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.

Autophagy suppresses tumorigenesis through elimination of p62.

Allelic 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-kappaB 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.

The xc- cystine/glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine.

PURPOSE: To determine whether the xc- cystine transporter could be a useful therapeutic target for small-cell lung cancer (SCLC). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Immortalized mouse epithelial cell models to study the role of apoptosis in cancer.

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.

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.

Autophagy suppresses tumor progression by limiting chromosomal instability.

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.

Sulfasalazine-induced cystine starvation: potential use for prostate cancer therapy.

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(c)(-) cystine transporter. METHODS: Cultures were evaluated for growth dependence on exogenous cystine, x(c)(-) 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(c)(-) transporter and were growth inhibited by SASP (IC(50)s: 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.

Identification of HRPAP20: a novel phosphoprotein that enhances growth and survival in hormone-responsive tumor cells.

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 (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 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 (P < 0.05) 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 (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.