Gene therapy for prostate cancer

By Live Dr - Mon Jul 04, 12:29 pm

Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths in men in the US (Damber and Aus, 2008). It is estimated that 217,730 new cases of prostate cancer will have been diagnosed in 2010 alone. The therapeutic options for patients with prostate cancer include radiotherapy and treatment with cytotoxic chemotherapeutic agents. Despite a palliative benefit, these approaches do not engender a long-term beneficial effect on the overall survival of patients. Late stage prostate cancer patients may benefit from hormone therapy, which removes a primary factor mediating tumor growth, male hormones (androgens) (Di Lorenzo and De Placido, 2006; Sternberg, 2002). Unfortunately, after a few years most patients become non-responsive to this treatment, resulting in uncontrolled disease and patient death. In these contexts, there is a pressing need to develop more effective therapeutic approaches for end-stage prostate cancer patients and genetic therapies represent promising approaches for the treatment of this neoplasm.

The prostate gland is not vital for survival and is accessible by ultrasound, potential therapeutic genes can be injected directly into the primary tumor, tissue-specific promoters exist that can target therapeutic gene expression or viral replication uniquely in the prostate gland, and disease progression can be monitored by measuring prostate-specific antigen (PSA) (Anderson, 1998; Gopalkrishnan et al., 2001; Mabjeesh et al., 2002). Since prostate cancer is commonly a relatively slow-growing disease, it may be necessary to use repeated gene therapy approaches, with single or multiple genes, over the lifespan of the patient. Adenoviruses (Ads) are the most commonly used vehicle for gene therapy approaches because the technology for virus production at high titers is established and Ad structure, genome, and replication cycle are well characterized thus facilitating the engineering of these viruses for therapeutic purposes. Although promising in vitro and in vivo results have been achieved using Ad vectors, administering unmodified serotype 5 Ads (Ad5) for gene delivery faces a number of clinical limitations. These include down-regulation of Coxsackie-adenovirus (CAR) receptors in cancer cells resulting in failure to transduce the majority of tumor cells by Ad5 (Anderson, 1998; Haviv et al., 2002; Mabjeesh et al., 2002).

An effective systemic gene delivery method is required to ensure safe and targeted delivery as administration of Ad-based gene therapy results in hepatic sequestration of the Ad so that very little reaches the target tumor tissue and clearance of the Ad from the circulation by the immune system. To develop safe and more efficient systemic delivery systems, we are focusing on ultrasound (US) contrast agents (microbubbles) (UTMD: ultrasound-targeted microbubble destruction) to enhance delivery of molecules in vivo. This review will summarize unique and novel aspects of effective gene therapy for prostate cancer that offers significant promise for moving basic science studies in the laboratory into the clinic to hopefully develop a ‘cure’ for advanced prostate cancer.

Using subtraction hybridization combined with induction of cancer cell terminal differentiation, our laboratory cloned mda-7/IL-24 (Jiang et al., 1995), a novel member of the IL-10-related cytokine gene family (Dash et al., 2010a; Gupta et al., 2006; Pestka et al., 2004; Sauane et al., 2003). Subsequent experiments documented that mda-7/IL-24 had nearly ubiquitous antitumor properties in vitro and in vivo, which led to its successful entry into the clinic in an unprecendented 5 years after discovery where acceptable safety and clinical efficacy, when administered by Ad (Ad.mda-7; INGN 241), has been demonstrated in Phase I clinical trials in humans with advanced carcinomas and melanomas (Cunningham et al., 2005; Eager et al., 2008; Emdad et al., 2009; Fisher, 2005; Fisher et al., 2003; Fisher et al., 2007; Lebedeva et al., 2007; Lebedeva et al., 2005; Tong et al., 2005). Its mechanism of action involves preferential induction of autophagy and apoptosis in prostate cancer without exerting harmful effects to normal cells (Bhutia et al., 2010; Dash et al., 2010b; 2010c; Gao et al., 2008; Miyahara et al., 2006; Sarkar et al., 2007a; Sauane et al., 2003; Su et al., 2005a; Su et al., 2001a; Su et al., 2006; Yacoub et al., 2008). Additional targets of mda-7/IL-24 action have also been investigated supporting its considerable potential as a gene therapeutic for cancer. Forced mda-7/IL-24 expression in cancer cells inhibits angiogenesis, stimulates an anti-tumor immune response (Gao et al., 2008; Miyahara et al., 2006), sensitizes cancer cells to radiation-, chemotherapy-, and antibody-induced killing (McKenzie et al., 2004; Su et al., 2005a; Su et al., 2001a; Su et al., 2006), and elicits potent “antitumor bystander activity” (Chada et al., 2004; Sauane et al., 2008; Su et al., 2001; Su et al., 2005a). MDA-7/IL-24 protein induces a sustained ER stress response as evidenced by expression of ER stress markers (BiP/GRP78, GADD153, GRP94, and phospho-eIF2α) and production of reactive oxygen species (ROS), indicating that both intracellular and secreted proteins activate similar signaling pathways to induce apoptosis (Sauane et al., 2008).

