Expression profile of COL2A1 and the pseudogene SLC6A10P predicts tumor recurrence in high-grade serous ovarian cancer.

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.

Differing von hippel lindau genotype in paired primary and metastatic tumors in patients with clear cell renal cell carcinoma.

In sporadic clear cell renal cell carcinoma (CCRCC), the von Hippel Lindau (VHL) gene is inactivated by mutation or methylation in the majority of primary (P) tumors. Due to differing effects of wild-type (WT) and mutant (MT) VHL gene on downstream signaling pathways regulating angiogenesis, VHL gene status could impact clinical outcome. In CCRCC, comparative genomic hybridization analysis studies have reported genetic differences between paired P and metastatic (M) tumors. We thus sequenced the VHL gene in paired tumor specimens from 10 patients to determine a possible clonal relationship between the P tumor and M lesion(s) in patients with CCRCC. Using paraffin-embedded specimens, genomic DNA from microdissected samples (>80% tumor) of paired P tumor and M lesions from all 10 patients, as well as in normal tissue from 6 of these cases, was analyzed. The DNA was used for PCR-based amplification of each of the 3 exons of the VHL gene. Sequences derived from amplified samples were compared to the wild-type VHL gene sequence (GenBank Accession No. AF010238). Methylation status of the VHL gene was determined using VHL methylation-specific PCR primers after DNA bisulfite modification. In 4/10 (40%) patients the VHL gene status differed between the P tumor and the M lesion. As expected, when the VHL gene was mutated in both the P tumor and M lesion, the mutation was identical. Further, while the VHL genotype differed between the primary tumor in different kidneys or multiple metastatic lesions in the same patient, the VHL germline genotype in the normal adjacent tissue was always wild-type irrespective of the VHL gene status in the P tumor. These results demonstrate for the first time that the VHL gene status can be different between paired primary and metastatic tissue in patients with CCRCC.

Association of VEGF and VEGFR2 single nucleotide polymorphisms with hypertension and clinical outcome in metastatic clear cell renal cell carcinoma patients treated with sunitinib.

PURPOSE: Biomarkers that predict response or toxicity to antiangiogenic therapy are sought to favorably inform the risk/benefit ratio. This study evaluated the association of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) genetic polymorphisms with the development of hypertension (HTN) and clinical outcome in metastatic clear cell renal cell carcinoma (MCCRCC) patients treated with sunitinib. PATIENT AND METHODS: Sixty-three MCCRCC patients receiving sunitinib (50 mg 4/2) with available blood pressure (BP) data and germline DNA were retrospectively identified. A panel of candidate VEGF and VEGFR2 single nucleotide polymorphisms (SNPs) were evaluated for associations with the development of hypertension and clinical outcome. RESULTS: VEGF SNP -634 genotype was associated with the prevalence and duration of sunitinib-induced hypertension (as defined by systolic pressure ≥150 mmHg and/or diastolic pressure ≥90 mmHg) in both univariable analysis (P = .03 and .01, respectively) and multivariable analysis, which adjusted for baseline BP and use of antihypertension medication (P = .05 and .02, respectively). Patients with the GG genotype were estimated to have a greater likelihood of being hypertensive during treatment compared with patients with the CC genotype (odds ratio of 13.62, 95% confidence interval [CI] 3.71-50.04). No single VEGF or VEGFR SNPs were found to correlate with clinical outcome. However, the combination of VEGF SNP 936 and VEGFR2 SNP 889 were associated with overall survival after adjustment for prognostic risk group (P = .03). CONCLUSIONS: In MCCRCC patients treated with sunitinib, VEGF SNP -634 is associated with hypertension and a combination of VEGF SNP 936 and VEGFR2 SNP 889 genotypes is associated with overall survival.

CCL2 expression in primary ovarian carcinoma is correlated with chemotherapy response and survival outcomes.

