Citations

Polycystic kidney (PKD) and liver (PLD) diseases cause significant morbidity and mortality. A large body of evidence indicates that cyclic AMP plays an important role in their pathogenesis. Clinical trials of drugs that reduce cyclic AMP levels in target tissues are now in progress. Secretin may contribute to adenylyl cyclase-dependent urinary concentration and is a major agonist of adenylyl cyclase in cholangiocytes. To investigate the role of secretin in PKD and PLD, we have studied the expression of secretin and the secretin receptor in rodent models orthologous to autosomal recessive ( PCK rat) and dominant ( Pkd2−/WS25mouse) PKD; the effects of exogenous secretin administration to PCK rats, PCK rats lacking circulating vasopressin ( PCKdi/di), and Pkd2−/WS25mice; and the impact of a nonfunctional secretin receptor on disease development in Pkd2−/WS25:SCTR−/−double mutants. Renal and hepatic secretin and secretin receptor mRNA and plasma secretin were increased in both models, and secretin receptor protein was increased in the kidneys and liver of Pkd2−/WS25mice. However, exogenous secretin administered subcutaneously via osmotic pumps had minimal or negligible effects and the absence of a functional secretin receptor had no influence on the severity of PKD or PLD. Therefore, it is unlikely that by itself secretin plays a significant role in the pathogenesis of PKD and/or PLD.

Rationale: Among the extracellular modulators of Bmp (bone morphogenetic protein) signaling, Bmper (Bmp endothelial cell precursor-derived regulator) both enhances and inhibits Bmp signaling. Recently we found that Bmper modulates Bmp4 activity via a concentration-dependent, endocytic trap-and–sink mechanism. Objective: To investigate the molecular mechanisms required for endocytosis of the Bmper/Bmp4 and signaling complex and determine the mechanism of Bmper's differential effects on Bmp4 signaling. Methods and Results: Using an array of biochemical and cell biology techniques, we report that LRP1 (LDL receptor-related protein 1), a member of the LDL receptor family, acts as an endocytic receptor for Bmper and a coreceptor of Bmp4 to mediate the endocytosis of the Bmper/Bmp4 signaling complex. Furthermore, we demonstrate that LRP1-dependent Bmper/Bmp4 endocytosis is essential for Bmp4 signaling, as evidenced by the phenotype of lrp1-deficient zebrafish, which have abnormal cardiovascular development and decreased Smad1/5/8 activity in key vasculogenic structures. Conclusions: Together, these data reveal a novel role for LRP1 in the regulation of Bmp4 signaling by regulating receptor complex endocytosis. In addition, these data introduce LRP1 as a critical regulator of vascular development. These observations demonstrate Bmper's ability to fine-tune Bmp4 signaling at the single-cell level, unlike the spatial regulatory mechanisms applied by other Bmp modulators.

Abstract Rare germline mutations in the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 are present in roughly 5 percent of ovarian cancer patients. Both genes play key roles in DNA damage repair but appear to have distinct, although often complementary, functions. The risks of breast and ovarian cancer have been shown to differ between BRCA1 and BRCA2 carriers, and mutation-specific effects have also been suggested for both groups. Published reports comparing the survival of ovarian cancer patients with and without BRCA1/2 mutations generally show a survival advantage among mutation carriers. This is thought to be related to an improved response of carriers to platinum-based therapies. Since it is not known whether BRCA1 and BRCA2 carriers show similar survival patterns, we have performed a large, multicenter study to investigate the impact of germline BRCA1 and BRCA2 mutations on ovarian cancer survival. 3,531 invasive epithelial ovarian cancer cases (1,178 BRCA1, 367 BRCA2 and 1,986 BRCA-negative) with survival time data from twenty-four studies in the U.S.A, Europe, Israel and Asia were included. In our main analysis, we excluded patients who were known or who were likely to have not received platinum-based therapy. Compared to non-carriers, BRCA1 and BRCA2 carriers were more likely to present with advanced stage (BRCA1; p=2×10−4, BRCA2; p=4×10−6), high grade (BRCA1; p=4×10−9, BRCA2; p=1×10−4), serous disease (BRCA1; p=3×10−6, BRCA2; p=0.003). BRCA1 carriers were younger at diagnosis (p=2×10−9) and BRCA2 slightly older at diagnosis (p=0.002) than non-carriers. BRCA1 and BRCA2 carriers did not differ in terms of tumor stage, grade or histology but did show differences in the age at diagnosis (p=2×10−14). In an unadjusted analysis, neither BRCA1 nor BRCA2 carriers differed from non-carriers in overall survival (BRCA1: HR=1.02, p=0.77, BRCA2: HR=0.89, p=0.36). After adjusting for stage, grade, histology and age at diagnosis, BRCA1 carriers showed a modest, non-significant, survival advantage (HR=0.84, p=0.12) while BRCA2 carriers showed a marked improvement (HR=0.57, p=6×10−4) compared to non-carriers. This survival advantage was also seen when comparing BRCA2 carriers to BRCA1 carriers after adjustment for age at diagnosis (HR=0.69, p=5×10−4). Similar results were obtained when we restricted the analysis to high grade, advanced stage, serous cases. The survival differences we observed between BRCA1 and BRCA2 carriers could be related to differences in tumor biology or chemo-sensitivity. Additional analyses investigating the later will be presented in addition to mutation-specific effects within BRCA1 and BRCA2. In this study, we provide strong evidence for genetic heterogeneity of survival effects in ovarian cancer patients with BRCA mutations. This could be applied to both clinical prediction and to aid our understanding of the complex biology of BRCA mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2752. doi:10.1158/1538-7445.AM2011-2752

