3, 20-dione; and Triamcinolone = 9α-fluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione.

Examples of methasone-type glucocorticoid receptor agonists, include but are not limited to: Alclometasone = 7α-chloro-11β, 17α, 21-trihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Beclometasone = 9α-chloro-11β, 17α, 21-trihydroxy-16β-methylpregna-1, 4-diene-3, 20-dione; Betamethasone = 9α-fluoro-11β, 17α, 21-trihydroxy-16β-methylpregna-1, 4-diene-3, 20-dione; Clobetasol = 9α-fluoro-11β, 17α-dihydroxy-16β-methyl-21-chloropregna-1, 4-diene-3, 20-dione; Clobetasone = 9α-fluoro-16β-methyl-17α-hydroxy-21-chloropregna-1, 4-diene-3, 11, 20-trione; Clocortolone = 6α-fluoro-9α-chloro-11β, 21-dihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Desoximetasone = 9α-fluoro-11β, 21-dihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Dexamethasone = 9α-fluoro-11β, 17α, 21-trihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Diflorasone = 6α, 9α-difluoro-11β, 17α, 21-trihydroxy-16β-methylpregna-1, 4-diene-3, 20-dione; Difluocortolone = 6α, 9α-difluoro-11β, 21-dihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Fluclorolone = 6α-fluoro-9α, 11β-dichloro-16α, 17α, 21-trihydroxypregna-1, 4-dien-3, 20-dione; Flumetasone = 6α, 9α-difluoro-11β, 17α, 21-trihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Fluocortin = 6α-fluoro-11β, 21-dihydroxy-16α-methylpregna-1, 4-diene-3, 20, 21-trione; Fluocortolone = 6α-fluoro- 11β, 21-dihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Fluprednidene = 9α-fluoro-11β, 17α, 21-trihydroxy-16-methylenepregna-1, 4-diene-3, 20-dione; Fluticasone = 6α, 9α-difluoro-11β, 17α-dihydroxy-16α-methyl-21-thia-21-fluoromethylpregna-1, 4-dien-3, 20-dione; Fluticasone furoate = 6α, 9α-difluoro-11β, 17α-dihydroxy-16α-methyl-21-thia-21-fluoromethylpregna-1, 4-dien-3, 20-dione 17α- (2-furoate) ; Halometasone = 2-chloro-6α, 9α-difluoro-11β, 17α, 21-trihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Meprednisone =16β-methyl-17α, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione; Mometasone = 9α, 21-dichloro-11β, 17α-dihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Mometasone furoate =9α,21-dichloro-11β, 17α-dihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione 17α- (2-furoate) ; Paramethasone = 6α-fluoro-11β, 17α, 21-trihydroxy-16α-methylpregna-1, 4-diene-3, 20-dione; Prednylidene = 11β, 17α, 21-trihydroxy-16-methylenepregna-1, 4-diene-3, 20-dione; Rimexolone = 11β-hydroxy-16α, 17α, 21-trimethylpregna-1, 4-dien-3, 20-dione; and Ulobetasol (halobetasol) = 6α, 9α-difluoro-11β, 17α-dihydroxy-16β-methyl-21-chloropregna-1, 4-diene-3, 20-dione.

Examples of acetonide related glucocorticoid receptor agonists, include but are not limited to: Amcinonide = 9α-fluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with cyclopentanone, 21-acetate; Budesonide = 11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with butyraldehyde; Ciclesonide = 11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with (R) -cyclohexanecarboxaldehyde, 21-isobutyrate; Deflazacort = 11β, 21-dihydroxy-2'-methyl-5'H-pregna-1, 4-dieno [17, 16-d] oxazole-3, 20-dione 21-acetate; Desonide = 11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with acetone; Formocortal (fluoroformylone) = 3- (2-chloroethoxy) -9α-fluoro-11β, 16α, 17α, 21-tetrahydroxy-20-oxopregna-3, 5-diene-6-carboxaldehyde cyclic 16α, 17α-acetal with acetone, 21-acetate; Fluclorolone acetonide (flucloronide) = 6α-fluoro-9α, 11β-dichloro-16α, 17α, 21-trihydroxypregna-1, 4-dien-3, 20-dione cyclic 16α, 17α-acetal with acetone; Fludroxycortide (flurandrenolone, flurandrenolide) = 6α-fluoro-11β, 16α, 17α, 21-tetrahydroxypregn-4-ene-3, 20-dione cyclic 16α, 17α-acetal with acetone; Flunisolide = 6α-fluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with acetone; Fluocinolone acetonide = 6α, 9α-difluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with acetone; Fluocinonide = 6α, 9α-difluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with acetone, 21-acetate; Halcinonide = 9α-fluoro-11β, 16α, 17α-trihydroxy-21-chloropregn-4-ene-3, 20-dione cyclic 16α, 17α-acetal with acetone; and Triamcinolone acetonide = 9α-fluoro-11β, 16α, 17α, 21-tetrahydroxypregna-1, 4-diene-3, 20-dione cyclic 16α, 17α-acetal with acetone.

Structure of Hydrocortisone

Structure of Dexamethasone

The structures of additional select glucocorticoid receptor agonists are shown in Figure 29.

In some embodiments, the glucocorticoid receptor agonists described herein can bind to one or more of glucocorticoid receptor isoforms. Upon binding of the glucocorticoid receptor agonists to the glucocorticoid receptor, the glucocorticoid receptor can be transported to the nucleus to transcriptionally activate or transcriptionally repress expression of target genes. Upon binding of the glucocorticoid receptor agonists to the glucocorticoid receptor, the glucocorticoid receptor can modulate the physicochemical properties of membrane lipids as well as Mitogen-Activated Protein Kinase (MAPK) and other signaling cascades. Upon binding of the glucocorticoid receptor agonists to the glucocorticoid receptor, the glucocorticoid receptor can translocate to the mitochondria and regulate the gene expression in the mitochondria.

In some embodiments, the glucocorticoid receptor agonist has a potency 0.01-1000 fold the potency of hydrocortisol or cortisol. In some embodiments, the glucocorticoid receptor agonist has a potency that is 0.01-0.1 fold, 0.1-1.0-fold, 1.0-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, or 500-100 fold the potency of hydrocortisol or cortisol.

In some embodiments, the glucocorticoid receptor agonist binds to the glucocorticoid receptor with an affinity that is 0.01-1000 fold the affinity of hydrocortisol or cortisol. In some embodiments, the glucocorticoid receptor agonist has an affinity to glucocorticoid receptor that is 0.01-0.1 fold, 0.1-1.0-fold, 1.0-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, or 500-1000 fold the affinity of hydrocortisol or cortisol to the glucocorticoid receptor.

