Replication-incompetent gammaretroviral and lentiviral vector-based insertional mutagenesis screens identify prostate cancer progression genes.

Victor M. Bii,Jonah D. Hocum,Casey P. Collins,Grant D. Trobridge

doi:10.18632/oncotarget.24503

Abstract

Replication-incompetent gammaretroviral (γRV) and lentiviral (LV) vectors have both been used in insertional mutagenesis screens to identify cancer drivers. In this approach the vectors stably integrate in the host cell genome and induce cancers by dysregulating nearby genes. The cells that contain a retroviral vector provirus in or near a proto-oncogene or tumor suppressor are preferentially enriched in a tumor. γRV and LV vectors have different integration profiles and genotoxic potential, making them potentially complementary tools for insertional mutagenesis screens. We performed screens using both γRV and LV vectors to identify driver genes that mediate progression of androgen-independent prostate cancer (AIPC) using a xenotransplant mouse model. Vector transduced LNCaP cells were injected orthotopically into the prostate gland of immunodeficient mice. Mice that developed tumors were castrated to create an androgen-deficient environment and metastatic tumors that developed were analyzed. A high-throughput modified genomic sequencing PCR (MGS-PCR) approach identified the positions of vector integrations in these metastatic tumors. OR2A14, FER1L6, TAOK3, MAN1A2, MBNL2, SERBP1, PLEKHA2, SPTAN1, ADAMTS1, SLC30A5, ABCC1, SLC7A1 and SLC25A24 were identified as candidate prostate cancer (PC) progression genes. TAOK3 and ABCC1 expression in PC patients predicted the risk of recurrence after androgen deprivation therapy. Our data shows that γRV and LV vectors are complementary approaches to identify cancer driver genes which may be promising potential biomarkers and therapeutic targets.

Highlights

Replication-incompetent retroviral vectors have the ability to stably integrate into the host cell genome and dysregulate nearby proto-oncogenes or tumor suppressor genes, leading to vector-induced cancer
The LV vector contains SIN long terminal repeats (LTRs) that have the enhancer and promoter elements deleted in the LTR U3 region
All established cultures had > 90% transduced cells after G418 selection as assessed by flow cytometry for enhanced green fluorescent protein (EGFP) (Figure 2A and 2B). This approach of transducing at a low multiplicity of infection (MOI) followed by selection to remove untransduced cells minimizes multiple vector insertions that can make it difficult to identify driver genes from passengers [14]

Summary

Introduction

Replication-incompetent retroviral vectors have the ability to stably integrate into the host cell genome and dysregulate nearby proto-oncogenes or tumor suppressor genes, leading to vector-induced cancer. Other vector-mediated mechanisms of genotoxicity have been reported [5, 6], enhancer-mediated activation of gene promoters is the most common [7, 8] Following these studies replicationincompetent retroviral vectors have been developed as a powerful tool to identify driver genes [9,10,11,12,13,14]. Replication-incompetent retroviral vectors have the ability to stably infect many mammalian cell types without causing secondary integrations that may impede the identification of the primary retroviral integration sites (RISs) This makes them a powerful tool to identify driver genes in different human cancers [14]

Methods

Results

Conclusion