A cluster of cooperating tumor-suppressor gene candidates in chromosomal deletions Wen Xuea,1,2, Thomas Kitzinga,1,3, Stephanie Roesslerb, Johannes Zubera, Alexander Krasnitza, Nikolaus Schultzc, Kate Revilla, Susann Weissmuellerd,3, Amy R. Rappaporta, Janelle Simona,e,3, Jack Zhanga, Weijun Luoa, James Hicksa, Lars Zendera,4, Xin Wei Wangb, Scott Powersa, Michael Wiglera,5, and Scott W. Lowea,e,3,5 aCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; bLaboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; cComputational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; dWatson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; and eHoward Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Contributed by Michael Wigler, April 12, 2012 (sent for review January 13, 2012) The large chromosomal deletions frequently observed in cancer the extent of chromosome 8p deletions from cancer genome genomes are often thought to arise as a “two-hit” mechanism in the datasets derived from array-based comparative genomic hybrid- process of tumor-suppressor gene (TSG) inactivation. Using a murine ization (aCGH) performed at Cold Spring Harbor Laboratory and model system of hepatocellular carcinoma (HCC) and in vivo RNAi, the Cancer Genome Atlas (TCGA) project, totaling 1411 primary we test an alternative hypothesis, that such deletions can arise from tumor samples and cell lines of HCC and breast, colon, and lung selective pressure to attenuate the activity of multiple genes. By cancers (Fig. 1A and Materials and Methods). According to these targeting the mouse orthologs of genes frequently deleted on hu- data, approximately half of these tumors harbor heterozygous man 8p22 and adjacent regions, which are lost in approximately deletions of human chromosome 8p, often encompassing a large half of several other major epithelial cancers, we provide evidence portion of or even the entire chromosome arm (Fig. 1A). Focusing suggesting that multiple genes on chromosome 8p can coopera- on 8p deletions in HCC, we noted that the most frequently deleted tively inhibit tumorigenesis in mice, and that their cosuppression region on 8p centered around the DLC1 gene (Fig. 1A), that in can synergistically promote tumor growth. In addition, in human HCC these deletions occur more frequently than those on chro- HCC patients, the combined down-regulation of functionally vali- mosome 17p encompassing TP53 (3). However, this chromosome dated 8p TSGs is associated with poor survival, in contrast to the arm contains other candidate TSGs (5–8), and indeed, most dele- down-regulation of any individual gene. Our data imply that large tions encompass regions adjacent to DLC1, including the whole cancer-associated deletions can produce phenotypes distinct from 8p22 cytoband or even the whole chromosome 8p arm (Fig. 1A). those arising through loss of a single TSG, and as such should be To identify a relevant genetic context in which to study 8p loss, considered and studied as distinct mutational events. we analyzed 197 primary HCCs (3, 9, 10) for copy number aber- rations associated with 8p deletions (Fig. 1B). Amplifications of cancer genomics | chromosome 8p deletion | RNAi screen chromosome 1q, 5p, 6p, and 8q (involving MYC) and losses in- cluding TP53 on 17p were significantly associated with 8p dele- ost cancer genomes contain large heterozygous deletions of tions (Fig. 1B). In addition, unsupervised hierarchical linkage Muncertain biological significance. Early studies on the tu- clustering of 197 primary HCCs revealed that they fell within 12 mor-suppressor genes (TSGs) RB and TP53 suggested that such groups, and that the 8p loss, 8q gain, and 17p loss cancers were clustered mainly within one subgroup that represents ∼40% of all deletions can arise as a single mechanism for loss of heterozygosity fi and, consequently, it is often assumed that they provide a “second- HCCs (Fig. 1C). These data con rm that genotypes involving hit” event to inactivate a single TSG (1). However, genomic MYC overexpression and TP53 loss are a valid genetic context in approaches have not conclusively identified a definitive TSG which to study candidate 8p TSGs. within some cancer-associated deletions, raising the possibility Chromosome 8p Harbors Multiple Genes That Inhibit Tumorigenesis in that they occur through genomic instability or selection for the Mice. To identify TSGs located on 8p, we tested whether RNAi- reduced activity of multiple genes. Even in chromosomal regions mediated suppression of various 8p genes would promote tu- fi “ ” fi where a bona de two-hit TSG has been identi ed, the large morigenesis in a mouse HCC model used previously for TSG deletions