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Supplementary Data from Bcl-2 Modulation to Activate Apoptosis in Prostate Cancer

Apoptosis resistance is a hallmark of cancer linked to disease progression and treatment resistance, which has led to the development of anticancer therapeutics that restore apoptotic function. Antiapoptotic Bcl-2 is frequently overexpressed in refractory prostate cancer and increased following standard hormonal therapy and chemotherapy; however, the rationally designed Bcl-2 antagonist, ABT-737, has not shown single agent apoptosis-promoting activity against human prostate cancer cell lines. This is likely due to the coordinate expression of antiapoptotic, Bcl-2–related Mcl-1 that is not targeted by ABT-737. We developed a mouse model for prostate cancer in which apoptosis resistance and tumorigenesis were conferred by Bcl-2 expression. Combining ABT-737 with agents that target Mcl-1 sensitized prostate cancer cell lines with an apoptotic block to cell death in vitro. In mice in vivo, ABT-737 showed single agent efficacy in prostate tumor allografts in which tumor cells are under hypoxic stress. In human prostate cancer tissue, examined using a novel tumor explant system designated Tumor Tissue Assessment for Response to Chemotherapy, combination chemotherapy promoted efficient apoptosis. Thus, rational targeting of both the Bcl-2 and Mcl-1 mechanisms of apoptosis resistance may be therapeutically advantageous for advanced prostate cancer. (Mol Cancer Res 2009;7(9):1487–96)

Supplementary Video 3 from Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

Supplementary Video 5 from Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

Supplementary Video 6 from Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity, and contact inhibition while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. [Cancer Res 2008;68(11):4105–15]

Supplementary Video 1 from Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

Apoptosis resistance is a hallmark of cancer linked to disease progression and treatment resistance, which has led to the development of anticancer therapeutics that restore apoptotic function. Antiapoptotic Bcl-2 is frequently overexpressed in refractory prostate cancer and increased following standard hormonal therapy and chemotherapy; however, the rationally designed Bcl-2 antagonist, ABT-737, has not shown single agent apoptosis-promoting activity against human prostate cancer cell lines. This is likely due to the coordinate expression of antiapoptotic, Bcl-2–related Mcl-1 that is not targeted by ABT-737. We developed a mouse model for prostate cancer in which apoptosis resistance and tumorigenesis were conferred by Bcl-2 expression. Combining ABT-737 with agents that target Mcl-1 sensitized prostate cancer cell lines with an apoptotic block to cell death in vitro. In mice in vivo, ABT-737 showed single agent efficacy in prostate tumor allografts in which tumor cells are under hypoxic stress. In human prostate cancer tissue, examined using a novel tumor explant system designated Tumor Tissue Assessment for Response to Chemotherapy, combination chemotherapy promoted efficient apoptosis. Thus, rational targeting of both the Bcl-2 and Mcl-1 mechanisms of apoptosis resistance may be therapeutically advantageous for advanced prostate cancer. (Mol Cancer Res 2009;7(9):1487–96)

To determine whether the xc- cystine transporter could be a useful therapeutic target for small-cell lung cancer (SCLC).Human SCLC cell cultures were examined for growth dependence on extracellular cystine, xc- expression, glutathione levels and response to highly specific xc- inhibitors, i.e., monosodium glutamate (MSG) and the anti-inflammatory drug, sulfasalazine (SASP). In studying tumor growth inhibition by SASP, use was also made of a novel SCLC tissue xenograft model, LU6-SCLC, derived from a chemoresistant patient's SCLC specimen.Growth of NCI-H69 and NCI-H82 SCLC cells greatly depended on xc- -mediated uptake of cystine. SASP substantially reduced their glutathione levels (70%; 0.3 mM SASP; 24 h) and growth (72 h) with IC(50)s of 0.21 and 0.13 mM, respectively; MSG also inhibited growth markedly. Both SASP- and MSG-induced growth arrests were largely prevented by cystine uptake-enhancing 2-mercaptoethanol (66 approximately microM) indicating they were primarily due to cystine starvation. Without major side-effects, SASP (i.p.) restrained growth of NCI-H69 cell xenografts (approximately 50%) and, importantly, substantially inhibited growth of the clinically more relevant LU6-SCLC tissue xenografts (approximately 70% by stereological analysis), reducing tumor glutathione contents.The xc- cystine/glutamate antiporter is potentially useful as a target for therapy of SCLC based on glutathione depletion. Sulfasalazine may be readily used for this approach, especially in combination chemotherapy.

