Citations

Abstract Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-186.

Abstract In order to identify novel targets and clinically exploitable vulnerabilities, we performed high-throughput RNA-interference (RNAi) synthetic lethality screening using myeloid cell lines TF-1 and ML-2 in combination with the hypomethylating agent 5-Azacytidine. A custom small interfering RNA (siRNA) library targeting nearly 1000 cancer-associated genes, including the human kinome, was utilized. Bcl-XL emerged as the strongest 5-Azacytidine sensitizing target in the erythroid cell line TF-1 but not in ML-2 cells. Additional siRNA sequences targeting Bcl-XL were transfected into a panel of myeloid cell lines and tested in drug dose response experiments using a ten-point serial dilution of 5-Azacytidine. The erythroid differentiated myeloid cell lines TF-1 and HEL both exhibited a 3 to 4-fold reduction in the IC50 of 5-Azacytidine upon knockdown of Bcl-XL, whereas IC50 values for non-erythroid, myeloid cell lines ML-2, THP-1 and MDS-L were unchanged. Furthermore, we tested lentiviral delivery of short-hairpin RNA (shRNA) targeting Bcl-XL in additional cell lines. Lentiviral knockdown of Bcl-XL confirmed 2 to 3-fold 5-Azacytidine sensitization in TF-1 and HEL cells while U937, HL-60, ML-2, THP-1 and MDS-L were not sensitized by Bcl-XL knockdown. Interestingly, while Bcl-XL knockdown with shRNA potentiated the effects of 5-Azacytidine in TF-1 and HEL, Bcl-XL knockdown alone resulted in considerable reductions in cell viability in TF-1 and HEL cells. These results demonstrate erythroid lineage-specific responses to Bcl-XL knockdown and suggest that Bcl-XL may represent an erythroid lineage-specific therapeutic vulnerability. To begin to explore this possibility we examined the putative BH3 mimetics Gossypol, Obatoclax and ABT-737 with 5-Azacytidine drug dose response experiments in myeloid cell lines. Neither Gossypol nor Obatoclax potentiated the effects of 5-Azacytdine. In striking contrast, ABT-737 resulted in a 2 to 4-fold reduction in the IC50 of 5-Azacytidine in all cell lines tested to date (TF-1, HEL, ML-2 and THP-1). ABT-737 sensitization to 5-Azacytidine was dose dependent. Furthermore, Combination Index (CI) values calculated with Calcusyn software demonstrate synergy between ABT-737 and 5-Azacytidine at clinically relevant concentrations. These results suggest that inhibition of anti-apoptotic Bcl-2 family members with a BH3 mimetic may represent an effective strategy for 5-Azacytidine combination therapy that can be directly translated into design of combination clinical trials in patients with myeloid leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-128.

Abstract Objectives. To assess chemo-response of patient derived ovarian cancer cells to first line chemotherapeutic agents as well as targeted therapies. Methods. Ascites was obtained at laparotomy from 14 patients with epithelial ovarian cancer. Short term cultures were established in 11 of these and the cells characterized by morphology and immuno-staining to cytokeratin AE1/AE3 and E-cadherin. MTT assays were done on the 11 samples after 48 hours of exposure to Cisplatin, Paclitaxel, Vandetanib and Flavopiridol. Results. Three samples did not grow well and ultimately showed senescence or were over-run by fibroblasts. In the remaining 11 samples, morphology showed the cells to be arranged in a cobble-stone or rosette pattern. Most of the cells stained positive for cytokeratin AE1/AE3 and E-cadherin. The majority of samples (8/11, 73%) were sensitive to either Cisplatin or Paclitaxel. Of the three resistant samples, one was sensitive to Vandetanib, one to Flavopiridol and one was resistant to all four drugs. Two patients did not receive chemotherapy due to morbidity or mortality. Among those who were treated, 4 (50%) had complete response and are currently free of disease. Conclusions. Short term cultures may be a good method of studying chemo-response of tumor cells ex-vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3544.