Abstract Phospholipase C epsilon (PLCε or PLC∈ 1) is a phosphoinositide-specific enzyme phospholipase C that converts phosphatidylinositol 4,5-biphosphate to two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which regulate protein kinase C activity and calcium mobilization, respectively. These responses are involved in the regulation of cell growth, differentiation and oncogenesis. However, the involvement of PLCε in human carcinogenesis, particularly in esophageal cancer, was not been recognized until a susceptible SNP (rs2274223: A5780G:His1927Arg) was recently identified in a large genome-wide association study (GWAS). This SNP, located within exon 26 and C2 domain of the PLCε gene at chromosome 10q23, leads to a nonsynonymous alteration from CAC (A allele, encoding histidine) to CGC (G allele, encoding arginine), which was associated with an increased risk of both squamous cell carcinoma and esophagus-cardia gastric adenocarcinoma. In the current report, we demonstrate that the presence of the G allele (AG or GG) at this locus leads to increased PLCε mRNA and protein expression in human esophageal cancer tissues and in human esophageal cancer cell lines. The present of G allele also leads o a higher enzyme activity in esophageal cancer cells in vitro. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance is strongly associated with esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects from high- and low-risk areas of China demonstrated that the presence of the G allele is strongly associated with moderate to severe esophagitis in the subjects from the high-risk areas of China: 77% of the severe esophagitis individuals in high-risk areas had AG/GG genotypes vs. only 37% of these subjects in low-risk areas (OR 6.03 with 95% CI 1.59-22.9 vs. OR 0.74 with 95% CI 0.33-1.64; p=0.008). These data suggest that the interaction of potential environmental factors with PLCε, particularly in individuals with AG or GG allele, not only exists in high-risk areas for esophageal cancer development in China, but also correlates with the severity of esophagitis. In conclusion, PLCε is likely to play a pivotal role in esophageal carcinogenesis: the presence of the 5780G allele may not only predict a high risk of future esophageal cancer development, but may also participate in esophageal cancer growth and progression by upregulating levels of PLCε mRNA, protein, and enzyme activity, ultimately leading to augmentation of the inflammatory process in esophageal epithelium. Thus, the 5780G allele in PLCε may constitute a promising biomarker for esophageal squamous carcinoma risk stratification, early detection, and progression prediction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4704. doi:10.1158/1538-7445.AM2011-4704