As prostate cancer requires repeated gene therapy approaches, the use of replication defective Ads to administer therapeutic and cytotoxic genes and conditionally replication competent Ads (CRAds) to selectively induce cytolysis in prostate tumor cells represent feasible treatment options (Anderson, 1998; Mabjeesh et al., 2002). Using subtraction hybridization we cloned a novel rodent gene, progression elevated gene-3 (PEG3), in the context of tumor progression in transformed rat embryo cells (Su et al., 1997). PEG-3 (i) displays elevated expression as a function of oncogenic transformation (by diverse oncogenes) (Park et al., 2001; Su et al., 1997), (ii) induces an aggressive cancer phenotype without promoting transformation when expressed in normal cells (Emdad et al., 2005a; Emdad et al., 2005b; Park et al., 2001; Su et al., 1999; Su et al., 2002; Su et al., 1997), and (iii) is regulated by a gene promoter (PEG-Prom) shown to display elevated expression in both rodent and human tumors (including prostate carcinomas) with negligible expression in normal cells (including human prostate epithelium) (Park et al., 2001; Su et al., 2000; Su et al., 2001b; Su et al., 2005b).

To test the cancer specificity of the PEG-Prom for tumor imaging in vivo, we used a firefly luciferase reporter PGL3-PEG-prom-Luc (pPEG-Luc) (Bhang et al., 2011; Su et al., 2005b). After confirmation of the presence of metastatic nodules in the lung by computed tomography (CT) at 4-6 weeks after intravenous administration of the human malignant breast cancer cell line (MDA-MB-231) or melanoma (MeWo), animals (athymic nude mice) received an intravenous dose of pPEG-Luc/PEI polyplex (PolyplusTransfection). Forty-eight hours after plasmid DNA (pDNA) delivery, PEG-Prom-driven gene expression was assessed by bioluminescence imaging (BLI). Quantification of the BLI signal intensity from the thoracic cavity, which represents Luc expression mainly in lung, showed significantly higher PEG-Prom activity in the model of melanoma or breast cancer metastasis as compared to controls, which did not show a detectable signal (Bhang et al., 2011). Additionally, it was possible to use repeat administrations of pPEG-Luc/PEI in tumor-bearing animals, which permitted us to follow growth and development of new metastatic lesions over time (Bhang et al., 2011).

Figure 1. Schematic representation of cancer terminator viruses (CTVs). In the CTVs the PEG-Prom drives the expression of E1A and E1B genes thus ensuring cancer-specific replication while the CMV-Prom regulates the expression of either mda-7/IL-24 or IFNγ in the E3 region of the Ad. These conditionally replication competent adenoviruses (CRCA) do not harm normal cells but induce oncolysis by Ad replication and diverse tumor-suppressor effects of the expressed transgene. (Reproduced with permission of the publisher, from Sarkar et al., 2005).Figure 1. Schematic representation of cancer terminator viruses (CTVs). In the CTVs the PEG-Prom drives the expression of E1A and E1B genes thus ensuring cancer-specific replication while the CMV-Prom regulates the expression of either mda-7/IL-24 or IFNγ in the E3 region of the Ad. These conditionally replication competent adenoviruses (CRCA) do not harm normal cells but induce oncolysis by Ad replication and diverse tumor-suppressor effects of the expressed transgene. (Reproduced with permission of the publisher, from Sarkar et al., 2005).