In some embodiments, the glucocorticoid receptor agonist promotes a glucocorticoid conformation that favors the monomer form of the glucocorticoid and inhibits or partially inhibits dimerization of the glucocorticoid receptor. In some embodiments, the glucocorticoid receptor agonist promotes a glucocorticoid conformation that favors the dimerized form of the glucocorticoid and promotes dimerization of the glucocorticoid receptor. In some embodiments, the glucocorticoid receptor agonists trigger heterodimerization between glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) . In some embodiments, the glucocorticoid receptor agonists is specific for the GR and activates GR/GR homodimers and not GR/MR heterodimers.

HIV Protease Inhibitors

HIV protease inhibitors of the present disclosure can be any known in the art. In certain embodiments the HIV protease inhibitor comprises a hydroxyl group of the HIV protease inhibitor interacts with the carboxyl group of the protease active site amino acid Asp25 by a hydrogen bond. In certain embodiments, the HIV protease inhibitor contacts one or more amino acids of HIV protease selected from the group consisting of Gly27, Asp29, Asp30, and Gly48. In certain embodiments, the HIV protease inhibitor inhibits activation of Sterol Regulatory Element Binding Transcription Factor (SREBP-1) . In certain embodiments, the HIV protease inhibitor inhibits proteasome activity. In certain embodiments, the HIV protease inhibitor inhibits the proteasomal 20S subunit. In certain embodiments, the HIV protease inhibitor inhibits AKT Serine Threonine Kinase (AKT/PKB) . In certain embodiments, the HIV protease inhibitor comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.

Structure of saquinavir

Structure of lopinavir

Structure of nelfinavir mesylate

Pharmaceutical compositions

The glucocorticoid receptor agonists and HIV protease inhibitors of the present disclosure can be formulated in one or more pharmaceutical compositions. These compositions can comprise, in addition to one or more of the glucocorticoid receptor agonists and HIV protease inhibitors, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.

The glucocorticoid receptor agonists and HIV protease inhibitors according to the present disclosure that is to be given to an individual, administration is preferably in a “therapeutically effective amount” that is sufficient to show benefit to the individual. A “prophylactically effective amount” can also be administered, when sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Methods of Treating Cancer

In certain aspects, disclosed herein are methods of treating cancer in a subject, comprising administration to the subject of (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitors. In some embodiments, the administration of the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitors results in a reduction in cancer proliferation, increased cancer cell death, or combinations thereof, compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitors.

In some embodiments, the one or more glucocorticoid receptor agonists are selected from the group consisting of: a natural glucocorticoid receptor agonist, a synthetic glucocorticoid receptor agonist, a hydrocortisone-type glucocorticoid receptor agonist, a methasone-type glucocorticoid receptor agonist and an acetonide related glucocorticoid receptor agonist.

In some embodiments, the glucocorticoid receptor agonist comprises hydrocortisone. In some embodiments, the glucocorticoid receptor agonist comprises dexamethasone. In some embodiments, the HIV protease inhibitors comprises saquinavir, lopinavir, and nelfinavir mesylate. In some embodiments, the one or more glucocorticoid receptor agonist comprises hydrocortisone and the more HIV protease inhibitor comprises saquinavir, lopinavir, and nelfinavir mesylate, or combinations thereof.

In some embodiments, the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor are administered to the subject or contacted with the cancer cells concurrently. In some embodiments, the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor are administered to the subject or contacted with the cancer cells sequentially. In some embodiments, wherein the one or more glucocorticoid receptor agonists are administered to the subject or contacted with the cancer cells prior to the administration of or contact with the one or more HIV protease inhibitor. In some embodiments, the one or more glucocorticoid receptor agonists are administered to the subject or contacted with the cancer cells after administration of or contact with the one or more HIV protease inhibitor.

In some embodiments, the cancer cells overexpress MYC compared to an otherwise identical non-cancer cell. In some embodiments, the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell. Myc-nick is a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of the full-length Myc. Myc-nick inhibits apoptosis, promotes anchorage-independent growth, and renders cancer cells resistance to a variety of chemotherapeutics in part by promoting autophagy. Moreover, Myc-nick increases acetylation of α-tubulin through the recruitment of GCN5, an acetyltransferase, and promotes formation of filopodia and migration of cancer cells by induction of the actin-bundling protein fascin. In some embodiments, the cancer cells express BRAF ^V600E. In some embodiments, the cancer cells exhibit loss of TP53 expression. Any method known in the art can be used for determining the expression of a gene in the cancer cell (e.g., immunohistochemistry and the like) , and for determining the presence of a mutation of a gene can be used (e.g., sequencing methods) .

In some embodiments, the administration to the subject of (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor results in reduced tumor volume in the subject compared to subjects not treated with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitors, or compared to historical controls.

In some embodiments, the cancer is lung cancer. In come embodiments, the cancer is liver cancer. In some embodiments, the cancer is metastatic.

Methods of Reducing Cancer Cell Proliferation

In certain aspects, described herein are methods of reducing cancer cell proliferation, comprising administration to the subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the cell proliferation is reduced compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor. In some embodiments, the administration of or contacting with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor results in a 2-fold or greater reduction in cancer cell proliferation as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor.

In some embodiments, the administration of or contacting with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor results in a 5-fold or greater reduction in cancer cell proliferation as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor.

In certain aspects, described herein are methods of reducing cancer cell proliferation in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor. In some embodiments, there is increased cell proliferation in the cancer cells when contacted with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor when the cells are grown in sub-confluent conditions as compared to otherwise identical cancer cells that are contacted with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor when grown in confluent conditions of about 100%or more confluency.

In some embodiments, the glucocorticoid receptor agonist comprises hydrocortisone. In some embodiments, the glucocorticoid receptor agonist comprises dexamethasone. In some embodiments, the HIV protease inhibitor is selected from the group consisting of saquinavir, lopinavir, and nelfinavir mesylate. In some embodiments, the one or more glucocorticoid receptor agonist comprises hydrocortisone and the HIV protease inhibitor is selected from the group consisting of saquinavir, lopinavir, and nelfinavir mesylate.

In some embodiments, cancer cells that have reduced proliferation after being administered to the subject or contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor also have restored localization of intercellular junction proteins to the intercellular junctions. In some embodiments, cancer cells that stopped proliferating after contact with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor have re-established formation of intercellular junctions.