Abstract Lung cancers are often characterized by numerical chromosome instability. Hexavalent chromium (Cr(VI)) is a well-known human lung carcinogen; however, its carcinogenic mechanism remains unknown. Studies show that solubility plays a key role in its carcinogenicity, with the particulate, or insoluble, compounds being the most potent carcinogens. Epidemiological and whole animal studies pinpoint particulate zinc chromate as the most potent Cr(VI) carcinogen; however, few studies have investigated its carcinogenic mechanism. Therefore, the goal of this study was to determine if zinc chromate induces numerical chromosome instability in human lung cells. We found that exposure to zinc chromate for 24 h did not induce aneuploidy but longer exposures induced both concentration- and time-dependent increases in aneuploid metaphases. For example, exposure to 0.1, 0.15 and 0.2 ug/cm2 zinc chromate for 120 h induced 28, 40 and 44 percent aneuploid metaphases, respectively, with increases in hypodiploid, hyperdiploid and tetraploid metaphases. Consistent with the aneuploidy data, a 24 h exposure to zinc chromate did not induce centrosome amplification but more chronic exposures induced concentration- and time-dependent increases in centrosome amplification in both interphase and mitotic cells. Exposure to 0.1, 0.15 and 0.2 ug/cm2 zinc chromate for 120 h induced centrosome amplification in 13.9, 18 and 21.3 percent of interphase cells and 21, 33 and 46 percent of mitotic cells. In order to determine the mechanism of centrosome amplification, we investigated the number of centrioles in each centrosome and found that cells with amplified centrosomes exhibited centrosomes with normal centriole pairs and centrosomes with centriolar defects indicating multiple mechanisms are involved in zinc chromate-induced centrosome amplification. Lastly, we found that chronic exposure to zinc chromate induced a prolonged G2 arrest. All together, these data indicate that chronic exposure to zinc chromate induces numerical chromosome instability via centrosome amplification in human lung cells. This work was supported by NIEHS grant ES016893 (J.P.W.), cooperative agreement #EP-08-01 through the Maine Space Grant Consortium (J.P.W.), EPA GRO Fellowship MA-91685401 (A.L.H.) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3473.

Abstract Cancers of the head and neck rank as the 6th most commonly observed cancers worldwide with an average 5-year survival rate of only 50% for advanced disease. Nearly 95% of head and neck cancers are squamous cell carcinomas (SSC) and these are frequently highly invasive metastatic tumors. The newly developing technologies of Next Generation Sequencing have allowed for the most comprehensive genetic analysis of these cancers to date. We have taken 3 tumor/normal pairs from patients with oral squamous cell carcinoma and performed full transcriptome and mate-pair DNA sequencing analysis using the AB SOLiD system. These runs generated 129-256 million 50bp long sequence reads per sample, with 21 to 56 million reads per sample uniquely aligning to human genome sequence. Analysis of these samples has revealed pathways with aberrant expression in tumor versus normal tissue. Our results show up-regulation of some metalloproteinase genes, HGMA2, which is a prognostic marker for oral carcinoma, and extracellular matrix, adhesion, and motility gene classes in tumors. Our functional analysis also indicates extensive down-regulation, in the tumors, of genes functioning in epidermal development. We were also able to measure allelic specific expression for the 50% most highly expressed genes and detect examples of allelic imbalance linked to pathways with dysregulated gene expression. Additionally, the depth of sequencing that was achieved (5-16X coverage of RefSeq Exons) also allowed us to detect mutations occurring in the transcripts. We have also detected evidence of RNA editing associated with point mutations, which we are validating in a larger group of samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2957.

