Sambasiva Rao

Identification of Protein Arginine Methyltransferase 2 as a Coactivator for Estrogen Receptor α

In an attempt to isolate cofactors capable of influencing estrogen receptor α (ERα) transcriptional activity, we used yeast two-hybrid screening and identified protein arginine methyltransferase 2 (PRMT2) as a new ERα-binding protein. PRMT2 interacted directly with three ERα regions including AF-1, DNA binding domain, and hormone binding domain in a ligand-independent fashion. The ERα-interacting region on PRMT2 has been mapped to a region encompassing amino acids 133–275. PRMT2 also binds to ERβ, PR, TRβ, RARα, PPARγ, and RXRα in a ligand-independent manner. PRMT2 enhanced both ERα AF-1 and AF-2 transcriptional activity, and the potential methyltransferase activity of PRMT2 appeared pivotal for its coactivator function. In addition, PRMT2 enhanced PR, PPARγ, and RARα-mediated transactivation. Although PRMT2 was found to interact with two other coactivators, the steroid receptor coactivator-1 (SRC-1) and the peroxisome proliferator-activated receptor-interacting protein (PRIP), no synergistic enhancement of ERα transcriptional activity was observed when PRMT2 was coexpressed with either PRIP or SRC-1. In this respect PRMT2 differs from coactivators PRMT1 and CARM1 (coactivator-associated arginine methyltransferase). These results suggest that PRMT2 is a novel ERα coactivator.

Peroxisome proliferator-activated receptors, fatty acid oxidation, steatohepatitis and hepatocarcinogenesis

Fatty acids are metabolized in the liver by beta-oxidation in mitochondria and peroxisomes and by omega-oxidation in microsomes. Peroxisomal beta-oxidation is responsible for the metabolism of very long chain fatty acids and mitochondrial beta-oxidation is responsible for the oxidation of short, medium and long chain fatty acids. Very long chain fatty acids are also metabolized by the cytochrome P450 CYP4A omega-oxidation system to dicarboxylic acids. Both peroxisomal beta-oxidation and microsomal omega-oxidation lead to the generation of H2O2. The genes encoding peroxisomal, microsomal and some mitochondrial fatty acid metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha). Sustained activation of PPAR alpha by peroxisome proliferators has been shown to induce hepatocellular carcinomas in rats and mice. The peroxisome proliferator-induced carcinogenic effect has been attributed to transcriptional activation of PPAR alpha regulated genes and the resulting excessive generation of H2O2. Evidence from mice lacking fatty acyl-CoA oxidase (AOX), PPAR alpha and PPAR alpha/AOX has confirmed the role of PPAR alpha in the development of hepatocellular carcinomas. In addition, mice lacking AOX developed steatohepatitis and provided clues regarding the molecular mechanism responsible for steatosis and steatohepatitis and the role of unmetabolized AOX substrates in the activation of PPAR alpha.

The use of antikeratin antiserum as a diagnostic tool: Thymoma versus lymphoma

Indirect immunofluorescence staining in two thymomas, one case of thymic hyperplasia, 10 malignant lymphomas and three seminomas was done with an antibody prepared against keratins from human epidermis. Staining was observed only in the epithelial cells of the thymomas and thymic hyperplasia and correlated well with electron microscopic studies. Immunofluorescence staining of thymic tumors with antikeratin antibody provides a simple, specific, and sensitive method for distinguishing thymoma from lymphoma and seminoma. The method may also prove to be useful in other instances in the distinction between epithelial and nonepithelial tumors.

Identification of the MLL2 Complex as a Coactivator for Estrogen Receptor α

A novel estrogen receptor (ER)α coactivator complex, the MLL2 complex, which consists of MLL2, ASH2, RBQ3, and WDR5, was identified. ERα directly binds to the MLL2 complex through two LXXLL motifs in a region of MLL2 near the C terminus in a ligand-dependent manner. Disrupting the interaction between ERα and the MLL2 complex with small interfering RNAs specific against MLL2 or an MLL2 fragment representing the interacting region with ERα significantly inhibited the ERα transcription activity. The MLL2 complex was recruited on promoters of ERα target genes along with ERα upon estrogen stimulation. Inhibition of MLL2 expression decreased the estrogen-induced expression of ERα target genes cathepsin D and to a lesser extent pS2. In addition, MCF-7 cell growth was also inhibited by the depletion of MLL2. These results demonstrate that the ERα signaling pathway is critically dependent on its direct interaction with the MLL2 complex and suggest a central role for the MLL2 complex in the growth of ERα-positive cancer cells.