In some embodiments, cancer cells grown in vitro that have stopped proliferating after contact with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor have re-established formation of intercellular junctions and/or are growing in a monolayer of confluent cells. Conversely, in some embodiments, the cells growing above the monolayer of confluent cells (in multilayer areas) , exhibit increased cell proliferation as compared to the cells growing as a monolayer of confluent cells. In some embodiments, the cells growing above the monolayer of confluent cells (in multilayer areas) , exhibit increased phosphorylation of Ser10 of Histone 3 compared to the cells growing as a monolayer of confluent cells.

In some embodiments, the cancer cells overexpress MYC compared to an otherwise identical non-cancer cell. In some embodiments, the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell. In some embodiments, the cancer cells express BRAF ^V600E. In some embodiments, the cancer cells exhibit loss of TP53 expression. In some embodiments, the cancer is lung cancer. In come embodiments, the cancer is liver cancer. In some embodiments, the cancer is metastatic.

Methods of Restoring Contact Inhibition of Cancer Cells

In certain aspects, disclosed herein are methods of restoring contact inhibition, comprising administration to the subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the cell proliferation is reduced compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor. In some embodiments, the cancer cells exhibit increased intracellular junctions as compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor.

In some embodiments, the cancer cells are grown in vitro. In some embodiments, the cancer cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor when grown to confluence, exhibit a greater area of cells growing in a single monolayer compared to an area of cells growing in a multilayer, as compared to otherwise identical cancer cells grown to confluence and not treated with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor. In some embodiments, greater than 50%of the area of the cancer cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor and grown to confluence, exhibit growth in a monolayer. In some embodiments, the cells growing above the monolayer of confluent cells exhibit increased phosphorylation of Ser10 of Histone 3 compared to the cells growing as a monolayer. In some embodiments, the cells growing above the monolayer of confluent cells exhibit increased vacuolization.

In some embodiments, the cancer cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor when grown to confluence have increased intercellular junctions as compared to normal cells. Increased intercellular junctions refers to cells that exhibit increased localization of markers of one or more intercellular junctions (e.g., tight junctions and adherens junctions) compared to otherwise identical cancers cells not contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor when grown to confluence.

In some embodiments, the population of cancer cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitors when grown to confluence exhibit reduced anchorage independent growth when grown in soft agar in vitro, as compared to the population of cells that has not been contacted with the test compound. In some embodiments, the population of cancer cells contacted with the test compound exhibits an increase in the area of monolayer growth compared to the area of multilayer growth relative to cells not treated with the test compound.

In some embodiments, the glucocorticoid receptor agonist comprises hydrocortisone. In some embodiments, the glucocorticoid receptor agonist comprises dexamethasone. In some embodiments, the HIV protease inhibitor is selected from the group consisting of saquinavir, lopinavir, and nelfinavir mesylate. In some embodiments, the one or more glucocorticoid receptor agonist comprises hydrocortisone and the more HIV protease inhibitors is selected from the group consisting of saquinavir, lopinavir, and nelfinavir mesylate.

Methods of Inducing Vacuolization

In certain aspects, described herein are methods of inducing vacuolization of cancer cells, comprising administration to the subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the vacuolization is characterized by an increase in the presence of vacuoles compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor.

In certain aspects, described herein are methods of inducing vacuolization of cancer cells in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor. In some embodiments, there is decreased vacuolization in the cancer cells when contacted with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor when the cells are grown in sub-confluent conditions as compared to otherwise identical cancer cells that are contacted with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor when grown in confluent conditions of about 100%or more confluency.

In some embodiments, the increase in the presence of vacuoles is at least 50 fold or more greater than the presence of vacuoles in cancer cells treated with the one or more HIV protease inhibitor alone. In some embodiments, the increase in the presence of vacuoles is at least 5-fold or greater than the presence of vacuoles in cancer cells treated with the one or more glucocorticoid receptor agonists alone. In some embodiments, the vacuoles have a maximum diameter of about 50 μm. In some embodiments, one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick TFEB or ERM1 are localized to the vacuoles of the cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor.

In some embodiments, the increase in the presence of vacuoles is at least 50 fold or greater than the presence of vacuoles in cancer cells treated with the one or more HIV protease inhibitor alone. In some embodiments, the increase in the presence of vacuoles is at least 5-fold or greater than the presence of vacuoles in cancer cells treated with the one or more glucocorticoid receptor agonists alone.

In some embodiments, the glucocorticoid receptor agonist comprises hydrocortisone. In some embodiments, the glucocorticoid receptor agonist comprises dexamethasone. In some embodiments, the HIV protease inhibitor is selected from the group consisting of saquinavir, lopinavir, and nelfinavir mesylate. In some embodiments, the one or more glucocorticoid receptor agonist comprises hydrocortisone and the more HIV protease inhibitor is selected from the group consisting of saquinavir, lopinavir, and nelfinavir mesylate.

Methods of Inducing Non-Apoptotic Cancer Cell Death

In certain aspects, described herein are methods of inducing cell death of one or more cancer cells, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor. In some embodiments, the cancer cell death is non-apoptotic cell death.

In certain aspects, described herein are methods of inducing cell death of one or more cancer cells in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor. In some embodiments, there is decreased cell death in the cancer cells when contacted with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor when the cells are grown in sub-confluent conditions as compared to otherwise identical cancer cells that are contacted with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor when grown in confluent conditions of about 100%or more confluency.

In some embodiments, the non-apoptotic cell death is characterized by an increase in the presence of vacuoles compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor. In some embodiments, the administration of or contacting with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor results in a 2-fold or greater increase in cancer cell death as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor.

In some embodiments, described herein is a method of inducing non-apoptotic cell death of one or more cancer cells, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor; wherein the cancer cells, following contact with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor, are characterized by the presence of vacuoles; wherein the vacuoles: (i) have a maximum diameter of about 50 μm; (ii) one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick, TFEB or ERM1 are localized to the vacuoles; and (iii) comprise condensed and minimized nuclei at inner periphery of vacuoles. In some embodiments, the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell.

In some embodiments, the glucocorticoid receptor agonist comprises hydrocortisone. In some embodiments, the glucocorticoid receptor agonist comprises dexamethasone. In some embodiments, the HIV protease inhibitor comprises saquinavir, lopinavir, and nelfinavir mesylate In some embodiments, the one or more glucocorticoid receptor agonist comprises hydrocortisone and the one or more HIV protease inhibitor comprises saquinavir, lopinavir, and nelfinavir mesylate.