Abstract Because numerous chemotherapeutic agents exert their cytotoxic effects through generation of reactive oxygen species, the use of antioxidant supplements during treatment is controversial. There are published recommendations to suspend antioxidant supplement use during treatment, despite lack of empirical data from clinical studies; however, it is unclear if patients follow these recommendations. We examined use of multivitamins and vitamin supplements (MV/SP) during treatment for breast cancer, particularly in relation to physician recommendations. High-risk breast cancer patients (n=855) were queried regarding physician recommendations and use of MV/SP via self-administered questionnaires in an ancillary study embedded in a phase III trial of dose-dense compared to metronomic administration of doxorubicin/cyclophosphamide + paclitaxel (SWOG S0221). Of the 658 patients who reported discussing use of supplements during treatment with their physicians, 390 (59%) received no counseling regarding MV/SP use, 85 (13%) received recommendations not to take MV/SP, and 183 (28%) received recommendations to use MV/SP. MV/SP use during treatment was compared to use prior to diagnosis in relation to physician recommendations, with the reference group comprised of those receiving no recommendations. Of those using MV ≥ once/week before diagnosis, recommendations not to take MV/SP were associated with a tenfold reduction in MV use to < once/week during treatment. (OR=10.0, 95% CI=3.7-27.0). MV use was less likely to decrease among patients receiving recommendations to use MV/SP (OR=0.23, 95% CI=0.12-0.44). Similar findings were observed among patients who reported use of vitamin C and vitamin E supplements ≥ once/week before diagnosis, with patients who were advised to take no supplements other than a MV more than 5 times as likely to decrease use of antioxidant supplements. MV use during treatment among previous non-users increased almost sixfold among those whose physicians recommended MV/SP use during treatment (OR=5.92, 95% CI=3.29-10.6). For those reporting vitamin C or vitamin E use < once/week before diagnosis, patients whose physicians recommended MV/SP during treatment were more likely to increase their use of vitamin C (OR=2.05, 95% CI=1.00-4.19) and vitamin E (OR=5.28, 95% CI=2.24-12.4). In this study, physician recommendations were strongly associated with patient use of MV/SP during chemotherapy, regardless of the patient's frequency of use prior to diagnosis. These findings highlight the critical need to better understand the mechanisms by which antioxidants may influence chemotherapeutic outcomes, and to determine if supplement use affects toxicity and/or recurrence. Yearly follow-up in this ongoing study will allow us to assess the impact of MV/SP use treatment outcomes, providing a basis for physician recommendations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4681. doi:10.1158/1538-7445.AM2011-4681

Abstract Breast cancer is the most common form of cancer diagnosed and the second leading cause of cancer death among women in the United States. Cancer metastases are the major cause of morbidity and mortality among breast cancer patients. The central importance of the HER2/RAS signaling pathway has been well established in promoting tumor growth, invasion and metastasis of human breast cancer. Even though oncogenic K-RAS mutations are rare in breast cancer, activation of the growth-promoting ERBB/RAS pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated HER2/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the ERBB/RAS pathway – the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These results suggest that SIAH2 may be an attractive and new drug target for novel breast cancer therapy. Developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. By attacking the growth-promoting HER2/EGFR/RAS pathway at multiple signaling modules simultaneously – a multi-pronged synergistic inhibition at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel, we may be in a position to halt the genesis, progression and metastasis of human breast cancer. Thus, SIAH-dependent proteolysis may be an Achilles heel for breast cancer. SIAH inhibition may represent an innovative and logical way to inhibit HER2/EGFR/K-RAS activation, alleviate tumor burdens, halt cancer metastasis, and improve the efficacy for the future treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2011-1659

Abstract Hyperactive K-RAS signaling is a major menace that drives aggressive tumor growth and metastasis in human cancer. Currently, there are no effective ways to treat human cancers that have oncogenic K-RAS mutations that confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS is an urgent goal and the major challenge in cancer therapy and treatment. Instead of targeting an upstream signaling module such as EGFR/HER2/KRAS/B-RAF/MEK/MAPK/AKT/mT0R, we targeted the most downstream signaling module in the EGFR/HER2/K-RAS pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of seven-in-absentia (SINA), an evolutionarily conserved RING E3 ligase, an essential downstream signaling module, and a critical gatekeeper required for proper ERBB/RAS/RAF signal transduction. Guided by the insights and principles learned from Drosophila RAS signaling pathway, we conducted preclinical studies to dissect SIAH function in the context of ERBB/K-RAS-mediated tumorigenesis and metastasis in human cancer cells and tumor specimens. We found that: 1) SIAH is a novel biomarker for human cancer, i.e., SIAH specifically decorates proliferating tumor cells (not stromal or immune cells in the tumor microenvironment). SIAH expression is progressively and markedly upregulated as human cancer progresses to the advanced stages in human pancreatic, lung, breast, and prostate cancers; and 2) inhibiting SIAH function is highly effective to abolish tumorigenesis and metastasis in multiple of the most aggressive human cancer cell lines known in the literature that include pancreatic cancer cells (MiaPaCa and Panc-1), lung cancer cells (A549, H727, and UMC11), invasive breast cancer cells (MDA-MB-231, MDA-MB-468, and MDA-MB-435), colorectal cancer cells (SW620 and HCT116) and hormonal-refractory prostate cancer cells (PC-3 and DU-145) in our preclinical studies. These findings demonstrate that SIAH is an attractive and logical new therapeutic target for novel anti-K-RAS and anticancer therapy in human cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic K-RAS activation in human cancer cells. Using anti-SIAH molecules to block K-RAS signaling in human cancer is an excellent example of science going from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials). As a highly evolutionarily conserved E3 ligase and the most downstream gatekeeper of EGFR/HER2/K-RAS/B-RAF signaling pathway, SIAH is uniquely and logically positioned to become a next-generation anti-K-RAS drug target. Our preclinical studies have demonstrated that SIAH-dependent proteolysis is an Achilles' heel for human cancer cells. Albeit at an early preclinical stage, knowledge gained from our study has great promise that has generated many excitements with immediate translational values. Anti-SIAH-based small molecule inhibitors may aid in expanding our arsenal of novel anticancer therapies. By attacking the oncogenic ERBB/K-RAS/B-RAF pathway using multipronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR), and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a great position to halt the genesis, progression, and metastasis of the most aggressive and the deadliest forms of human cancers. If successful, we hope to translate our findings to the cancer clinics to treat many cancer patients with metastatic diseases in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B14.