Abstract INTRODUCTION: The centrosome is the primary microtubule organizing center of the cell. Numerical or functional defects of the centrosome can result in improper chromosome segregation, leading to aneuploidy and polyploidy. Because many cancer cells have aberrant numbers of chromosomes, many have suggested a link between centrosomes and cancer. We conducted an association study to determine whether common genetic variations in 100 genes involved in maintaining and regulating centrosome structure and function account in part for the contribution of the pathway to breast cancer risk. METHODS: Incident cases of BC were identified at the Mayo Clinic in Rochester, MN from 2001 through 2005. Controls (N=843) were frequency matched to cases (N=798) on age and region of residence. Seven hundred-eighty-two tagging and candidate functional SNPs were selected from 100 genes involved in the structure and/or function of the centrosome. Genotyping was performed on an Illumina BeadLab using the Illumina GoldenGate genotyping assay. Logistic regression was used to calculate odds ratios and 95% confidence intervals for BC. Women having two copies of the most common allele were defined as the referent category. Linear regression was used to estimate mean and 95% CI for PD for each additional copy of the variant allele. All models were adjusted for age, residence, and confounding factors. RESULTS: Forty-eight SNPs from 29 genes (out of 782 SNPs examined) showed evidence of significant associations with breast cancer risk in our population in the log-additive (1 degree of freedom (d.f.)) model (Ptrend< 0.05). One SNP, rs1374468 SNP in TACC3, displayed the most significant association with risk in the overall analyses (Ptrend = 0.001). Two SNPs from each of seven genes (AJUBA, CHUK, MCPH1, NEK7, PAK1, PIK3CB, and PINS) were significantly associated with risk of breast cancer (Ptrend < 0.05). Three genes, AXIN2, NIN, NUMA1, had four or more SNPs that were significantly associated with breast cancer risk (Ptrend < 0.05). All of the SNPs in these four genes were in strong linkage disequilibrium (r2>0.6). No single SNP association was significant after adjustment for multiple comparisons via permutation testing. Haplotype analyses showed 20 genes for which global haplotypes were significantly associated with breast cancer risk. Of these, haplotype blocks in six genes (14-3-3 Epsilon, CDC16, CD-TOG, KIF2, NEK9, NPM2) were significantly associated with risk (global P<0.05), although none of the individual SNPs in these six genes displayed significant associations. This suggests that the associations of these specific combinations of alleles with risk were not due to the effects of individual SNPs. CONCLUSION: These analyses provide some evidence to support an association between genetic variation in centrosomal genes and risk of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2852.

Abstract Mitochondria have been implicated in carcinogenesis because of their central role in apoptosis, free radical production, and cellular energy metabolism. Although studies have identified somatic mitochondrial DNA mutations in ovarian cancers, little attention has been directed to the possible role of germline mitochondrial variants in mitochondrial dysfunction and subsequent ovarian cancer development. Through an ongoing epithelial ovarian cancer genome-wide association study of 1,856 invasive cases (59% with serous carcinomas) and 1,912 controls (frequency-matched on age, race, and study site), we genotyped 138 SNPs tagging variation in the maternally-inherited mitochondrial genome (mtDNA) to evaluate the hypothesis that single nucleotide polymorphisms (SNPs) in mtDNA are associated with ovarian cancer risk. All subjects were non-Hispanic, non-Jewish Caucasians, and were genotyped with the Illumina 610-quad Beadchip. Samples and SNPs with call rates less than 95% were excluded. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were excluded. For each SNP, logistic regression was used to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI) between carriers of the minor versus major maternally-inherited allele and case status. Subgroup analysis was conducted to estimate allele-specific risks between serous-only cases and controls. Haplotype analysis was performed for regions containing statistically significant SNPs (p<0.05). Two rare SNPs, C16329T in the displacement loop gene (MT-D-loop) (OR: 6.72, 95%CI: 1.5-29.8, p=0.004; minor allele frequency (MAF)=0.001) and C15905T in the tRNA threonine gene (MT-TT) (OR: 1.60, 95%CI: 1.1-2.3, p=0.009; MAF=0.027), were associated with increased ovarian cancer risk; whereas T6777C in the cytochrome c oxidase subunit 1 gene (MT-CO1) (OR: 0.68, 95%CI: 0.51-0.91, p=0.009; MAF=0.063) was associated with decreased risk. All three SNPs remained statistically significant after adjustment for multiple comparisons within the mitochondrial SNPs using permutation testing. C16329T and T6777C were also significantly associated among cases with serous histology, with ORs (95% CIs) of 6.13 (1.2-29.6) and 0.61 (0.43-0.87), respectively. Haplotype analysis for each of the three regions investigated revealed significant haplotype-disease associations; however, findings suggest that the respective associations were driven by the most significant SNP(s) listed above. This is the first large-scale epidemiologic study to provide evidence that SNPs in mitochondrial genes may be novel risk factors for ovarian cancer. Future investigation of additional mitochondrial variants (i.e. nuclear-encoded mitochondrial proteins) is necessary to more comprehensively investigate this association. Furthermore, replication of the most promising SNPs in a larger population is warranted to validate these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2835.