Methods using predicative biomarkers for the therapeutic efficacy of glucocorticoids and/or HIV protease inhibitors

In some aspects, described herein are methods of predicting therapeutic efficacy of glucocorticoids and/or HIV protease inhibitor for treatment of cancer. In certain embodiments, Myc-Nick is a predictive biomarker for the therapeutic efficacy of at least one glucocorticoid (s) and/or at least one HIV protease inhibitor. In some embodiments, the methods described herein comprise determining Myc-Nick expression in cancer cells obtained from a subject. In some embodiments, the expression or amount of expression of Myc-Nick informs the decision to whether to treat a subject harboring cancer cells with at least one glucocorticoid (s) and/or at least one HIV protease inhibitor. The expression of Myc-Nick can be determined by any method known in the art for detection of protein, and/or nucleic acid sequences encoding Myc-Nick.

In some embodiments, high or elevated calpain activity greater than appropriate controls predicts therapeutic efficacy of glucocorticoids and/or HIV protease inhibitor for treatment of cancer. In certain embodiments, the appropriate control for the determination of elevated calpain activity is non-cancer tissue from the same individual. In certain embodiments, high or elevated calpain activity is a predictive biomarker for the therapeutic efficacy of at least one glucocorticoid (s) and/or at least one HIV protease inhibitor. In certain embodiments, both expression of Myc-Nick and elevated calpain activity is a predictive biomarker for the therapeutic efficacy of at least one glucocorticoid (s) and/or at least one HIV protease inhibitor. In some embodiments, the methods described herein comprise determining calpain activity in cancer cells obtained from a subject. In some embodiments, high or elevated calpain activity informs the decision to treat a subject harboring cancer cells with at least one glucocorticoid (s) and/or at least one HIV protease inhibitors. Calpain activity can be determined by any method known in the art, including but not limited to, fluorometric assays.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc. ) , but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993) ; A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition) ; Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989) ; Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc. ) ; Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990) ; Carey and Sundberg Advanced Organic Chemistry 3 ^rd Ed. (Plenum Press) Vols A and B (1992) .

Methods

Cell culture

The murine liver cancer cell line T2pMig was derived from a mouse liver cancer model initiated by a transgene of MYC. The MycNick cell line was generated by stably expressing MycNick, the N-terminal fragment of Myc in the T2pMig cell line. The murine lung cancer cell line MM was generated from a mouse model of lung cancer collaboratively initiated by BRAF ^V600E and homozygous loss of the tumor suppressor TP53. All cell lines were cultured in DMEM (Gibco, Cleveland, TN, USA) supplemented with 5%fetal bovine serum (Gibco) , penicillin (100 U/mL) -streptomycin (100 μg/mL) (Gibco, Cat. No. 15140-122) , 2 mM L-glutamine (Gibco, 200 mM solution, Cat. No. 25030081) , and 1 mM sodium pyruvate (Gibco, 100 mM solution, Cat. No. 11360070) at 37 ℃ in a humidified incubator that was maintained at 5%CO2.

Chemicals

The compounds purchased from Aladdin were Hydrocortisone (H132728-1 g) , Lopinavir (L299096-1 g) , Nelfinavir (N137745-250 mg) , and Saquinavir (S126514-100 mg) . Immunofluorescent microscopy

Cells were cultured on collagen-coated coverslips in a 6-well plate and exposed to chemicals including hydrocortisone, its analogs, HIV protease inhibitors or a combination between hydrocortisone and a HIV protease inhibitors. Cells were transferred every three days to fresh media supplemented with the same drug or drug recombination. After the treatment lasted for 10-16 days, cells were fixed with either 4%paraformaldehyde or 4%paraformaldehyde followed with methanol treatment, and then permeabilized with 0.1% Triton X-100. After blocking with 5%BSA, cells were incubated with a primary antibody for 2 hours at room temperature.

The following are primary antibodies used in this study. Rabbit antibodies for β-Catenin (6B3) (Cat. #9582s) , H3Ser10P (Cat. #9701L) , cleaved caspase 3 (Asp175) (Cat. #9661L) , Rab5 (C8B1) (Cat. #2965S) , Rab7 (Cat. #9367P) , Rab11 (Cat. #5589P) , and LAMP1 (Cat. #9091P) were from Cell Signaling Technology. Mouse mAb for Calnexin (Cat. #ab2798-100) , Rabbit pAb for Lamp1 (Cat. #ab24170-100) , Rabbit mAb to Calpain S1 (EPR3324) (Cat. #ab92333) and Rabbit mAb for MYC (Y69) (Cat. #ab30072) were from Abcam. Rabbit anti-TFEB antibody (Cat. #A303-673A) was from Bethyl. Rabbit antibody for cyclin A (H-432) (Cat. #sc-751) was from Santa Cruz Biotechnology. Mouse mAb for ZO-1 (Cat. #66452-1-1g) was from Proteintech. The anti-Histone H3 (Ser10 P) antibody was used after a 1000-fold dilution and the rest of the antibodies were used at a dilution of 1: 100. Primary antibodies were detected with Rhodamine (TRITC) -conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L) (Cat. #711-025-152) , Fluorescein (FITC) -conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Cat. #111-095-003) , Alexa Fluor488 Affinipure Goat Anti-Mouse IgG(H+L) (Cat. #115-545-003 from Jackson ImmunoResearch at 1: 500 dilution. After immunostaining, cells were mounted on microscope slides with 4’, 6’-diamidino-2-phenylindole (DAPI) -containing Vectashield mounting solution (Vector Laboratories, Cat. #H1500) . For fluorescence detection, an EVOS FL Auto microscope (Thermo Fisher) was used.

Imaging intracellular acidic organelles

For determination of acidic subcellular compartments, cells were cultured on cover slips and exposed for 40 min at 37 ℃ with 1 nM of LysoSensor ^TM green DND189 (Thermo Fisher, Cat. #L7535) , a dye that is routinely used to measure the pH of acidic organelles such as lysosomes and becomes more fluorescent in acidic environments. Alternatively, cells were treated with 1 μM of Acridine Orange (AO) (Invitrogen, Cat. #A3568) . Although quite cell permeant in the neutral form, once protonated, AO dye tends to become trapped on the low pH side of the membrane barrier leading to its accumulation in acidic organelle structures, such as lysosomes. The effectiveness of this concentration process is sufficient to create intra-lysosomal concentrations leading to precipitation of the AO dye into aggregated granules. These oligomeric structures exhibit a red shift (640 nm) compared to the monomeric AO that emits at 525 nm. Lysosomes will appear yellowish green by illuminating cells with a blue light (488 nm) excitation filter and a green light (540-550 nm) emission/barrier filter. Alternatively, lysosomes will appear red when using an excitation filter of 550 nm (540-560 nm) and a long pass >610 nm emission/barrier filter. After fixation in 4%of paraformaldehyde, dye-stained cells were mounted on microscope slides with DAPI-containing Vectashield. An EVOS FL Auto microscope (Thermo Fisher) was used to detect fluorescence.

