The 19q12 amplicon can therefore be mapped to a region spanning 34

The 19q12 amplicon can therefore be mapped to a region spanning 34.435.4 of chromosome 19 and involving 5 annotated genes, each of which is over-expressed in OVCAR-3. == Cells lines with amplification at 19q12 are specifically sensitive toCCNE1knockdown == Short interfering RNA (siRNA) were used to knockdown the expression of the seven genes in and BABL adjacent to the high-level 19q12 Px-104 amplicon in OVCAR-3 and SK-OV-3 plusGAPDHand non-silencing (NS) controls. presence of a cooperative mutational network. These observations have implications for the application of targeted therapies inCCNE1dependent ovarian cancers. == Introduction == Advanced stage serous tumors account for the majority of invasive ovarian cancers and despite a generally good initial response to cytoreductive surgery and platinum-based chemotherapy, most women face a high risk of recurrence and poor long-term survival[1]. Platinum-based agents, such as cisplatin and carboplatin, are toxic to dividing cells due to the formation of DNA adducts that result in double strand breaks, activating DNA damage-mediated apoptotic signals[2]. Response to chemotherapy is, however, difficult to predict and there are currently no predictive biomarkers for serous ovarian cancers in clinical use. We have previously mapped a region of 19q12 amplification associated with treatment-resistant serous ovarian tumors by performing a genome-wide survey of copy number change[3]. These findings were consistent with previous reports of amplification being associated with poor overall survival[4],[5]. Similarly, recurrent amplification of 19q12 has been reported in a variety of cancers including esophageal[6], gastric[7], lung[8]and endometrial tumors[9]. The 19q12 amplification is a high-level focal amplification that targets a cluster of only several genes Px-104 on chromosome 19.CCNE1(Cyclin E) has previously been suggested as the target of amplification in ovarian cancer[4],[10],[11], however a systematic analysis of known genes within the amplicon has not been performed. Furthermore, whilstCCNE1amplification likely provides an oncogenic stimulus through activation of the cell cycle, it is not obvious how it may contribute to primary chemotherapy resistance. For example, over-expression ofCCNE1 in vitrorenders ovarian cancer cells more sensitive to platinum agents, presumably due to increased proliferation[12]. It is possible that the biological consequence of 19q12 amplification is not limited to over-expression ofCCNE1, and that other genes in the amplicon contribute to tumor growth or progression. Furthermore, other co-existing mutational events elsewhere in the cancer genome may cooperate or enhance the oncogenic effect ofCCNE1over-expression. We performed an siRNA knockdown screen of all annotated genes within and immediately flanking the 19q12 amplicon in ovarian cancer cell lines with or without regional amplification. We foundCCNE1to be the only gene target within the amplicon that reduced cell viability in the amplicon-containing OVCAR-3 cell line after siRNA knockdown.CCNE1knockdown induced cell cycle arrest and apoptosis, while also impairing clonogenic survival after cisplatin treatment, despite Px-104 increasingin vitrodrug resistance in a short-term cytotoxicity assay. In a disease setting, these results suggest that treatment failure inCCNE1amplified tumors may relate to rapid repopulation of the tumor after chemotherapy and not cellular drug resistance specifically. We also foundTPX2amplification and over-expression to be significantly correlated withCCNE1copy number status implying the presence of a cooperative mutational network between these genes. == Results == == Focal amplification of 19q12 is common to various tumor types == We first sought to compare the minimal region of chromosomal gain Px-104 at 19q12 across multiple tumor types. We obtained data from SNP-based high-resolution copy number studies including 15 tumor types[9],[13]for a comparison with our findings[3](Figure 1A). Minimal targeted regions of amplification were defined by GISTIC, an analysis tool that assesses the statistical significance of copy number events based on frequency and amplitude[14]. Significant amplification of 19q12 was present in one third of the cancer types analyzed. Of the tumor types with 19q12 amplification, approximately 25% of individual samples showed copy number gain, except for endometrial tumors where a higher frequency was observed (45%)[9]. Minimal amplicon boundaries were found to target a region less than 2 Mb in size, centered at approximately at 35.0 Mb on Px-104 chromosome 19. In both ovarian tumor data sets analyzed[3],[13]the minimal mapped region of gain incorporated the same five genes (POP4,PLEKHF1,C19orf12, CCNE1 and C19orf2), with similar overlapping regions detected in endometrial and breast tumors. In contrast, the minimal region mapped in non-small cell lung tumors incorporated onlyCCNE1while a broad region was mapped in esophageal tumors (9.5 Mb),.