Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/- rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/- myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/- rats. The ß-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/- myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/- cardiomyocytes.

Supervisor Support Buffers Daily Psychological and Physiological Reactivity to Work-to-Family Conflict.

Using a daily diary design, the current study assessed within-person associations of work-to-family conflict with negative affect and salivary cortisol. Furthermore, we investigated whether supervisor support moderated these associations. Over eight consecutive days, 131 working parents employed by an information technology company answered telephone interviews about stressors and mood that occurred in the previous 24 hours. On Days 2-4 of the study protocol, they also provided five saliva samples throughout the day that were assayed for cortisol. Results indicated a high degree of day-to-day fluctuation in work-to-family conflict, with employed parents having greater negative affect and poorer cortisol regulation on days with higher work-to-family conflict compared to days when they experience lower work-to-family conflict. These associations were buffered, however, when individuals had supervisors who offered support. Discussion centers on the use of dynamic assessments of work-to-family conflict and employee well-being.

High heregulin expression is associated with activated HER3 and may define an actionable biomarker in patients with squamous cell carcinomas of the head and neck.

PURPOSE: Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3. EXPERIMENTAL DESIGN: qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization. RESULTS: SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens. CONCLUSIONS: HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3.

DNA methylation profiling defines clinically relevant biological subsets of non-small cell lung cancer.

PURPOSE: Non-small cell lung cancers (NSCLC) comprise multiple distinct biologic groups with different prognoses. For example, patients with epithelial-like tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like tumors. Here, we test the hypothesis that epithelial-like NSCLCs can be distinguished from mesenchymal-like NSCLCs on the basis of global DNA methylation patterns. EXPERIMENTAL DESIGN: To determine whether phenotypic subsets of NSCLCs can be defined on the basis of their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative reverse transcriptase PCR and methylation-specific PCR in formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy. RESULTS: We show that patterns of methylation divide NSCLCs into epithelial-like and mesenchymal-like subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple differentially methylated regions, including one in ERBB2 and one in ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy. CONCLUSIONS: Our data show that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used as a platform for predictive biomarker discovery and development.

Serum fatty acid synthase as a marker of pancreatic neoplasia.

Markers of early pancreatic cancer and its precursors are needed to improve the uniformly poor prognosis of this disease. Fatty acid synthase (FAS) catalyzes the synthesis of long-chain fatty acids and is overexpressed in most human solid tumors. We therefore evaluated serum FAS as a marker of pancreatic adenocarcinoma. FAS expression patterns in primary pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis tissues were analyzed by immunohistochemistry. Serum FAS levels were determined by ELISA in 102 patients with pancreatic adenocarcinomas, in 42 patients with IPMNs, in 27 patients with chronic pancreatitis, and in 39 healthy control subjects. FAS protein was overexpressed in the ductal epithelium of 343 of 399 primary pancreatic adenocarcinomas (86.0%) and 28 of 30 IPMNs (93.3%), and in the islet and ductal cells in 3 of 54 chronic pancreatitis tissues (5.6%), whereas normal ductal epithelium lacked FAS expression. Serum FAS levels were significantly higher in patients with pancreatic ductal adenocarcinoma (first quartile median, 22.0; 4.5 ng/mL), in patients with IPMNs (20.7; 9.4 ng/mL), and in patients with chronic pancreatitis (31.1; 11.9 ng/mL) than in healthy controls (0; 0 ng/mL). FAS levels declined postoperatively in 8 of 9 patients with pancreatic adenocarcinoma and elevations of their preoperative serum FAS. In conclusion, serum FAS levels are elevated in patients with pancreatic cancer and IPMNs and are associated with neoplastic overexpression of FAS.

Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays.

Pancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca(++) homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3 sigma, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer.

Aberrant methylation of the 5' CpG island of TSLC1 is common in pancreatic ductal adenocarcinoma and is first manifest in high-grade PanlNs.

The recently identified tumor-suppressor gene TSLC1 on chromosome 11q23.2 is frequently inactivated in human non-small cell lung adenocarcinoma by DNA methylation-associated silencing. The aim of this study was to determine if TSLC1 is inactivated in adenocarcinoma of the pancreas. We analyzed 17 pancreatic cancer cell lines, 91 primary pancreatic adenocarcinoma, 46 pancreatic intraepithelial (PanIN) precursor lesions and 15 microscopically normal pancreata for methylation of the 5' CpG island of the TSLC1 gene through methylation-specific PCR. We observed 5' CpG methylation of TSLC1 in 4 of 17 cell lines (24%). In each cell line the aberrant methylation was associated with loss of TSLC1 expression by RT-PCR that was reversible after treatment with the DNA methyl-transferase inhibitor 5-aza-2'- deoxycytidine. Furthermore, we observed that TSLC1 was methylated in 25 of 91 primary pancreatic adenocarcinomas (27%), and in 2 of 7 highgrade PanIN-3 lesions (29%), but not in low-grade PanIN (0 of 9 PanlN-2 and 0 of 30 PanIN-1) lesions or in normal pancreata (n=15). We conclude that epigenetic silencing of TSLC1 expression through 5' CpG island associated methylation is common in pancreatic adenocarcinoma and is a late event in pancreatic neoplastic development.