CCL2, a chemokine, is expressed in normal human ovarian epithelium but down-regulated in ovarian adenocarcinomas. The association of CCL2 expression with chemotherapy response, invasion and survival outcomes was studied in patients with primary ovarian cancer (OC) and in ovarian cancer cell lines (OCCLs). Tumor specimens (>80% tumor) from patients with primary, advanced serous OC obtained at the time of cytoreductive surgery was used to isolate total RNA. The CCL2 gene expression evaluated by RT-PCR was investigated in relation to chemo-response/clinical outcomes in the OC patients and to sensitivity to cisplatin/paclitaxel in the OCCLs. In vitro invasion was measured by matrigel invasion and matrixmetallo-proteinase-9 (MMP-9) zymogram assays. Thirty-seven patients were included. In multivariable analyses that adjusted for the impact of debulking status, the CCL2 mRNA expression was correlated with objective complete response (p = 0.01), chemosensitivity (p = 0.04), and progression-free survival (PFS; p = 0.006). These findings were corroborated in vitro in the OCCLs. The cells expressing higher levels of CCL2 were more sensitive to paclitaxel and cisplatin as compared to those lines expressing lower levels of this chemokine. Up-regulation of CCL2 in the PAT-7 cell line further enhanced the response of these cells to paclitaxel (p = 0.0001) and led to decreased invasion (p = 0.0009). Increased ovarian tumoral expression of CCL2 is associated with improved chemoresponse and survival outcomes, and higher levels of CCL2 in ovarian cancer cell lines are associated with increased chemosensitivity and decreased invasion in vitro.

Vascular endothelial growth factor polymorphisms: role in response and toxicity of tyrosine kinase inhibitors.

Angiogenesis is central to the growth of normal tissues and tumors. Inhibiting this pathway has been a strategy for drug development for tumors not responsive to most agents used in chemotherapy. Notably, signaling mediated by vascular endothelial growth factor (VEGF) is a key target because aberrant signaling via this pathway is frequently associated with neoangiogenesis in tumors. The drug-discovery effort to blunt VEGF signaling has led to the approval of bevacizumab and several receptor tyrosine kinase inhibitors (TKIs) that have shown efficacy in the clinical management of breast, colorectal, lung, and kidney cancer. Understanding the genetic variability in VEGF and VEGF receptor has led to identifying genotypic variations (single nucleotide polymorphisms [SNPs]) associated with treatment outcome and toxicity. Notably, identification of SNPs in VEGF associated with angiogenesis inhibitor treatment-induced hypertension and outcome provides exciting opportunities for personalized medicine to improve outcome and reduced toxicity with these novel TKIs.

Inhibition of proteasome activity by bortezomib in renal cancer cells is p53 dependent and VHL independent.

BACKGROUND: Antiproliferative effects of proteasome inhibitors are suggested to be primarily due to effects on nuclear factor-kappaB (NF-kappaB)-dependent pathways and the induction of apoptosis. The objective of this study was to elucidate the mechanistic basis for the antiproliferative effects of the proteasome inhibitor, bortezomib, in human clear cell renal cell cancer cells (CCRCC). MATERIALS AND METHODS: von Hippel Lindau (VHL) mutation/methylation status and cytotoxic response to bortezomib was determined in a panel of CCRCC cell lines. Effects on target protein/gene expression and the role of p53 in bortezomib-mediated cytotoxicity, inhibition of proteasome activity, survivin transcript and protein expression as well as induction of p21 expression was determined in CCRCC that differed in their intrinsic sensitivity to bortezomib. RESULTS: VHL status was not associated with cytotoxic response to bortezomib treatment. Cytotoxicity in cell lines that differed in intrinsic sensitivity to bortezomib correlated with sustained inhibition of proteasome activity, survivin expression and induction of p21 expression. Stable down-regulation of p53 expression by siRNA led to attenuation of bortezomib effects, survivin down-regulation and p21 induction, suggesting that cellular effects are p53-dependent. CONCLUSION: These results demonstrate that the antiproliferative effects of bortezomib in CCRCC cells are VHL independent and dependent on pathways regulated by p53.

von Hippel-Lindau gene status and response to vascular endothelial growth factor targeted therapy for metastatic clear cell renal cell carcinoma.

PURPOSE: The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear. MATERIALS AND METHODS: Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points. RESULTS: A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status. CONCLUSIONS: To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.

Proteasome inhibition with bortezomib enhances activity of topoisomerase I-targeting drugs by NF-kappaB-independent mechanisms.