Testing compounds for their ability of restoring contact inhibition

Exponentially growing MycNick cells were passaged into 24-well plates at a final confluence of 90%and were allowed to attach for 48 hours before being exposed to a cortisol analog in the presence or absence of a HIV protease inhibitors. After the initiation of treatment, the cells were allowed to grow beyond confluence for 7 days before being subjected to morphological analysis and cell density. An inverted tissue culture microscope (Leica) was used to document vacuoles in live cells. Cell density was evaluated under an EVOS FL Auto microscope (Thermo Fisher) after fixation of the treated cells with 4%paraformaldehyde (PFA) in the presence of detergent 0.5%Triton X-100 and subsequently staining of DNA with 5 μg/ml of Propidium Iodide (PI) in the presence of 5 μg/ml of Ribonuclease A from bovine pancreas (RNase A) (Sigma, Cat. #R6513-50MG) .

The threshold concentration that was used to elicit no less than 50%of reduction in the cell density was determined. So was the minimal effective concentration that could induce vacuolation to an extent that covered no less than 10%of a random field documented under a bright field microscope. Quantification of the area with vacuolation, multilayer zones or multilayer zones was performed with the Image J software. More than 10 random fields were chosen for quantification for each data point in each of two independent experiments. The average of these data in was presented in the figures.

Xenograft assays

Xenografts were initiated in immunocompromised (Nu/Nu) mice with the murine lung adenocarcinoma cell line MM and liver cancer cell line MycNick. Five million cells were injected subcutaneously into each mouse and treatment was initiated when the average tumor volumes reached 150mm ³ (n = 5/groups) . Tumor-bearing mice were randomized into different groups to receive either vehicle or indicated compounds. The compounds were administered once a day. Hydrocortisone at 2.5 mg/kg was administered by injection subcutaneously near the tumors whereas HIV inhibitors at 10 mg/kg were given through oral gavage. For these experiments, clinically used Hydrocortisone injections (5 mg/ml, H20023069) were obtained from China National Pharmaceutical Group Co., Ltd. Drug solution was administered with each dose. Tumor volumes were determined once every three days and are calculated from digital caliper raw data by using the formula: Volume (mm ³) =(L x W ²) /2. The value W (Width) is the smaller of two perpendicular tumor axes and the value L (Length) is the larger of two perpendicular axes. Mean tumor volume growth curves and means are calculated for each treatment group.

Assessing therapeutic synergism between two drugs

The combination index (CI) was used to evaluate therapeutic synergism between two drugs. CI= (1-TGIab) / (1-TGIa) (1-TGIb) a, b: represents two different drugs. TG1ab: Tumor growth inhibition when both drugs a and b are used in combination. TGIa: Tumor growth inhibition when drug a is administered. TGIb: Tumor growth inhibition when drug b is administered. 0.9≤CI≤1.1: superposition effect; 0.8≤CI<0.9: low degree of synergy; 0.6≤CI<0.8: Moderate synergy; 0.4≤CI<0.6 : highly synergistic effect; CI<0.4: strong synergetic effect.

Statistical Analysis

Two or multiple group comparisons were conducted by using Graphpad Prism 7.0. Analysis of variance (ANOVA) provides a statistical test of whether two or more group means are equal. Tukey or Dunnett test are then carried out if ANOVA rejects the null hypothesis that two or more-group means are equal. Tukey’s test compares the means of every treatment to the means of every other treatment. Dunnett's test compares the means of every treatment to a single control.

Example 1: Hydrocortisone (HC) reduces cell density and multilayer cellular growth in HCC cells

Murine hepatocellular carcinoma (HCC) cells stably expressing MycNick were generated by expressing the Myc-Nick, the N-terminal of the Myc protein, in the T2 cell line ( “T2 MycNick” cells) , which was, in turn, derived from a MYC-driven mouse liver cancer (Shachaf et al., Nature 2004 Oct 28; 431 (7012) : 1112-7) . T2 MycNick HCC cells were incubated with 0.08 uM to 0.6 uM hydrocortisone (HC (Figure 1) . The percentage of cell growth area that exhibited multiple layers of cells ( “multilayer zone” ) decreased in a dose-dependent manner. These results demonstrate that hydrocortisone reduces cell density of HCC cells in vitro.

Example 2: Reduction in Cell Density by Hydrocortisone and RU486 in HCC cells expressing Myc-Nick

To determine if HC restores contact inhibition through interaction with the glucocorticoid receptor, T2 MycNick cells were incubated with hydrocortisone (HC) (5 uM) , the glucocorticoid receptor antagonist RU486 (2.5 uM) or both HC and RU486 (Figure 2) . The cells were subjected to daily analysis of viability and morphology under an inverted tissue culture microscope or GE InCell Analyzer 2000. Cell density was documented after staining of DNA with PI 7-10 days after the initiation of treatment. Figure 2 shows the relative number of cells after incubation with DMSO, the glucocorticoid receptor (GR) antagonist RU486, Hydrocortisone (HC) or both RU486 and HC. Both HC and RU486 reduced the final cell density, and their combination failed to further reduce the final cell density, indicating that HC mediated reduction in cell density occurs by a mechanism distinct from modulation of glucocorticoid receptor activity.

Example 3: Hydrocortisone (HC) causes vacuolization of HCC cells

T2 MycNick HCC cells were incubated with 0.08 uM to 0.6 uM hydrocortisone (HC (Figure 3) , and the area of cell growth that exhibited the presence of vacuoles was measured. The percent of cells exhibiting vacuoles increased in a dose-dependent manner. These results indicate that hydrocortisone induces vacuolization of HCC cells.

Example 4: The GR antagonist RU486 inhibits vacuolation induced by HC in multilayer

To evaluate the role of glucocorticoid receptor activity in HC-induced vacuolization of cells growing in multi-layers, murine HCC cells expressing Myc-Nick were incubated with HC, RU486 or both HC RU486 (Figure 4) . Treatment with HC induced vacuolation of the cells, while incubation with RU486 did not lead to vacuolization. Furthermore, RU486 inhibited HC-mediated vacuolization. These findings show that HC mediated induction of vacuolization in cells expressing Myc-Nick is mediated through activity of the glucocorticoid receptor.