BACKGROUND Autophagy is a starvation induced cellular process of self-digestion that allows cells to degrade cytoplasmic contents. The understanding of autophagy, as either a mechanism of resistance to therapies that induce metabolic stress, or as a means to cell death, is rapidly expanding and supportive of a new paradigm of therapeutic starvation. METHODS To determine the effect of therapeutic starvation in prostate cancer, we studied the effect of the prototypical inhibitor of metabolism, 2-deoxy-D-glucose (2DG), in multiple cellular models including a transfected pEGFP-LC3 autophagy reporter construct in PC-3 and LNCaP cells. RESULTS We found that 2DG induced cytotoxicity in PC-3 and LNCaP cells in a dose dependent fashion. We also found that 2DG modulated checkpoint proteins cdk4, and cdk6. Using the transfected pEGFP-LC3 autophagy reporter construct, we found that 2DG induced LC3 membrane translocation, characteristic of autophagy. Furthermore, knockdown of beclin1, an essential regulator of autophagy, abrogated 2DG induced autophagy. Using Western analysis for LC3 protein, we also found increased LC3-II expression in 2DG treated cells, again consistent with autophagy. In an effort to develop markers that may be predictive of autophagy, for assessment in clinical trials, we stained human prostate tumors for Beclin1 by immunohistochemistry (IHC). Additionally, we used a digitized imaging algorithm to quantify Beclin1 staining assessment. These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials. Prostate 68: 1743-1752 (c) 2008 Wiley-Liss, Inc. These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials.

Autophagy is a bulk degradation process that promotes survival under metabolic stress, but it can also be a means of cell death if executed to completion. Monoallelic loss of the essential autophagy gene beclin1 causes susceptibility to metabolic stress, but also promotes tumorigenesis. This raises the paradox that the loss of a survival pathway enhances tumor growth, where the exact mechanism is not known. Here, we show that compromised autophagy promoted chromosome instability. Failure to sustain metabolism through autophagy was associated with increased DNA damage, gene amplification, and aneuploidy, and this genomic instability may promote tumorigenesis. Thus, autophagy maintains metabolism and survival during metabolic stress that serves to protect the genome, providing an explanation for how the loss of a survival pathway leads to tumor progression. Identification of this novel role of autophagy may be important for rational chemotherapy and therapeutic exploitation of autophagy inducers as potential chemopreventive agents.

BACKGROUND Certain cancers depend for growth on uptake of cystine/cysteine from their environment. Here we examined advanced human prostate cancer cell lines, DU-145 and PC-3, for dependence on extracellular cystine and sensitivity to sulfasalazine (SASP), a potent inhibitor of the x cystine transporter. METHODS Cultures were evaluated for growth dependence on exogenous cystine, x transporter expression, response to SASP (growth and glutathione content). In vivo, effect of SASP was determined on subrenal capsule xenograft growth. RESULTS Cystine omission from culture medium arrested DU-145 and PC-3 cell proliferation; both cell lines expressed the x transporter and were growth inhibited by SASP (IC50s: 0.20 and 0.28 mM, respectively). SASP-induced growth inhibition was associated with vast reductions in cellular glutathione content—both effects based on cystine starvation. SASP (i.p.) markedly inhibited growth of DU-145 and PC-3 xenografts without major toxicity to hosts. CONCLUSIONS SASP-induced cystine/cysteine starvation leading to glutathione depletion may be useful for therapy of prostate cancers dependent on extracellular cystine. Prostate © 2006 Wiley-Liss, Inc.

The prolactin (PRL)-dependent rat Nb2 T lymphoma is a valuable model for investigation of molecular mechanisms that underlie tumor progression in hormone-dependent cancers. mRNA differential display was used to screen for novel gene products expressed in hormone-stimulated or differentiating agent-treated Nb2 sublines. From numerous transcripts identified, DNA sequencing and GenBank analysis revealed a novel 289-bp fragment. Using 5′-rapid amplification of complementary ends-PCR, this fragment was used to clone a unique 2117-bp cDNA, designated HRPAP20 (hormone-regulated proliferation-associated protein), in rat lymphoma cells. Computer-assisted sequence analysis revealed a single open reading frame that encoded a putative 20.2-kDa protein. The effect of hormone stimulation to alter expression of HRPAP20 was evaluated by Northern blot analysis of total RNA obtained from PRL-stimulated, lactogen-dependent Nb2-11 cells. Quiescent cells, synchronized in the G0-G1 phase of cell cycle, exhibited reduced HRPAP20 expression compared with exponentially proliferating cultures. The addition of mitogenic concentrations of PRL to stationary cells increased HRPAP20 mRNA accumulation within 4–6 h, corresponding to G1 cell cycle progression. Immunoblot analysis showed that PRL also increased HRPAP20 protein levels within 4 h. In addition, PRL stimulated serine phosphorylation of the HRPAP20 protein with a similar kinetic pattern. Stable transfection of the HRPAP20 cDNA into Nb2-11 cells significantly (P < 0.01) increased proliferation in the absence of hormonal stimulation and inhibited apoptosis induced by lactogen deprivation (P < 0.001). In the hormone-independent and highly malignant Nb2-SFJCD1 subline, the constitutive expression of HRPAP20 was markedly reduced by exposure of the cells to dietary differentiating agents (butyrate, retinoic acid, and vitamin D3). After removal of these substances, PRL stimulated its expression in a manner similar to that observed in PRL-dependent Nb2-11 cells. HRPAP20 expression was also evaluated in MCF-7 cells. Its expression was detectable in quiescent cultures; addition of PRL significantly (P < 0.05) increased HRPAP20 during G1 cell cycle progression. Exposure of the cells to butyrate or retinoic acid reduced HRPAP20 expression, similar to the effects of these substances in the malignant rat lymphoma. Stable transfection of HRPAP20 into MCF-7 cells significantly (P < 0.006) increased proliferation in the absence of hormone stimulation and augmented survival in the absence of serum (P < 0.05). We conclude that HRPAP20 is a phosphoprotein that is required for proliferation and survival of hormone-dependent tumor cells.