PURPOSE: The potentiation of topoisomerase (topo)-I-induced apoptosis by proteasome inhibitors is dependent on the treatment sequence, but not on NF-kappaB. In this study, alternate mechanisms modulating apoptosis induced with the topo I-targeting drug, SN-38, when followed by the proteasome inhibitor bortezomib (PS-341) were investigated. MATERIALS AND METHODS: Human non-small cell lung carcinoma (NSCLC-3) cells transfected with a control vector (NSCLC-3/neo) or a vector containing dominant negative IkappaBalpha (NSCLC-3/mIkappaBalpha) were treated with SN-38 for 1 h followed by PS-341 for 4 h (SN-38 --> PS-341), or with either drug alone. The functional role of the anti-apoptotic protein survivin was tested using NSCLC-3 transfected with myc-tagged wild-type (NSCLC-3/myc-survivin), or dominant negative mutant T34A survivin (NSCLC-3/myc-T34A). RESULTS: In NSCLC-3/neo or NSCLC-3/mIkappaBalpha cells, treatment with SN-38 --> PS-341 led to down-regulation of the survivin transcript and protein, enhanced apoptosis and reduced (> 3-fold) survival compared to SN-38 or PS-341 alone. In contrast to the cells transfected with wild-type survivin, or the control NSCLC-3/neo, those cells transfected with mutant survivin and treated with SN-38 --> PS-341 exhibited enhanced caspase 9 activity (> 2-fold), caspase 3 (> 2- to 3-fold) activity and cytotoxicity compared to the NSCLC-3/neo cells. CONCLUSION: In contrast to inhibition of NF-kappaB activity, down-regulation of the anti-apoptotic survivin was correlated with modulation of the sequence-dependent synergistic effects of PS-341 in SN-38-induced apoptosis.

Sensitization of DNA damage-induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3sigma and survivin.

Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-kappaB-independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I-targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38-->PS-341). The p53-dependent sensitization of DNA damage-induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341-->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341-->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38-->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38-->PS-341-treated cells compared with PS-341-->SN-38-treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3sigma and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3sigma or survivin in regulating the divergent function of p53 in response to SN-38-->PS-341 and PS-341-->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage-induced apoptosis via a p53-dependent pathway.

Increased CpG methylation of the estrogen receptor gene in BRCA1-linked estrogen receptor-negative breast cancers.

A distinctive feature of BRCA1-linked breast cancers is that they typically do not express estrogen receptor-alpha (ER(alpha)). Previous investigation suggests that methylation of CpGs within the ER(alpha) promoter mediates repression of gene expression in some ER(alpha)-negative breast cancers. To determine if methylation of CpGs within the ER(alpha) promoter is associated with BRCA1-linked breast cancers, we evaluated methylation in exon 1 of the ER(alpha) gene in 40 ER(alpha)-negative breast cancers, 20 of which were non BRCA1-linked and 20 BRCA1-linked. CpG methylation was evaluated by either methylation-sensitive restriction digest (HpaII), methylation-sensitive PCR (MSP), or direct sequencing of bisulfite-treated genomic DNA. Results from HpaII digests and MSP documented a high degree of methylation, the MSP data showing slightly higher methylation in the BRCA1-linked group. CpGs analysed by direct sequencing showed an overall average methylation of 25% among non BRCA1-linked cancers and 40% among BRCA1-linked cancers (P=0.0031). The most notable difference was found at five particular CpGs, each of which exhibited a greater than twofold increase in methylation in the BRCA1-linked group compared to the non BRCA1-linked group (P<0.03 for each CpG). Methylation of certain critical CpGs may represent an important factor in transcriptional repression of the ER(alpha) gene in BRCA1-linked breast cancers.

Inhibition of NF-kappaB and proteasome activity in tumors: can we improve the therapeutic potential of topoisomerase I and topoisomerase II poisons.

Activation of signaling pathways following DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. NF-kappaB, a major regulator of the stress response and a negative regulator of apoptosis is often activated following treatment with topoisomerase poisons. Since activation of NF-kappaB is generally considered to relay an anti-apoptotic signal, inactivation of this signaling molecule is considered to represent an important strategy to improve therapeutic efficacy. Although this strategy seems to be effective in some model systems, our results in human non-small cell lung cancers differed. In this review we will discuss the role of NF-kappaB in mediating topoisomerase poison-induced DNA damage and apoptosis and the consequence of inhibiting its activity. Newer insights about the importance of proteasome inhibitors and anti-apoptotic genes in topoisomerase poison-induced signaling mechanisms leading to apoptosis will also be reviewed. The knowledge obtained from these studies may be useful for translation to a clinical setting for development of more effective therapeutic strategies.