Considering the cancer-specific expression property of the PEG-Prom, we constructed a bipartite CRAd [called a Cancer Terminator Virus (CTV)] in which the expression of E1A and E1B genes of Ad, necessary for replication, is regulated by the PEG-Prom (Figure 1) (Sarkar et al., 2005). This novel biCRAd (CTV) also expressed mda-7/IL-24 in the E3 region (Ad.PEG-E1A-mda-7). To test our hypothesis of cancer-specific activity and therapeutic effectiveness of the Ad.PEG-E1A-mda-7, experiments were done in three prostate cancer cell lines, androgen-nonresponsive DU-145 and PC-3 cells, and androgen-responsive LNCaP cells and their Ad.mda-7-resistant variants (i.e., DU-145-Bcl-2, DU-145-Bcl-xL, PC3-Bcl-2 and PC-3-Bcl-xL, DU-145, and PC3 that stably expresses Bcl-2 and Bcl-xL). As a control, P69 normal prostate epithelial cells immortalized by SV40 T/t antigen were used (Sarkar et al., 2007b). From Western blot analysis it was evident that infection of normal immortal human P69 prostate epithelial cells with Ad.CMV-E1A (CRAd where E1A is driven under CMV promoter control) or Ad.CMV-E1A-mda-7 (bipartite CRAd where both E1A and mda-7/IL-24 are driven by the CMV promoter) but not with Ad.PEG-E1A (CRAd where E1A is driven under CMV promoter control) or Ad.PEG-E1A-mda-7 (CTV) resulted in production of E1A proteins; whereas in prostate cancer cells, infection with all four replication-competent Ads generated E1A proteins. In P69 cells, infection with Ad.CMV-E1A-mda-7 or Ad.CMV-mda-7 resulted in MDA-7/IL-24 protein production, whereas infection with Ad.PEG-mda-7 or Ad.PEG-E1A-mda-7 (CTV) resulted in barely detectable levels of MDA-7/IL-24 protein production. In prostate cancer cells, infection with Ad.CMV-mda-7, Ad.PEG-mda-7, Ad.CMV-E1A-mda-7, or Ad.PEG-E1A-mda-7 (CTV) generated significant MDA-7/IL-24 production. No MDA-7/IL-24 protein production could be detected in uninfected control cells or following infection with Ad.vec, Ad.CMV-E1A, or Ad.PEG-E1A (Sarkar et al., 2007b). These findings document that the PEG-Prom facilitates cancer cell-selective replication of Ads and concomitant mda-7/IL-24 expression. The effects of the engineered Ads on cell viability and apoptosis were evaluated in the various prostate cell lines. In P69 cells, infection with only Ad.CMV-E1A or Ad.CMV-E1A-mda-7, but not with Ad.PEG-E1A, Ad.CMV-mda-7, Ad.PEG-mda-7, or Ad.PEG-E1A-mda-7 (CTV), induced profound growth inhibition (Sarkar et al., 2007b). In contrast, in all prostate cancer cells, both parental and mda-7/IL-24-resistant, Ad.CMV-E1A-mda-7, Ad.PEG-E1A-mda-7 (CTV), Ad.CMV-E1A, and Ad.PEG-E1A infection resulted in significant growth inhibition, indicating potential therapeutic applications of the CTV in prostate cancer patients frequently showing Bcl-2 and Bcl-xL over-expression. Replication of Ad.PEG-E1A-mda-7 results in robust amounts of mda-7/IL-24 production resulting in a potent antitumor immune response. Moreover, in vivo assays in established melanoma, breast cancer, and therapy-resistant prostate cancer xenografts in athymic nude (immunocompromized) mice showed that injection of Ad.PEG-E1A-mda-7 completely eradicated not only the primary tumors but also distant tumors (Sarkar et al., 2007b; Sarkar et al., 2008; Sarkar et al., 2005).

Therapy of cancer using Ads has been restricted for a number of reasons, particularly when utilizing systemic administration routes. These include: limitations in tumor transduction efficiency that are frequently mediated by a reduction in the number of CAR that regulate Ad entry into cancer cells (Paul et al., 2008; Tsuruta et al., 2007); sequestering of Ads in the liver limiting virus delivery to disseminated tumors (Koizumi et al., 2007); neutralization of viruses by the immune system (Koizumi et al., 2007); and absence of broad-spectrum anti-tumor agents capable of selectively killing cancer cells and provoking elimination of disseminated metastatic tumors through potent “bystander’“anti-tumor activity (Fisher, 2005; Sarkar et al., 2007b; Sarkar et al., 2008; Sarkar et al., 2005). We have attempted to overcome these barriers to achieve effective systemic therapy of cancer using a number of innovative approaches. We have modified the infectivity tropism of Ad by producing chimeric viruses containing regions of both Ad type 5 and Ad type 3, Ad.5/3, which allow CAR-independent transduction of tumor cells. Ad.5/3 shows superiority in transducing genes in a CAR-independent manner in prostate cancer and is effective in cells with both low and high CAR receptors (Dash et al., 2010b; Hamed et al., 2010; Park et al., 2010; Yacoub et al., 2010). To prevent trapping of Ads in the liver and elimination of viruses by the immune system, we have developed a novel approach in which Ads, both replication incompetent and conditionally replication competent, are incorporated in a perfluorocarbon microbubble that is treated with complement (to inactivate and mask viruses on the surface of the microbubble from the immune system) and then administered systemically and released in the tumor microenvironment through ultrasound, i.e., the UTMD approach (Greco et al., 2010). Early phase clinical studies suggest that mda-7/IL-24 may be an effective agent for gene therapy of primary and metastatic cancers (Cunningham et al., 2005; Tong et al., 2005). This novel IL-10-family member cytokine selectively kills cancer cells without harming normal cells, displays potent systemic “bystander antitumor effects, inhibits tumor angiogenesis, stimulates the immune system resulting in long term antitumor effects, and potentiates the therapeutic activity of currently used modalities of therapy, including radiation, chemotherapy, and monoclonal antibodies (Emdad et al., 2007; Gupta et al., 2008; Lebedeva et al., 2007; Su et al., 2006; Dash et al., 2010b). We have now generated Ad.5/3-CTV (tropism modified CTV), which we intend to evaluate this virus for delivery into the prostate of immunocompetent prostate cancer transgenic mice (Hi-Myc) by the UTMD technique. Successful completion of our proposed studies using tropism-modified viruses, including the CTV, and the UTMD technology will provide a direct path for translation into the clinic for potentially improving the therapy for advanced prostate cancer and other difficult to treat neoplastic diseases.


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