Example 5: The HIV protease inhibitor saquinavir moderately enhances the vacuolation elicited by hydrocortisone

To investigate the role of HIV protease inhibitors in restoration of contact inhibition and vacuolization by HC, T2 MycNick HCC cells were incubated with hydrocortisone (HC) , the HIV protease inhibitor saquinavir, or both saquinavir and HC (Figure 5) . The percentage of area of cell growth with vacuoles ( “the vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) were measured, and the vacuole zone moderately increased when cells were exposed to both saquinavir and HC compared to HC alone. T2 cells MycNick HCC cells incubated with saquinavir or both saquinavir and hydrocortisone were also stained with Lysosensor ^TM Green DNA-189 to determine if the vacuoles induced by HC and saquinavir were acidic (Figure 6) . The periphery of the vacuoles exhibited acidic vesicles. These results suggest that HIV protease inhibitors promotes HC-induced cellular vacuolization in HCC cells.

Example 6: Saquinavir and hydrocortisone synergistically and potently reduce cell density and promote the formation of monolayer zones

To determine if saquinavir promotes HC induced reduction in cell density, T2 MycNick HCC cells were incubated with HC, saquinavir, or both saquinavir and HC, and stained with propidium iodide (Figure 7) . The monolayer zone increased synergistically in cells incubated with both saquinavir and HC as compared to either saquinavir or HC alone. These results indicate that saquinavir greatly increases the effects of HC on reducing cell density of HCC cells.

Example 7: The HIV protease inhibitor lopinavir moderately enhances the vacuolation elicited by hydrocortisone when used at a low concentration

To determine if additional HIV protease inhibitors promote HC induction of vacuolation, T2 MycNick HCC cells were incubated with HC and the HIV protease inhibitor lopinavir, or both lopinavir and HC (Figure 8) . The percentage of area of cell growth with vacuoles ( “the vacuoles zone” ) and the percentage of area of cell growth that does not exhibit vacuoles ( “non-vacuoles zone” ) were measured, and the vacuole zone moderately increased when cells were exposed to both lopinavir and HC compared to HC alone. These results suggest that lopinavir at low concentrations moderately promotes HC-induced vacuolation in HCC cells.

T2 MycNick HCC cells were incubated with lopinavir at a high concentration of 2.5 uM, HC, or both lopinavir (at 2.5 uM) and HC (Figure 9) . At high concentrations, lopinavir was sufficient to elicit low level of vacuolation, but did not promote vacuolation induced by HC. These results indicate that lopinavir is a moderate inducer of HC-mediated vacuolation at lower concentrations but not at high concentrations.

Example 8: Combination treatment with lopinavir and hydrocortisone reduces the cell density more severely than either of two individual treatments

To determine if lopinavir promotes HC induced reduction in cell density, T2 MycNick HCC cells were incubated with HC, lopinavir, or both lopinavir and HC, and stained with propidium iodide (Figures 10-12) . The number of cells, and the area of the monolayer zone increased in cells incubated with both lopinavir and HC as compared to either lopinavir or HC alone (Figures 10 and 11) . Furthermore, combination treatment with HC and lopinavir at a high concentration reduced cell density more severely than either HC or lopinavir alone (Figure 12) . Like HC001, CI024 reduced cell density when cells are allowed to overgrow beyond confluence. Both compounds achieved a similar extent of reduction in the cell number, implying that they may act through the same pathway. These results indicate that lopinavir promotes the effects of HC on reducing cell density of HCC cells.

Example 9: Acidic vehicles are concentrated around both large and small vacuoles elicited by either lopinavir alone or lopinavir in combination with hydrocortisone

T2 MycNick HCC cells were incubated with HC, lopinavir (at a concentration of 2.5 uM) or both lopinavir and HC and stained with Lysosensor ^TM Green DNA-189 (Figures 13 and 14) . Similar to cells treated with HC alone, the vacuoles exhibited acidic vesicles in the periphery of the vacuoles in cells incubated with lopinavir or lopinavir and HC. These results suggest that lopinavir induction of vacuolation in HCC cells has similar morphological characteristics and acidity as observed in vacuoles indued by HC.

Example 10: Nelfinavir mesylate and hydrocortisone synergistically promote the formation of vacuoles with acidic vescicles

To determine if the HIV protease inhibitor, nelfinavir mesylate, induces vacuolation in HCC cells, T2MycNick HCC cells were incubated with HC, nelfinavir mesylate or both HC and nelfinavir mesylate (Figure 15) . The area with vacuoles increased synergistically in cells incubated with both HC and nelfinavir mesylate compared to either HC or nelfinavir mesylate alone. T2 MycNick HCC cells were incubated with HC, nelfinavir mesylate (at a concentration of 5 uM) or both nelfinavir mesylate and HC and stained with Lysosensor ^TM Green DNA-189 (Figure 15) . Similar to cells treated with HC alone, the vacuoles exhibited acidic vesicles in the periphery of the vacuoles in cells incubated with nelfinavir mesylate or nelfinavir mesylate and HC. These results suggest that nelfinavir mesylate induction of vacuolation in HCC cells has similar morphological characteristics and acidity as observed in vacuoles indued by HC and that nelfinavir mesylate can synergistically increase vacuolation induced by HC in HCC cells.

Example 11: Combination treatment with nelfinavir mesylate and hydrocortisone reduces the cell density more severely than either of two individual treatments

To determine if nelfinavir mesylate can promote HC-induced reduction in cell density, T2 MycNick HCC cells were incubated with nelfinavir mesylate, HC, or both nelfinavir mesylate and HC, and cells were stained with propidium iodine to determine cell number (Figure 17) . The monolayer zone area was increased when cells were incubated with nelfinavir mesylate and HC vs when cells were incubated with either nelfinavir mesylate or HC alone. These results indicate that the HIV protease nelfinavir mesylate promotes HC-induced reduction of cell density in HCC cells.

Example 12: Saquinavir and HC synergistically suppress the growth of a lung cancer model driven by BRAF ^V600E

The murine lung cancer cell line MM was generated from a mouse model of lung cancer collaboratively initiated by BRAF ^V600E and homozygous loss of the tumor suppressor TP53. To determine the effects of HIV protease inhibitor and HC treatment of lung cancer, mice bearing MM lung tumors were treated with saquinavir (10 mg/kg) and HC (2.5 mg/kg) or either saquinavir or HC alone (Figure 18) . Mice treated with both HC and saquinavir synergistically reduced tumor burden compared to mice treated with only HC or saquinavir alone. Furthermore, HC and saquinavir neither individually nor in combination had no impact on mouse body weight (Figure 19) . These results show that saquinavir and HC combination therapy can be used to treat lung cancer.