Autophagy is a crucial component of the cellular stress adaptation response that maintains mammalian homeostasis. Autophagy protects against neurodegenerative and inflammatory conditions, aging, and cancer. This is accomplished by the degradation and intracellular recycling of cellular components to maintain energy metabolism and by damage mitigation through the elimination of damaged proteins and organelles. How autophagy modulates oncogenesis is gradually emerging. Tumor cells induce autophagy in response to metabolic stress to promote survival, suggesting deployment of therapeutic strategies to block autophagy for cancer therapy. By contrast, defects in autophagy lead to cell death, chronic inflammation, and genetic instability. Thus, stimulating autophagy may be a powerful approach for chemoprevention. Analogous to infection or toxins that create persistent tissue damage and chronic inflammation that increases the incidence of cancer, defective autophagy represents a cell-intrinsic mechanism to create the damaging, inflammatory environment that predisposes to cancer. Thus, cellular damage mitigation through autophagy is a novel mechanism of tumor suppression.

SummaryAllelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumor-prone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulated p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-κB regulation and gene expression and to promote tumorigenesis. Thus, defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62-controlling pathways critical for oncogenesis.

Human cancer cell lines are widely used to model cancer but also have serious limitations. As an alternate approach, we have developed immortalized mouse epithelial cell model systems that are applicable to different tissue types and involve generation of immortalized cell lines that are genetically defined. By applying these model systems to mutant mice, we have extended the powerful approach of mouse genetics to in vitro analysis. By use of this model we have generated immortal epithelial cells that are either competent or deficient for apoptosis by different gain- and loss-of-function mutations that have revealed important mechanisms of tumor progression and treatment resistance. Furthermore, we have derived immortalized, isogenic mouse kidney, mammary, prostate, and ovarian epithelial cell lines to address the issues of tissue specificity. One of the major advantages of these immortalized mouse epithelial cell lines is the ability to perform biochemical analysis, screening, and further genetic manipulations. Moreover, the ability to generate tumor allografts in mice allows the integration of in vitro and in vivo approaches to delineate the mechanistic aspects of tumorigenesis. These model systems can be used effectively to determine the molecular requirements of epithelial tumorigenesis and tumor-promoting functions. This approach provides an efficient way to study the role of apoptosis in cancer and also enables the interrogation and identification of potential chemotherapeutic targets involving this pathway. Applying this technology to other mouse models can provide insight into additional aspects of oncogenesis.

Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity, and contact inhibition while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. [Cancer Res 2008;68(11):4105–15]

Human cancer cell lines are widely used to model cancer but also have serious limitations. As an alternate approach, we have developed immortalized mouse epithelial cell model systems that are applicable to different tissue types and involve generation of immortalized cell lines that are genetically defined. By applying these model systems to mutant mice, we have extended the powerful approach of mouse genetics to in vitro analysis. By use of this model we have generated immortal epithelial cells that are either competent or deficient for apoptosis by different gain- and loss-of-function mutations that have revealed important mechanisms of tumor progression and treatment resistance. Furthermore, we have derived immortalized, isogenic mouse kidney, mammary, prostate, and ovarian epithelial cell lines to address the issues of tissue specificity. One of the major advantages of these immortalized mouse epithelial cell lines is the ability to perform biochemical analysis, screening, and further genetic manipulations. Moreover, the ability to generate tumor allografts in mice allows the integration of in vitro and in vivo approaches to delineate the mechanistic aspects of tumorigenesis. These model systems can be used effectively to determine the molecular requirements of epithelial tumorigenesis and tumor-promoting functions. This approach provides an efficient way to study the role of apoptosis in cancer and also enables the interrogation and identification of potential chemotherapeutic targets involving this pathway. Applying this technology to other mouse models can provide insight into additional aspects of oncogenesis.

