A novel approach to identify driver genes involved in androgen-independent prostate cancer

Abstract

BackgroundInsertional mutagenesis screens have been used with great success to identify oncogenes and tumor suppressor genes. Typically, these screens use gammaretroviruses (γRV) or transposons as insertional mutagens. However, insertional mutations from replication-competent γRVs or transposons that occur later during oncogenesis can produce passenger mutations that do not drive cancer progression. Here, we utilized a replication-incompetent lentiviral vector (LV) to perform an insertional mutagenesis screen to identify genes in the progression to androgen-independent prostate cancer (AIPC).MethodsProstate cancer cells were mutagenized with a LV to enrich for clones with a selective advantage in an androgen-deficient environment provided by a dysregulated gene(s) near the vector integration site. We performed our screen using an in vitro AIPC model and also an in vivo xenotransplant model for AIPC. Our approach identified proviral integration sites utilizing a shuttle vector that allows for rapid rescue of plasmids in E. coli that contain LV long terminal repeat (LTR)-chromosome junctions. This shuttle vector approach does not require PCR amplification and has several advantages over PCR-based techniques.ResultsProviral integrations were enriched near prostate cancer susceptibility loci in cells grown in androgen-deficient medium (p < 0.001), and five candidate genes that influence AIPC were identified; ATPAF1, GCOM1, MEX3D, PTRF, and TRPM4. Additionally, we showed that RNAi knockdown of ATPAF1 significantly reduces growth (p < 0.05) in androgen-deficient conditions.ConclusionsOur approach has proven effective for use in PCa, identifying a known prostate cancer gene, PTRF, and also several genes not previously associated with prostate cancer. The replication-incompetent shuttle vector approach has broad potential applications for cancer gene discovery, and for interrogating diverse biological and disease processes.

Functional and physiopathological implications of TRP channels

“…Initial evidence of TRPM4 and TRPM5 involvement in cancer was limited to observations of alterations in their expression levels in CD5+ B-cell lymphomas and Wilms tumours and rhabdomyosarcomas (Beckwith-Wiedemann syndrome) respectively [170,171]. Later, changes in the expression level of TRPM4 were found to be important for the proliferation of the prostate cancer cells in the androgenindependent phase and cervical cancer-derived HeLa cells [172][173][174]. However the majority of evidence in the TRPM family concentrates on the TRPM7 channel, which is implicated in enhancing proliferative potential of the grade III breast tumour cells [175] as well as participating in gastric and pancreatic cancers [176][177][178], human hypopharyngeal squamous cell and nasopharyngeal carcinomas [179,180], prostate cancer [181] and enhances invasive properties of neuroblastoma [182][183][184].…”

Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

“…Pseudotyped retroviral vectors with VSV-G have broad tropism, and are stable allowing concentration to high titers by centrifugation. Thus, high titer VSV-G pseudotyped γRV (gammaretroviral) and LV (lentiviral) vectors have been used for prostate cancer and breast cancer screens that have utilized human cells in mouse xenotransplant models [21,23,24]. …”

“…These LTR modifications have reduced retroviral vector genotoxicity but self-inactivating vectors are still capable of dysregulating nearby genes when a strong internal promoter is used. For example, a self-inactivating LV vector with a strong internal spleen focus forming virus (SFFV) promoter was capable of dysregulating nearby genes in a prostate cancer mutagenesis screen [23,24] (Figure 3). In addition, a highly genotoxic γRV vector with a MLV-LTR and an internal SFFV promoter was used in a breast cancer mutagenesis screen [21] (Figure 3).…”

“…In addition, a highly genotoxic γRV vector with a MLV-LTR and an internal SFFV promoter was used in a breast cancer mutagenesis screen [21] (Figure 3). The use of either LTR-driven or self-inactivating retroviral vector has proven to be effective in studies performed to identify potential candidate driver genes in different types of cancer, such as prostate cancer [24], hepatocellular carcinoma [22], breast cancer [21,25] and pancreatic adenocarcinoma [25] as we describe later in this review.…”

“…Thus, due to the broad tropism of current replication-incompetent vectors mediated by envelope pseudotyping, mutagenesis screens can be performed for essentially any cancer type. To date, replication-incompetent vectors have been used to successfully identify cancer driver genes in breast, prostate, liver and pancreatic cancers [21,22,23,24,25]. Replication-incompetent retroviral vectors integrate into the host genome and dysregulate proto-oncogenes via well-known mutagenic mechanisms including enhancer-mediated activation of nearby gene promoters [26].…”