Example 13: Nelfinavir mesylate and HC synergistically suppress the growth of a lung cancer model driven by BRAF ^V600E

To determine the effects of HIV protease inhibitor and HC treatment of lung cancer, mice bearing MM lung tumors were treated with nelfinavir mesylate (10 mg/kg) and HC (2.5 mg/kg) or either nelfinavir mesylate or HC alone (Figure 20) . Mice treated with both HC and nelfinavir mesylate synergistically reduced tumor burden compared to mice treated with only HC or saquinavir alone. Furthermore, HC and s neither individually nor in combination had no impact on mouse body weight (Figure 21) . These results show that nelfinavir mesylate and HC combination therapy can be used to treat lung cancer.

Examle 14: Lopinavir and HC promote the formation of similar monolayer zones

H2 MycNick HCC cells were incubated with HC or lopinavir ( “CI024” ) and visualized by microscopy (Figure 22) . The vacuoles induced by HC and by CI024 when cells were allowed to grow beyond 100%confluence exhibited similar morphology. Furthermore, Like HC, CI024 has no appreciable effect on proliferation and viability of MycNick cells in subconfluence when used at a concentration that suffices to elicit contact inhibition of proliferation. The cells proliferated and grew into confluence in the presence of CI024. Neither HC nor CI024 arrested proliferation or induced cell death when cells are cultured in sub-confluence. Like HC, high cell-density was required for CI024 to elicit vacuolation (Figure 23) . Treatment of confluent but not sub-confluent cells for 4 days by CI024 elicited vacuolation. Both HC and CI024 required high cell-density to elicit vacuolation. Manifestation of vacuolation also required at least a 2-day period of latency, indicating that this process might need new synthesis of proteins. These results indicate that HIV protease inhibitor CI024 induces vacuolation similar to HC in HCC cells.

Example 15: Mainenance of vacuoles requires continued exposure to lopinavir.

To determine whether CI024 induced vacuoles required continued CI024 exposure, T2 MycNick HCC cell lines were incubated with Hydrocortisone (HC001) or CI024 for 5 days, followed by withdrawal of the HC or CI024, where the cells were imaged after 4 days or 7 days following the withdrawal, and HCC cells were re-exposed to Hydrocortisone (HC001) or CI024 for three days (Figure 22) . The HCC cells regressed and fully developed vacuoles disappeared within 7 days after withdrawal of CI024, and re-appeared rapidly. Therefore, these results show that like the situation with HC, the maintenance of vacuoles requires the continued exposure to CI024.

Example 16: Lopinavir induces the formation of condensed andminimized nuclei in multilayer zones

To determine if CI024 can trigger the formation of condensed and minimized nuclei in multilayer zones, T2 MycNick cells were incubated with HC or CI024 and stained with propidium iodide to visualize nuclei (Figure 25) . Both HC and CI024 induced the condensation of nuclei at the multilayer zones but not at monolayer zones.

Example 17: Treatment of MycNick HCC cells with lopinavir diminishes the mitotic population.

To determine if CI024 proliferative arrest occurs in monolayer zones, T2 MycNick HCC cells were stained with ant-phosphorylated histone H3 Serine 10 antibody (Figure 26) . Proliferating cells were almost exclusively found in the multilayer zones. To determine if CI024 also reduces the number of cells in S phase, T2 MycNick HCC cells were stained with anti-cyclin A1 antibodies (Figure 27) . The number of cells in S phase were reduced in the cells treated with CI024, and cyclin A1-positive cells were mainly distributed in the multilayer zones.

These results show that there is a diminished population of mitotic cells growing in the monolayer induced by CI024.

Table 1

Example 18 : Lopinavir restores the entopic membrane location of β-catenin in monolayer zones

To determine if CI024 can restore membrane localization of β-catenin, T2

MycNick HCC cells were incubated with HC or CI024 for 12 days and stained with an anti-β-catenin antibody (Figure 28) . The distribution of β-catenin localization was measured using Image J software by measuring gray value and distance (pixels) of the image. The peak-valley grey value was increased in cells incubated with CI024 or HC to a similar extent. β-catenin membrane localization was not observed in the multilayer zones for cells incubated with either CI024 or β-catenin. These results show that CI024 restores β-catenin membrane localization in the monolayer zones.

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

A method of treating cancer in a subject, comprising administration to the subject of (1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) .
A method of reducing cancer cell proliferation, comprising administration to a subject (1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the cell proliferation is reduced compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor (s) .
A method of reducing cancer cell proliferation in vitro, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonists and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonists and one or more HIV protease inhibitor (s) .
A method of inducing vacuolization of cancer cells, comprising administration to a subject (1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the vacuolization is characterized by an increase in the presence of vacuoles compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
A method of inducing vacuolization of cancer cells in vitro, comprising contacting the cancer cells with (1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonist (s) and one or more HIV protease inhibitor (s) .
A method of inducing cell death of cancer cells, comprising administration to a subject (1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the number of cells undergoing cell death is increased compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
A method of inducing cell death of one or more cancer cells in vitro, comprising contacting the cancer cells with (1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells are grown at about 100 percent or more confluence prior to contacting the cancer cells with the one or more glucocorticoid receptor agonist (s) and one or more HIV protease inhibitor (s) .
The method of any one of claims 1 -7, wherein the one or more glucocorticoid receptor agonist (s) are selected from the group consisting of: a natural glucocorticoid receptor agonist, a synthetic glucocorticoid receptor agonist, a hydrocortisone-type glucocorticoid receptor agonist, a methasone-type glucocorticoid receptor agonist and an acetonide related glucocorticoid receptor agonist.
The method of any one of claims 1 -8, wherein the glucocorticoid receptor agonist comprises hydrocortisone.
The method of any one of claims 1 -9, wherein the glucocorticoid receptor agonist comprises dexamethasone.
The method of any one of claims 1 -10, wherein a hydroxyl group of the HIV protease inhibitor interacts with the carboxyl group of the protease active site amino acid Asp25 by a hydrogen bond.
The method of any one of claims 1 -11, wherein the HIV protease inhibitor contacts one or more amino acids of HIV protease selected from the group consisting of Gly27, Asp29, Asp30, and Gly48.
The method of any one of claims 1 -12, wherein the HIV protease inhibitor inhibits activation of SREBP-1.
The method of any one of claims 1 -13, wherein the HIV protease inhibitor inhibits proteasome activity.
The method of claim 14, wherein the HIV protease inhibitor inhibits the proteasomal 20S subunit.
The method of any one of claims 1 -15, wherein the HIV protease inhibitor inhibits AKT/PKB.
The method of any one of claims 1 -16, wherein the HIV protease inhibitor comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.
The method of any one of claim 1 -17, wherein the one or more glucocorticoid receptor agonist (s) comprises hydrocortisone and the one or more HIV protease inhibitor comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.
The method of any one of claims 1 -18, wherein the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) are administered to the subject or contacted with the cancer cells concurrently.
The method of any one of claims 1 -18, wherein the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) are administered to the subject or contacted with the cancer cells sequentially.
The method of claim 20, wherein the one or more glucocorticoid receptor agonist (s) are administered to the subject or contacted with the cancer cells prior to the administration of or contact with the one or more HIV protease inhibitor (s) .
The method of claim 20, wherein the one or more glucocorticoid receptor agonist (s) are administered to the subject or contacted with the cancer cells after administration of or contact with the one or more HIV protease inhibitor (s) .
The method of any one of the above claims, wherein the cancer cells overexpress

