Daniel J. Brackett

Expression profiling identifies microRNA signature in pancreatic cancer

microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real‐time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR‐155, miR‐21, miR‐221 and miR‐222) as well as those not previously reported in cancer (miR‐376a and miR‐301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real‐time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR‐221, ‐376a and ‐301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma.

Association of MicroRNA Expression in Hepatocellular Carcinomas with Hepatitis Infection, Cirrhosis, and Patient Survival

Purpose: MicroRNA (miRNA) is a new class of small, noncoding RNA. The purpose of this study was to determine if miRNAs are differentially expressed in hepatocellular carcinoma (HCC). Experimental Design: More than 200 precursor and mature miRNAs were profiled by real-time PCR in 43 and 28 pairs of HCC and adjacent benign liver, respectively, and in normal liver specimens. Results: Several miRNAs including miR-199a, miR-21, and miR-301 were differentially expressed in the tumor compared with adjacent benign liver. A large number of mature and precursor miRNAs were up-regulated in the adjacent benign liver specimens that were both cirrhotic and hepatitis-positive compared with the uninfected, noncirrhotic specimens (P < 0.01). Interestingly, all of the miRNAs in this comparison had increased expression and none were decreased. The expression of 95 randomly selected mRNAs was not significantly altered in the cirrhotic and hepatitis-positive specimens, suggesting a preferential increase in the transcription of miRNA. Comparing the miRNA expression in the HCC tumors with patients survival time revealed two groups of patients; those with predominantly lower miRNA expression and poor survival and those with predominantly higher miRNA expression and good survival (P < 0.05). A set of 19 miRNAs significantly correlated with disease outcome. A number of biological processes including cell division, mitosis, and G1-S transition were predicted to be targets of the 19 miRNAs in this group. Conclusion: We show that a global increase in the transcription of miRNA genes occurs in cirrhotic and hepatitis-positive livers and that miRNA expression may prognosticate disease outcome in HCC.

σ Receptors: potential medications development target for anti-cocaine agents

The ability of cocaine to interact with sigma receptors suggests a viable target for medications development. Recently, numerous novel compounds and antisense oligodeoxynucleotides targeting sigma receptors have been synthesized and shown to prevent the behavioral toxicity and psychomotor stimulant effects of cocaine in animals. Protective doses of sigma receptor antagonists have also been shown to prevent changes in gene expression that are induced by cocaine. Together, the studies provide insight and promising future directions for the development of potential medications for the treatment of cocaine addiction and overdose.

Mesothelin Is Overexpressed in Pancreaticobiliary Adenocarcinomas but Not in Normal Pancreas and Chronic Pancreatitis

Mesothelin, a cell surface glycoprotein present on normal mesothelial cells, has been reported to be expressed in pancreatic adenocarcinomas. We conducted this study to fully characterize mesothelin expression in surgically resected, formalin-fixed, paraffin-embedded tissue specimens of 18 pancreatic adenocarcinomas, 9 adenocarcinomas of the ampulla of Vater, 12 adenocarcinomas of the common bile duct, and 17 cases of chronic pancreatitis. Mesothelin immunostaining was performed using the antimesothelin monoclonal antibody 5B2. All 18 cases (100%) of pancreatic adenocarcinomas showed mesothelin expression, as did 8 (89%) of 9 cases of ampullar adenocarcinoma and all 12 cases (100%) of common bile duct adenocarcinoma. In all cases of pancreaticobiliary adenocarcinoma, the adjacent normal pancreas did not stain for mesothelin. Of 17 specimens of chronic pancreatitis, 16 were negative for mesothelin expression, and 1 case showed weak mesothelin staining of fewer than 5% of normal pancreatic ducts. Our results demonstrated mesothelin expression in the majority of pancreaticobiliary adenocarcinomas and no expression in normal pancreatic tissues and in chronic pancreatitis.

DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism

Pancreatic cancer is an exceptionally aggressive disease in great need of more effective therapeutic options. Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion and metastasis, and there is a gain of stem cell properties during EMT. Here we report increased expression of the putative pancreatic stem cell marker DCAMKL-1 in an established KRAS transgenic mouse model of pancreatic cancer and in human pancreatic adenocarcinoma. Colocalization of DCAMKL-1 with vimentin, a marker of mesenchymal lineage, along with 14-3-3 σ was observed within premalignant PanIN lesions that arise in the mouse model. siRNA-mediated knockdown of DCAMKL-1 in human pancreatic cancer cells induced microRNA miR-200a, an EMT inhibitor, along with downregulation of EMT-associated transcription factors ZEB1, ZEB2, Snail, Slug, and Twist. Furthermore, DCAMKL-1 knockdown resulted in downregulation of c-Myc and KRAS through a let-7a microRNA-dependent mechanism, and downregulation of Notch-1 through a miR-144 microRNA-dependent mechanism. These findings illustrate direct regulatory links between DCAMKL-1, microRNAs, and EMT in pancreatic cancer. Moreover, they demonstrate a functional role for DCAMKL-1 in pancreatic cancer. Together, our results rationalize DCAMKL-1 as a therapeutic target for eradicating pancreatic cancers.

miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor

Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target the retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G(2)/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the β2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The β2 adrenergic pathway may play an important role in this novel mechanism.

Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study

Autophagy is the regulated process cells use to recycle nonessential, redundant, or inefficient components and is an adaptive response during times of stress. In addition to its function in enabling the cell to gain vital nutrients in times of stress, autophagy can also be involved in elimination of intracellular microorganisms, tumor suppression, and antigen presentation. Because of difficulty in diagnosing autophagy, few clinical studies have been performed. This study examined whether autophagy occurs in hepatocytes during sepsis. Electron microscopy (EM) was performed on liver samples obtained from both an observational clinical cohort of six septic patients and four control patients as well as liver specimens from mice with surgical sepsis (by cecal ligation and puncture) or sham operation. EM demonstrated increased autophagic vacuoles in septic vs nonseptic patients. Randomly selected fields (3000 μm2) from control and septic patients contained 1.2±1.5 vs 5.3±3.3 (mean±s.d.) complex lysosomal/autophagolysosomal structures per image respectively (P<0.001). In rare instances, hepatocytes with autophagic vacuoles appeared to be unequivocally committed to death. Membrane alterations (membrane vacuoles, invagination into adjacent organelles, and myelin figure-like changes) occur in a subpopulation of mitochondria in sepsis, but other hepatocyte organelles showed no consistent ultrastructural injury. Findings in murine sepsis paralleled those of patients, with 7.2±1.9 vs 38.7±3.9 lysosomal/autophagolysosomal structures in sham and septic mice, respectively (P=0.002). Quantitative RT-PCR demonstrated that sepsis induced the upregulation of select apoptosis and cytokine gene expression with minimal changes in the core autophagy genes in liver. In conclusion, hepatocyte autophagic vacuolization increases during sepsis and is associated with mitochondrial injury. However, it is not possible to determine whether the increase in autophagic vacuolization is an adaptive response or a harbinger of cell death.

Heart rate reactivity as a predictor of neuroendocrine responses to aversive and appetitive challenges.

&NA; The present paper examines the neuroendocrine influences of aversive and reward incentives (noise and shock versus monetary bonuses) presented during reaction time tasks administered to 71 healthy men (ages 21 to 35) classified as being high (N = 30) or low (N = 41) in heart rate reactivity. High heart rate reactivity was defined as a peak heart rate increase or greater than 19 bpm to a cold pressor test administered on a different day. Independent groups of subjects worked on one of two visual reaction time tasks: either to avoid exposure to noise (115‐dBA bursts) and shock (3.5 mV, 2 sec), or to earn monetary bonuses (

Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe

0.50). High heart rate reactors showed significant plasma norepinephrine rises from baseline both to aversive incentives and to reward, although they showed significant cortisol responses only during aversive incentives. In contrast, the low heart rate reactors were unresponsive in cortisol and norepinephrine during either type of incentive. These results support psychoendocrine models which view the norepinephrine response as being nonspecifically related to expenditure of effort regardless of the emotional connotations of the challenge, while cortisol is seen as being secreted primarily during periods of distress. The present data further suggest that cardiovascular reactivity is linked to neuroendocrine reactivity, possibly within the central nervous system.

Shock after blast wave injury is caused by a vagally mediated reflex.

RNA-binding proteins play a key role in post-transcriptional regulation of mRNA stability and translation. We have identified that RBM3, a translation regulatory protein, is significantly upregulated in human tumors, including a stage-dependent increase in colorectal tumors. Forced RBM3 overexpression in NIH3T3 mouse fibroblasts and SW480 human colon epithelial cells increases cell proliferation and development of compact multicellular spheroids in soft agar suggesting the ability to induce anchorage-independent growth. In contrast, downregulating RBM3 in HCT116 colon cancer cells with specific siRNA decreases cell growth in culture, which was partially overcome when treated with prostaglandin E2, a product of cyclooxygenase (COX)-2 enzyme activity. Knockdown also resulted in the growth arrest of tumor xenografts. We have also identified that RBM3 knockdown increases caspase-mediated apoptosis coupled with nuclear cyclin B1, and phosphorylated Cdc25c, Chk1 and Chk2 kinases, implying that under conditions of RBM3 downregulation, cells undergo mitotic catastrophe. RBM3 enhances COX-2, IL-8 and VEGF mRNA stability and translation. Conversely, RBM3 knockdown results in loss in the translation of these transcripts. These data demonstrate that the RNA stabilizing and translation regulatory protein RBM3 is a novel proto-oncogene that induces transformation when overexpressed and is essential for cells to progress through mitosis.