Abstract Purpose: miRNA plays an important role in human disease and cancer. We seek to investigate the expression status, clinical relevance, and functional role of miRNA in non–small cell lung cancer. Experimental Design: We conducted miRNA expression profiling in matched lung adenocarcinoma and uninvolved lung using 56 pairs of fresh-frozen (FF) and 47 pairs of formalin-fixed, paraffin-embedded (FFPE) samples from never smokers. The most differentially expressed miRNA genes were evaluated by Cox analysis and log-rank test. Among the best candidate, miR-708 was further examined for differential expression in two independent cohorts. Functional significance of miR-708 expression in lung cancer was examined by identifying its candidate mRNA target and through manipulating its expression levels in cultured cells. Results: Among the 20 miRNAs most differentially expressed between tested tumor and normal samples, high expression level of miR-708 in the tumors was most strongly associated with an increased risk of death after adjustments for all clinically significant factors including age, sex, and tumor stage (FF cohort: HR, 1.90; 95% CI, 1.08–3.35; P = 0.025 and FFPE cohort: HR, 1.93; 95% CI, 1.02–3.63; P = 0.042). The transcript for TMEM88 gene has a miR-708 binding site in its 3′ UTR and was significantly reduced in tumors high of miR-708. Forced miR-708 expression reduced TMEM88 transcript levels and increased the rate of cell proliferation, invasion, and migration in culture. Conclusions: miRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly downregulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer. Clin Cancer Res; 18(13); 3658–67. ©2012 AACR.

Abstract A61 Background Patients with Barrett's esophagus (BE) and dysplasia are ideal candidates for chemopreventive strategies to reduce cancer risk. Difluoromethylornithine (DMFO) is a promising chemopreventive agent that inhibits polyamine synthesis involved in the regulation of cell growth and differentiation. We conducted a pilot study to examine the effects of DFMO on esophageal histopathology, mucosal polyamine content, and gene expression. Materials and Methods Patients (n=10) with histologically confirmed BE and low grade dysplasia were enrolled in a single-arm study of DFMO (0.5 g/m2/d) given continuously for 6 months. Esophagoscopy with biopsies (4 quadrants every 1 cm) was performed at baseline, 3 and 6 months, and at 12 months (where available). Grading of dysplasia was performed by a GI pathologist blinded to clinical/biomarker data. Audiology was also performed. Mucosal polyamine content was measured by high-performance liquid chromatography, and analyzed using paired t-tests. Gene expression profiling was performed using a cDNA two-color chip, and was compared between baseline and 6 months. Expression data was normalized using the two-channel fastlo routine and differential expression was computed using a Wilcoxon signed-rank test. Results After 6 months of DFMO treatment, histopathological response data demonstrate regression of dysplasia (n= 1), stable disease (n= 8), and progression of dysplasia (n= 1). Within the stable category, 2 patients with extensive low grade dysplasia showed only focal dysplasia based upon 4 or more biopsies at 6 months. Histolopathological improvements seen at 6 months were maintained at 12 months. Overall, DFMO was well tolerated. One patient had subclinical, unilateral ototoxicity. Modulation of polyamines (spermine, spermidine, putrescine, histamine) was shown only for putrescine which decreased with treatment (6 mo., p=0.07) and showed recovery to baseline at 12 months (p= 0.01). Gene expression profiling revealed modulated genes of potential biological importance (KLF5, KAL1, RFC5, TIMM8a, TC10) and of these, Krüppel-like factor 5 (KLF5) was downregulated whereas the others were upregulated. Conclusion >Further study of the chemopreventive effect of DFMO upon BE with dysplasia is suggested. DFMO treatment suppressed mucosal putrescine levels and downregulated KLF5, an important mediator of cell proliferation. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A61.