The prolactin (PRL)-dependent rat Nb2 T lymphoma is a valuable model for investigation of molecular mechanisms that underlie tumor progression in hormone-dependent cancers. mRNA differential display was used to screen for novel gene products expressed in hormone-stimulated or differentiating agent-treated Nb2 sublines. From numerous transcripts identified, DNA sequencing and GenBank analysis revealed a novel 289-bp fragment. Using 5'-rapid amplification of complementary ends-PCR, this fragment was used to clone a unique 2117-bp cDNA, designated HRPAP20 (hormone-regulated proliferation-associated protein), in rat lymphoma cells. Computer-assisted sequence analysis revealed a single open reading frame that encoded a putative 20.2-kDa protein. The effect of hormone stimulation to alter expression of HRPAP20 was evaluated by Northern blot analysis of total RNA obtained from PRL-stimulated, lactogen-dependent Nb2-11 cells. Quiescent cells, synchronized in the G(0)-G(1) phase of cell cycle, exhibited reduced HRPAP20 expression compared with exponentially proliferating cultures. The addition of mitogenic concentrations of PRL to stationary cells increased HRPAP20 mRNA accumulation within 4-6 h, corresponding to G(1) cell cycle progression. Immunoblot analysis showed that PRL also increased HRPAP20 protein levels within 4 h. In addition, PRL stimulated serine phosphorylation of the HRPAP20 protein with a similar kinetic pattern. Stable transfection of the HRPAP20 cDNA into Nb2-11 cells significantly (P0.01) increased proliferation in the absence of hormonal stimulation and inhibited apoptosis induced by lactogen deprivation (P0.001). In the hormone-independent and highly malignant Nb2-SFJCD1 subline, the constitutive expression of HRPAP20 was markedly reduced by exposure of the cells to dietary differentiating agents (butyrate, retinoic acid, and vitamin D(3)). After removal of these substances, PRL stimulated its expression in a manner similar to that observed in PRL-dependent Nb2-11 cells. HRPAP20 expression was also evaluated in MCF-7 cells. Its expression was detectable in quiescent cultures; addition of PRL significantly (P0.05) increased HRPAP20 during G(1) cell cycle progression. Exposure of the cells to butyrate or retinoic acid reduced HRPAP20 expression, similar to the effects of these substances in the malignant rat lymphoma. Stable transfection of HRPAP20 into MCF-7 cells significantly (P0.006) increased proliferation in the absence of hormone stimulation and augmented survival in the absence of serum (P0.05). We conclude that HRPAP20 is a phosphoprotein that is required for proliferation and survival of hormone-dependent tumor cells.

10530 The metabolic fragility of cancer cells is magnified with the preferential utilization of glycolysis that metabolizes glucose inefficiently rather than oxidative phosphorylation (the “Warburg effect”) and from impaired mechanisms of survival. In fact, one pathway by which cells survive metabolic stress is thought to be autophagy, a catabolic process of organelle digestion, which creates ATP during periods on nutrient limitation. Remarkably, autophagy is often impaired in human prostate cancers, due to either activation of the PI-3 kinase/Akt/mTOR pathway, which normally inhibits autophagy, or through allelic loss of the essential autophagy gene beclin1. This suggests that prostate cancers may be susceptible to metabolic stress that can be exploited therapeutically. Using immortalized mouse epithelial prostate cells, as well as PC-3 and LNCaP cell lines, we demonstrated the cytotoxic effect of 2-deoxyglucose (2DG), as an inhibitor of glycolysis. We found that 2DG induced membrane translocation in cells characteristic of autophagy using a transfected pEGFP-LC3 autophagy marker construct. We then demonstrated that induction of autophagy was dependent on Beclin1 expression in these cell models using Beclin1 siRNA. Based on these data, we initiated a phase I/II clinical trial with 2DG in patients with advanced malignancies and prostate cancer, which is now ongoing. In an effort to develop markers of autophagy for assessment in a clinical trial, we stained a human prostate TMA (>35 patient cores) for Beclin1 by IHC. Beclin1 staining was increased in tumor tissue compared to normal tissue. Staining was imaged and digitized using a 40x volume scan on a high- throughput MedMicro whole slide scanner. The imaged specimens were stored in multi-tiled TIFF format on a redundant array of independent devices (RAID); staining will be analyzed using a color decomposition algorithm and compared to tumor characteristics as we have previously done (Foran DJ et al., IEEE Trans Inf Technol Biomed, 8:89–96, 2004). These data further support the rationale to inhibit metabolism in cancer and the potential importance of autophagy with metabolic approaches to therapy. Further imaging assessment and analysis of markers of autophagy such as Beclin1 are ongoing. No significant financial relationships to disclose.