A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene

Abstract

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.[Keywords: Metastasis suppressor gene; melanoma; GAS1; RNAi screen; three-dimensional cell culture system] Supplemental material is available at http://www.genesdev.org. Received July 10, 2008; revised version accepted September 8, 2008. Metastatic dissemination of a primary tumor to a secondary site is the major cause of deaths from solid tumors (for review, see Gupta and Massague 2006;Nguyen and Massague 2007). The progression to metastasis involves a series of discrete steps, commonly known as the metastatic cascade, which minimally includes: invasion of the tumor border, intravasation into vascular structures, survival during transport to the secondary site, extravasation, and colonization of the secondary site (for review, see Gupta and Massague 2006;Steeg 2006). The complex process of metastasis is controlled by multiple genes that either increase or decrease metastatic potential (Berger et al. 2005;Nguyen and Massague 2007). Although many genes have been identified that promote metastasis, a relatively small number of metastasis suppressor genes have been documented (Rinker-Schaeffer et al. 2006). This is due, at least in part, to a lack of experimental approaches for the systematic identification of genes that specifically inhibit metastasis.Three-dimensional (3D) cell culture systems comprising cancer cell lines grown in matrices of collagen and fibrin provide an ex vivo model system for studying tumor cell invasion and expansion into the extracellular matrix (Doillon et al. 2004). Using this bicomposite gel technology system, it has been shown that a vari...

Lentiviral transfer of an inducible transgene expressing a soluble form of Gas1 causes glioma cell arrest, apoptosis and inhibits tumor growth

“…31,32 Another strategy to obtain a more complete and homogeneous distribution of the therapeutic agent is to use soluble molecules that can be released from cells expressing the transgene, 33 and in this manner more freely diffuse within the tumor, thus enhancing the beneficial effect of the treatment. Gas1 is a molecule that can induce cell arrest and apoptosis of different tumor cells, [34][35][36] including glioma cell lines and human primary gliomas. [7][8][9][10] Bioinformatics analysis uncovered significant structural homology between Gas1 and GFRas.…”

Effects of Gas1 on gliomas: a review on current preclinical studies

“…[126,127] Additionally the loss of GAS1 increases the metastasis of breast, prostate and gastric cancers. [124,128] On the other hand, GAS1 has been proposed as a molecular marker for prostate cancer. [129] The mechanisms that regulate the expression of GAS1 in gliomas have not been identified yet; however these tumors express several transcription factors that negatively regulate GAS1 such as c-Myc and v-Src.…”

Microenvironmental Influences on Metastasis Suppressor Expression and Function during a Metastatic Cell’s Journey

“…Gas1 encodes a GPI-anchored membrane protein [129] that is a cell cycle inhibitor (G 0 to S phase transition) since Gas1 over-expressing cells do not incorporate BrdU. Gas1 was first identified as a metastatic suppressor in a genome wide shRNA screen in B16-F10 melanoma cells [130]. Gas1 plays a role in several tumors, including colorectal, prostate, bladder, and melanoma.…”