Lentiviral vector‐mediated insertional mutagenesis screen identifies genes that influence androgen independent prostate cancer progression and predict clinical outcome

Abstract

Prostate cancer (PC) is the second leading cause of cancer related deaths in US men. Androgen deprivation therapy (ADT) improves clinical outcome, but tumors often recur and progress to androgen independent prostate cancer (AIPC) which no longer responds to ADT. The progression to AIPC is due to genetic alterations that allow PC cancer cells to grow in the absence of androgen. Here we performed an insertional mutagenesis screen using a replication-incompetent lentiviral vector (LV) to identify the genes that promote AIPC in an orthotopic mouse model. Androgen sensitive PC cells, LNCaP, were mutagenized with LV and injected into the prostate of male mice. After tumor development, mice were castrated to select for cells that proliferate in the absence of androgen. Proviral integration sites and nearby dysregulated genes were identified in tumors developed in an androgen deficient environment. Using publically available datasets, the expression of these candidate androgen independence genes in human PC tissues were analyzed. A total of 11 promising candidate AIPC genes were identified: GLYATL1, FLNA, OBSCN, STRA13, WHSC1, ARFGAP3, KDM2A, FAM83H, CLDN7, CNOT6 and B3GNT9. Seven out the 11 candidate genes; GLYATL1, OBSCN, STRA13, KDM2A, FAM83H, CNOT6 and B3GNT6, have not been previously implicated in PC. An in vitro clonogenic assay showed that knockdown of KDM2A, FAM83H and GLYATL1 genes significantly inhibited the colony forming ability of LNCaP cells. Additionally, we showed that a combination of four genes, OBSCN, FAM83H, CLDN7 and ARFGAP3 could significantly predicted the recurrence risk in PC patients after prostatectomy (P=5.3×10-5).

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).…”

“…Typically, to identify RIS, the genomic DNA is obtained from tumors that have developed from transduced cancer cells in an insertional mutagenesis screen. The LTR-chromosomal junctions are then amplified using well-established approaches such as shuttle vector rescue [21,24], linear amplified mediated-PCR (LAM-PCR) [22,25] or high-throughput modified genomic sequencing-PCR (MGS-PCR) [23] as described below. These candidate tagged driver genes can be oncogenes that require only a single retroviral insertion to be activated, or a tumor suppressor gene that requires only a single viral insertion to cause loss of function of one allele resulting in haplo-insufficiency.…”

“…A γRV shuttle vector approach identified previously known ( WWTR1, RIN1 ) and also novel ( SHARPIN) breast cancer metastasis genes [21]. However, despite these successes shuttle vector rescue approach can lack sensitivity to detect RIS in tumors with low vector copy number [23]. …”

“…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].…”