MYC compared to an otherwise identical non-cancer cell.
The method of any one of claims 1 -22, wherein the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell.
The method of any one of the above claims, wherein the cancer cells express BRAF ^V600E.
The method of claim 25, wherein the cancer cells exhibit loss of TP53 expression.
The method of any one of the above claims, wherein the cancer is metastatic.
The method of any one of the above claims, wherein the cancer is lung cancer.
The method of any one of the above claims, wherein the administration of or contacting with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) results in a reduction in cancer proliferation, increased cancer cell death, or combinations thereof, compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
The method of claim 29, wherein the administration of or contacting with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) results in a 2-fold or greater reduction in cancer cell proliferation as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
The method of claim 29, wherein the administration of or contacting with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) results in a 2-fold or greater increase in cancer cell death as compared to otherwise identical cancer cells not contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
The method of any one of claims 5, 29 or 31, wherein the cancer cell death is non-apoptotic cell death.
The method of claim 32, wherein the non-apoptotic cell death is characterized by an increase in the presence of vacuoles compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
The method of claim 33, wherein the increase in the presence of vacuoles is at least 2-fold or more greater than the presence of vacuoles in cancer cells treated with the one or more HIV protease inhibitor (s) alone.
The method of claim 33, wherein the increase in the presence of vacuoles is at least 5-fold or more greater than the presence of vacuoles in cancer cells treated with the one or more glucocorticoid receptor agonist (s) alone.
The method of any one of claims 33 -35, wherein the vacuoles have a maximum diameter of about 50 μm.
The method of any one of claims 33 -36, wherein the vacuoles comprise have regions of high acidity in the periphery of the vacuoles.
The method of any one of claims 33 -37, wherein the vacuoles comprise condensed and minimized nuclei at inner periphery of vacuoles.
The method of any one of claims 33 -37, wherein one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick TFEB or ERM1 are localized to the vacuoles of the cells contacted with the one or more glucocorticoid receptor agonists and the one or more HIV protease inhibitor (s) .
The method of any one of the above claims, wherein the cancer cells exhibit increased intercellular junctions as compared to cancer cells that have not been contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
The method of any one of the above claims, wherein the cancer cells exhibit increased plasma membrane localization of B-catenin.
The method of any one of the above claims, wherein the cancer cells exhibit increased plasma membrane localization of ZO-1.
The method of any one of claims 3, 5, 7, and 9-42, wherein the cancer cells are grown in vitro.
The method of claim 43 wherein the cancer cells contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) when grown to confluence, exhibit a greater area of cells growing in a single monolayer compared to an area of cells growing in a multi-layer, as compared to otherwise identical cancer cells grown to confluence and not treated with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) .
The method of claim 44, wherein greater than 50%of the area of the cancer cells contacted with the one or more glucocorticoid receptor agonist (s) and the one or more HIV protease inhibitor (s) and grown to confluence, exhibit growth in a monolayer.
A method of inducing vacuolation of one or more cancer cells, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells, following contact with the one or more glucocorticoid receptor agonist (s) and one or more HIV protease inhibitor (s) , are characterized by the presence of vacuoles; wherein the vacuoles:

(i) have a maximum diameter of about 50 μm; and

(ii) one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick, TFEB or ERM1 are localized to the vacuoles.
A method of inducing non-apoptotic cell death of one or more cancer cells, comprising contacting the cancer cells with 1) one or more glucocorticoid receptor agonist (s) and (2) one or more HIV protease inhibitor (s) ; wherein the cancer cells, following contact with the one or more glucocorticoid receptor agonist (s) and one or more HIV protease inhibitor (s) , are characterized by the presence of vacuoles; wherein the vacuoles:

(i) have a maximum diameter of about 50 μm;

(ii) one or more of Calpain, Rab5, Rab11, LAMP1, MYC, MYC-Nick, TFEB or ERM1 are localized to the vacuoles; and

(iii) comprise condensed and minimized nuclei at inner periphery of vacuoles.
The method of claims 46 or 47, wherein a hydroxyl group of the HIV protease inhibitor interacts with the carboxyl group of the protease active site amino acid Asp25 by a hydrogen bond.
The method of any one of claims 46 -48, wherein the HIV protease inhibitor contacts one or more amino acids of HIV protease selected from the group consisting of Gly27, Asp29, Asp30, and Gly48.
The method of any one of claims 46 -49, wherein the HIV protease inhibitor inhibits activation of SREBP-1.
The method of any one of claims 46 -50, wherein the HIV protease inhibitor inhibits proteasome activity.
The method of claim 51, wherein the HIV protease inhibitor inhibits the proteasomal 20S subunit.
The method of any one of claims 46 -52, wherein the HIV protease inhibitor inhibits AKT/PKB.
The method of any one of claims 46 -53, wherein the one or HIV protease inhibitor (s) comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.
The method of any one of claims 46-54, wherein the one or more glucocorticoid receptor agonist (s) comprises hydrocortisone.
The method of any one of claims 46-55, wherein the one or more glucocorticoid receptor agonist (s) comprises dexamethasone.
The method of claim 55, wherein the one or more glucocorticoid receptor agonist (s) comprises hydrocortisone and the one or more HIV protease inhibitor (s) comprises an inhibitor selected from the group consisting of: saquinavir, lopinavir, and nelfinavir mesylate.
The method of any one of claims 46-57 wherein the cancer cells overexpress MYC-Nick compared to an otherwise identical non-cancer cell.
The method of claim 58, wherein the cancer cells overexpress MYC-Nick compared to otherwise identical non-cancer cells.
The method of any one of claims 46-59, wherein the cancer cells express BRAF ^V600E.
The method of claim 60, wherein the cancer cells exhibit loss of TP53 expression.
The method of any one of claims 46-61, wherein the cancer is metastatic.
The method of any one of claims 46-62, wherein the cancer is lung cancer.
The method of any one of claims 46-62, wherein the cancer is liver cancer.