Abstract DNA topoisomerase IIα (Topo IIα) is a critical target for cancer therapy and its protein expression, however, is frequently depleted by its inhibitors and strategies to maintain Topo IIα expression and thereby to sustain the Topo II drug sensitivity are still lacking. Here we show that p38γ MAPK is specifically activated by Topo II inhibitors and activated p38γ in turn phosphorylates Topo IIα, leading to increased Topo IIα protein expression through attenuating its proteasome-dependent degradation. The sensitivity of a panel of human breast cancer cell lines to Topo II inhibitors was found not to correlate with the intrinsic but with inducible Topo IIα protein expression and with endogenous protein expression of p38γ MAPK that is specifically phosphorylated in response to Topo II toxins. p38γ activation and inhibition alone were shown to increase and decrease Topo IIα expression and Topo drug toxicities. Mechanistically, p38γ MAPK phosphorylates Topo IIα at Ser 1524, leading to a decreased proteasome-dependent Topo IIα degradation. These results reveal a novel mechanism by which p38γ MAPK specifically confers Topo II drug toxicity in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 317.

Abstract Growth factor and K-ras-induced generation of reactive oxygen species have important roles in mediating pancreatic ductal adenocarcinoma. Pancreatic cancer cells also express high levels of active transcription factor Nuclear Factor-κB (NF-κB), which is responsible for cell survival and resistance to chemotherapeutics. The serine/threonine kinase Protein Kinase D1 (PKD1) is a sensor for oxidative stress and protects cancer cells by inducing anti-apoptotic and anti-oxidant genes. Here we provide evidence that PKD1 is the means of how high NF-κB activity levels are achieved in pancreatic cancer cells. We also show the contribution of K-ras, which is upregulated in its activity in over 90% of PDAC (pancreatic ductal adenocarcinoma). We found that K-ras in pancreatic ductal HPDE cells increases PKD1 activity and that the depletion of the pancreatic ductal adenocarcinoma cell line Panc-1 from K-ras leads to decreased PKD1 activity and NF-κB activation. As a mechanism we found that K-ras mediates activation of PKD1 via Src and that these signaling events relay to high NF-κB levels. Our data reveal K-ras as an activator of PKD1 and show that both oncogenes, Src and K-ras cooperate to activate NF-κB in PDAC through PKD1. This suggests the K-ras/Src/PKD1/NF-κB pathway as a new potential drug target for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3143.

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL), a member of lipocalin family, is a 25-kDa secreted glycoprotein. NGAL modulates multiple biological functions including cell proliferation, invasion, and metastasis. NGAL is upregulated in multiple gastrointestinal cancers including colorectal, pancreatic, and esophageal adenocarcinoma (EAC). Our group recently showed that NGAL over-expression significantly blocked metastasis of pancreatic adenocarcinoma cells [Cancer Research 68(15): 6100-6108, 2008]. However, the biological role of NGAL in EAC is not known. Aim: To examine the biological role of NGAL in human EAC cells both in vitro and in vivo. Methods and Results: We used OE33 (well-differentiated) and Flo-1 (poorly differentiated) EAC cell lines as our model system. We observed that OE33 cells have significantly higher levels of NGAL expression than Flo-1 cells both by qPCR and western blot analyses. To further study the biological role of NGAL in EAC, we created stable NGAL over-expression clones in Flo-1 (Flo1-NGAL) and NGAL under-expression clones in OE33 (OE33-shNGAL) using lentiviral system. We observed that under-expression of NGAL in OE33 significantly increased cell proliferation as determined by clonogenic assays. In contrast, NGAL over-expression in Flo-1 significantly suppressed cell proliferation. We also observed that NGAL under-expression increased cyclin D1 expression, a known cell cycle regulator. This, in part, explained increased EAC cell proliferation secondary to NGAL under-expression. In addition, NGAL over-expression potently suppressed and conversely, NGAL under-expression significantly increased invasion of EAC clones through MatrigelTM in vitro. Furthermore, to confirm our in vitro findings, we observed that OE33-shNGAL clones strikingly increased tumor volume in a subcutaneouos heterotopic xenograft model in nude mice. Conclusion: We demonstrated for the first time that NGAL under-expression increased cell proliferation and invasion in vitro, which could be in part secondary to regulation of cyclin D1 expression. NGAL under-expression also enhanced tumor growth in vivo. Thus, modulation of NGAL can be a novel therapeutic target in progression of EAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3074.