often associated with loss of heterozygosity reduce the discovery (11). Initially focusing in an unbiased approach on the dosages of neighboring genes, which could in principle contribute 8p22 region surrounding DLC1, we transduced pools of three fi to tumorigenesis in a haploinsuf cient manner. shRNAs individually targeting each mouse ortholog of all 21 Large deletions encompassing regions of chromosome 8p are very common in human tumors (2, 3) and often occur together with 8q gains encompassing MYC (4). Previously, we validated the 8p Author contributions: W.X., T.K., J. Zuber, L.Z., M.W., and S.W.L. designed research; W.X., gene DLC1, encoding a Rho GAP, as a TSG using a mouse model T.K., and J.S. performed research; J. Zuber, N.S., and A.R.R. contributed new reagents/ of hepatocellular carcinoma (HCC), confirming that its attenua- analytic tools; W.X., T.K., S.R., A.K., N.S., K.R., S.W., J. Zhang, W.L., J.H., X.W.W., and S.P. tion can serve as a driving oncogenic event (3). Although DLC1 is analyzed data; and W.X., T.K., M.W., and S.W.L. wrote the paper. at an epicenter of deletions, these deletions are frequently much The authors declare no conflict of interest. 1 larger and reduce the dosages of tens or hundreds of genes, often W.X. and T.K. contributed equally to this work. encompassing the entire 8p22 cytoband and beyond (2, 5, 6). In- 2Present address: Koch Institute for Integrative Cancer Research and Department of Bi- deed, multiple candidate TSGs have been proposed in the region ology, Massachusetts Institute of Technology, Cambridge, MA 02139. 3 (5–8). Here we explore the hypothesis that chromosome 8p dele- Present address: Department of Cancer Biology and Genetics, Sloan-Kettering Institute, New York, NY 10065. tions arise owing to selection for the attenuation of multiple genes. 4Present address: Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany and Department of Gastroenterology and Hepatology, University of Hannover Medical Results School, 30625 Hannover, Germany. Chromosome 8p Deletions Are Frequently Large and Co-Occur with 8q 5To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. fi Gains and 17p Loss. To better de ne regions affected by the 8p This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deletions frequently occurring in human cancers, we determined 1073/pnas.1206062109/-/DCSupplemental. 8212–8217 | PNAS | May 22, 2012 | vol. 109 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1206062109 Downloaded by guest on September 26, 2021 Fig. 1. Chromosome 8p deletion characteristics and co-occurring genomic aberrations. (A) Size and extent of chromosome 8p deletions (in blue) and amplifications (in red) from individual HCCs, breast cancers, colon cancers, and lung adenocarcinomas based on aCGH data analysis (Materials and Methods). The 8p22 cytoband is highlighted by a dashed line, with the organization of the 8p22 genes indicated on the right. (B) Chromosome 8p deletions co-occur with genomic aberrations in HCC, including amplifications (red) of 1q, 5p, 6p, and 8q and deletions (blue) of 17p. Fisher’s exact test was used for statistical calculations. (C) Unsupervised hierarchical clustering of genomic aberrations indicates 12 groups within the HCC dataset (n = 197). Occurrence of 8p deletion (dark red), 8q amplification (dark blue), and 17p deletion (dark orange) within the individual samples is highlighted below the dendrogram. − − annotated 8p22 protein-coding genes into p53 / liver progenitors promoted tumorigenesis over background, although with sub- overexpressing Myc, thereby approximating a relevant genetic stantial variability in tumor incidence and size (Fig. 2B and Table context in human HCC progression. The resulting cell populations S2). Five of these 14 genes demonstrated a statistically significant were then assessed for their tumorigenic potential (Fig. 2A). increase over background at the time of tumor harvest (Fig. 2B and Whereas the parental cells transduced with control shRNA were Table S2). only weakly tumorigenic, cells harboring three of the 8p22 pools, For further validation of the original candidates, we sub- including one pool containing shRNAs targeting Dlc1, sub- sequently tested the individual hairpins against the genes that stantially promoted tumorigenesis above background (Fig. 2A). showed significant tumor acceleration (i.e., Fgl1, Vps37a, Arh- fi The two other scoring shRNA pools targeted brinogen-like 1 gef10, Bin3, Bnip3l, Scara5, and Trim35) plus one more (Fbxo25) fi (Fgl1), a secreted protein of the brinogen family that is a candi- that, although not statistically significant, yielded large tumors date TSG in human HCC (12), and vacuolar