Sequencing of candidate chromosome instability genes in endometrial cancers reveals somatic mutations in ESCO1, CHTF18, and MRE11A.

Most endometrial cancers can be classified histologically as endometrioid, serous, or clear cell. Non-endometrioid endometrial cancers (NEECs; serous and clear cell) are the most clinically aggressive of the three major histotypes and are characterized by aneuploidy, a feature of chromosome instability. The genetic alterations that underlie chromosome instability in endometrial cancer are poorly understood. In the present study, we used Sanger sequencing to search for nucleotide variants in the coding exons and splice junctions of 21 candidate chromosome instability genes, including 19 genes implicated in sister chromatid cohesion, from 24 primary, microsatellite-stable NEECs. Somatic mutations were verified by sequencing matched normal DNAs. We subsequently resequenced mutated genes from 41 additional NEECs as well as 42 endometrioid ECs (EECs). We uncovered nonsynonymous somatic mutations in ESCO1, CHTF18, and MRE11A in, respectively, 3.7% (4 of 107), 1.9% (2 of 107), and 1.9% (2 of 107) of endometrial tumors. Overall, 7.7% (5 of 65) of NEECs and 2.4% (1 of 42) of EECs had somatically mutated one or more of the three genes. A subset of mutations are predicted to impact protein function. The co-occurrence of somatic mutations in ESCO1 and CHTF18 was statistically significant (P = 0.0011, two-tailed Fisher's exact test). This is the first report of somatic mutations within ESCO1 and CHTF18 in endometrial tumors and of MRE11A mutations in microsatellite-stable endometrial tumors. Our findings warrant future studies to determine whether these mutations are driver events that contribute to the pathogenesis of endometrial cancer.

Predisposition to cancer caused by genetic and functional defects of mammalian Atad5.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

A unique spectrum of somatic PIK3CA (p110alpha) mutations within primary endometrial carcinomas.

PURPOSE: The goal of this study was to comprehensively define the incidence of mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC) and nonendometrioid endometrial cancer (NEEC). EXPERIMENTAL DESIGN: We resequenced all coding exons of PIK3CA and PTEN, and exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations were confirmed by sequencing matched normal DNAs. The biochemical properties of a subset of novel PIK3CA mutations were determined by exogenously expressing wild type and mutant constructs in U2OS cells and measuring levels of AKT(Ser473) phosphorylation. RESULTS: Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of 66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons 1-7 and half were in exons 9 and 20. The exons 1-7 mutations localized to the ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93, Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93, and Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E, -delP449-L455, and -E453K mutants led to increased levels of phospho-AKT(Ser473) compared to wild-type p110α. Overall, 62% of exons 1-7 PIK3CA mutants and 64% of exons 9-20 PIK3CA mutants were activating; 72% of exon 1-7 mutations have not previously been reported in endometrial cancer. CONCLUSIONS: Our study identified a new subgroup of endometrial cancer patients with activating mutations in the amino-terminal domains of p110α; these patients might be appropriate for consideration in clinical trials of targeted therapies directed against the PI3K pathway.

Ginger inhibits cell growth and modulates angiogenic factors in ovarian cancer cells.

BACKGROUND: Ginger (Zingiber officinale Rosc) is a natural dietary component with antioxidant and anticarcinogenic properties. The ginger component [6]-gingerol has been shown to exert anti-inflammatory effects through mediation of NF-kappaB. NF-kappaB can be constitutively activated in epithelial ovarian cancer cells and may contribute towards increased transcription and translation of angiogenic factors. In the present study, we investigated the effect of ginger on tumor cell growth and modulation of angiogenic factors in ovarian cancer cells in vitro. METHODS: The effect of ginger and the major ginger components on cell growth was determined in a panel of epithelial ovarian cancer cell lines. Activation of NF-kappaB and and production of VEGF and IL-8 was determined in the presence or absence of ginger. RESULTS: Ginger treatment of cultured ovarian cancer cells induced profound growth inhibition in all cell lines tested. We found that in vitro, 6-shogaol is the most active of the individual ginger components tested. Ginger treatment resulted in inhibition of NF-kB activation as well as diminished secretion of VEGF and IL-8. CONCLUSION: Ginger inhibits growth and modulates secretion of angiogenic factors in ovarian cancer cells. The use of dietary agents such as ginger may have potential in the treatment and prevention of ovarian cancer.

Genistein-induced apoptosis and autophagocytosis in ovarian cancer cells.

OBJECTIVE: Genistein, a naturally occurring isoflavenoid abundant in soy products, has anti-neoplastic activity in multiple tumor types. There are several mechanisms reported for genistein's anti-neoplastic activity. In the present study, we studied the mechanism of genistein-induced cell death in ovarian cancer cells. METHODS: The effect of genistein on the induction of apoptosis, autophagy, and inhibition of glucose uptake in ovarian cancer cells was determined. The effect of genistein on the expression of phosphorylated Akt was determined by immunoblotting. RESULTS: Genistein is cytotoxic to ovarian cancer cells. The mechanism of genistein-induced cell death includes both apoptosis and autophagy. Because autophagy is typically an adaptive response to nutrient starvation, we hypothesized that genistein could induce a starvation-like signaling response. We show here that genistein treatment results in caspase-independent cell death with hallmarks of autophagy. Genistein treatment dramatically inhibits glucose uptake in ovarian cancer cells, and methyl pyruvate, a cell-permeable 3-carbon substrate for oxidative phosphorylation and fatty acid synthesis, rescues cells from genistein-induced autophagy. In addition, genistein treatment results in reduced levels of phosphorylated Akt, which may contribute towards a mechanism to limit glucose utilization. CONCLUSIONS: Most conventional chemotherapeutic agents induce apoptotic cell death. Because genistein can induce both apoptotic and autophagic cell death, it has the potential to circumvent chemoresistance due to alterations in apoptotic signaling.

Expression of Bcl-xL in ovarian carcinoma is associated with chemoresistance and recurrent disease.

OBJECTIVE: The long-term survival of patients with epithelial ovarian cancer is limited by the emergence of tumor cells that are resistant to chemotherapy. We hypothesized that expression of Bcl-x(L), a homologue of Bcl-2 that confers protection from chemotherapy-induced apoptosis, may be predictive of patients' clinical response to treatment, and that treatment with chemotherapy may result in the selection of tumor cells that overexpress this protein. METHODS: We determined the expression of Bcl-x(L) in epithelial ovarian cancers from 28 patients at the time of initial staging laparotomy and in recurrent tumors in the same patients following treatment with platinum-based chemotherapy. The data were analyzed to determine whether Bcl-x(L) expression was predictive of clinical outcome. A2780 ovarian cancer cells were stably transfected with Bcl-x(L) or control plasmid. Chemotherapy-induced apoptosis in these cell lines was determined in vitro and in a xenograft model. RESULTS: Bcl-x(L) expression in primary tumors was associated with a significantly shorter disease-free interval as compared to patients whose tumors did not express Bcl-x(L) (1.6 months as compared to 7.7 months). We found that Bcl-x(L) expression conferred resistance to chemotherapy-induced apoptosis resulting from treatment with cisplatin, paclitaxel, topotecan, and gemcitabine in vitro. In a xenograft model, Bcl-x(L) expressing tumors continued to grow following treatment with cisplatin, paclitaxel, topotecan, and gemcitabine, in contrast to control tumors, which disappeared. CONCLUSION: These results portray an important role for Bcl-x(L) as a key factor associated with chemotherapy failure in the treatment of ovarian cancer.