LETTERS High-density array comparative genomic hybridization (CGH) 1 showed amplification of chromosome 1q22 centered on the RAB25 small GTPase 2 , which is implicated in apical vesicle trafficking 3 , in approximately half of ovarian and breast cancers. RAB25 mRNA levels were selectively increased in stage III and IV serous epithelial ovarian cancers compared to other genes within the amplified region, implicating RAB25 as a driving event in the development of the amplicon. Increased DNA copy number or RNA level of RAB25 was associated with markedly decreased disease-free survival or overall survival in ovarian and breast cancers, respectively. Forced expression of RAB25 markedly increased anchorage-dependent and anchorage-independent cell proliferation, prevented apoptosis and anoikis, including that induced by chemotherapy, and increased aggressiveness of cancer cells in vivo. The inhibition of apoptosis was associated with a decrease in expression of the proapoptotic molecules, BAK and BAX, and activation of the antiapoptotic phosphatidylinositol 3 kinase (PI3K) and AKT pathway, providing potential mechanisms for the effects of RAB25 on tumor aggressiveness. Overall, these studies implicate RAB25, and thus the RAB family of small G proteins, in aggressiveness of epithelial cancers. Ovarian cancer remains the fifth most frequent cause of cancer death in women. An improved understanding of the genetic aberrations in ovarian cancer may identify new etiologic, prognostic or therapeutic targets that can improve patient management. Several genes located at sites of DNA copy number aberrations in ovarian cancer, including PIK3CA 4 , ERBB2 (ref. 5), MYC 6 , EEF1A2 (ref. 7), AKT2 (ref. 8) and NCOA3 (ref. 9), have been implicated in the pathophysiology of ovar- ian cancer. Importantly, chromosome CGH analyses reveal other regions of recurrent abnormality in ovarian cancer, which may encode additional genes contributing to tumor behavior 10–12 . In par- ticular, chromosome 1q is frequently increased in copy number in ovarian 11,12 and breast 13 cancers and Wilms 14 tumors; a high relapse rate in invasive ductal breast carcinomas 13 and Wilms tumors 14 are associated with increased DNA copy number on chromosome 1q, but the driver of the regional copy number increase at 1q has not been identified. Using array CGH 1 to more precisely define region(s) of recurrent copy number increase on chromosome 1q, we delineated an increase (at least 1.3-fold) in DNA copy number in a 1.1-Mb region located on chromosome 1q22 in 28 of 52 (54%) of advanced serous epithelial ovarian cancers (Fig. 1a). Notably, ovarian cancer patients with ele- vated 1q22 (at least 1.5 copies of RAB25) either did not enter a disease- free state following surgery and chemotherapy or showed very short disease-free survival, implicating gene(s) in this region as potential oncogenes regulating the behavior of ovarian cancers (Fig. 1b). The minimal region of copy number increase on 1q22 encompassed the region from position 152,377,895 to 153,495,551, which contains a total of 34 genes, including 22 known genes and 12 hypothetical pro- teins based on the July 2003 human reference sequence. Based on expression levels from our microarray data sets 15 , the Gene Expression Omnibus and the Stanford Microarray Database, we eliminated 18 can- didates that did not show a significant difference in RNA levels between normal ovarian epithelium (NOE) and ovarian cancers. We analyzed mRNA expression levels of the remaining 16 open reading frames located in this region using real-time quantitative PCR to identify potential drivers of the copy number increase at 1q22. Although mRNA levels of several of the genes in this region were modestly elevated in a fraction of ovarian cancers as compared to NOE or benign ovarian tumors, RAB25 mRNA levels were markedly increased in 55 of 62 (88.7%) of ovarian cancers (P < 0.001; Fig. 1c,d). This observation was confirmed in an independent ovarian cancer data set 16 wherein RAB25 transcript levels were increased in 35 of 44 (80%) ovarian cancer sam- ples compared to NOE. The increase in RAB25 expression was stage dependent, with stage III and stage IV cancers showing higher levels (P < 0.01; Fig. 1d) than early stage cancers, suggesting a potential role of RAB25 in tumor progression. Sequencing of the open reading frame in 8 ovarian cancer samples did not identify mutations in RAB25, suggest- ing that mutation of RAB25 rarely or never occurs in ovarian cancer. Linear regression analysis of 21 epithelial ovarian cancers for which both CGH and expression levels were available showed a direct rela- tionship between copy number and expression of RAB25 (Fig. 1e) with 1 Department of Molecular Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA. 2 Lawrence Berkley National Laboratory, Berkeley, California 94720, USA. 3 Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia V6H 3V5, Canada. 4 Department of Pathology, 6 Department of Gynecological Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas, 77030, USA. 5 Department of Obstetrics and Gynecology, University of California, San Francisco, San Francisco, California 94143, USA. 7 Department of Obstetrics and Gynecology, School of Medicine, New York University, New York, New York 10016, USA. Correspondence should be addressed to G.B.M. (gmills@mdanderson.org). Published online 24 October 2004; doi:10.1038/nm1125 The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers Kwai Wa Cheng 1 , John P Lahad 1 , Wen-lin Kuo 2 , Anna Lapuk 2 , Kyosuke Yamada 2 , Nelly Auersperg 3 , Jinsong Liu 4 , Karen Smith-McCune 5 , Karen H Lu 6 , David Fishman 7 , Joe W Gray 2 & Gordon B Mills 1 NATURE MEDICINE VOLUME 10 | NUMBER 11 | NOVEMBER 2004 1251 © 2004 Nature Publishing Group http://www.nature.com/naturemedicine