Overexpression of 5-Lipoxygenase in Colon Polyps and Cancer and the Effect of 5-LOX Inhibitors In vitro and in a Murine Model

Purpose: Arachidonic acid metabolism via the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways modulates cell growth and apoptosis. Many studies have examined the effects of COX inhibitors on human colorectal cancer, but the role of 5-LOX in colonic cancer development has not been well studied. The purpose of this study was to evaluate the expression of 5-LOX in colonic polyps and cancer and the effect of 5-LOX inhibition on colon cancer cell proliferation. Experimental Design: Colonic polyps, cancer, and normal mucosa were evaluated for 5-LOX expression by immunohistochemistry. Reverse transcription-PCR was used to establish 5-LOX expression in colon cancer cells. Thymidine incorporation and cell counts were used to determine the effect of the nonspecific LOX inhibitor Nordihydroguaiaretic Acid and the 5-LOX inhibitor Rev5901 on DNA synthesis. A heterotopic xenograft model in athymic mice using HT29 and LoVo human colon cancer cells was used to evaluate the effect of the 5-LOX inhibitor zileuton on tumor growth. Results: 5-LOX is overexpressed in adenomatous polyps and cancer compared with that of normal colonic mucosa. LOX inhibition and 5-LOX inhibition decreased DNA synthesis in a concentration- and time-dependent manner in the Lovo cell line (P < 0.05). Inhibition of 5-LOX in an in vivo colon cancer xenograft model inhibited tumor growth compared with that of controls (P < 0.05). Conclusions: This study showed that 5-LOX is up-regulated in adenomatous colon polyps and cancer compared with normal colonic mucosa. The blockade of 5-LOX inhibits colon cancer cell proliferation both in vitro and in vivo and may prove a beneficial chemopreventive therapy in colon cancer.

5-Lipoxygenase, a Marker for Early Pancreatic Intraepithelial Neoplastic Lesions

Pancreatic cancer has an abysmal prognosis because of late diagnosis. Therefore, it is important to identify risk factors if we are to be able to prevent and detect this cancer in an early, noninvasive stage. Pancreatic intraepithelial neoplasias (PanIN) are the precursor lesions which could be an ideal target for chemoprevention. This study shows up-regulation of 5-lipoxygenase (5-LOX) in all grades of human PanINs and early lesions of pancreatic cancer in two different animal models (EL-Kras mice and N -nitrosobis(2-oxopropyl)amine–treated hamsters) by immunohistochemistry. The results were consistent in all tissues examined, including seven chronic pancreatitis patients, four pancreatic cancer patients, one multiorgan donor, nine EL-Kras mice, and three N -nitrosobis(2-oxopropyl)amine–treated hamsters, all with PanINs. Overexpression of 5-LOX in NIH3T3 cells resulted in greater sensitivity of these cells to the growth inhibitory effects of the 5-LOX inhibitor Rev5901. These findings provide evidence that 5-LOX plays a key role in the development of pancreatic cancer. Furthermore, the lipoxygenase pathway may be a target for the prevention of this devastating disease.

Surgical outcomes of patients with gastric carcinoma: the importance of primary tumor location and microvessel invasion

BACKGROUND This study was done to identify clinicopathologic predictors of disease-free survival (DFS) and overall survival (OS) among patients undergoing potentially curative resections for gastric carcinoma. METHODS We reviewed 110 patients surgically treated between 1987 and 2001. There were 74 men and 36 women with a mean age of 65.2 years (range 27 to 87 years). Log-rank tests and Kaplan-Meier survival curves were generated to determine clinicopathologic factors influencing DFS and OS. Significant factors were then determined with Cox multivariate analysis. RESULTS Median survival for all patients was 38.2 months and the estimated 5-year OS was 42.3%. There were no significant differences in DFS or OS for female sex, black race, or age>65. Symptoms associated with lower DFS were weight loss and palpable abdominal mass (P<.05), although none were predictive for OS. Median survival was markedly worse in patients undergoing esophagogastrectomy versus gastrectomy (26.8 vs 52.3 months; P<.05), although there were no significant differences between patients undergoing total gastrectomy versus subtotal gastrectomy. As expected, OS was inversely proportional to the American Joint Committee on Cancers tumor stage. When compared with patients with partial-thickness tumors (T(1-2)), full-thickness (T(3-4)) tumors had a decreased median survival (63.8 vs 27.9 months; P<.01). Although N(2) stage was associated with decreased survival (20.6 months), patient outcomes were similar for N(0) and N(1) stages (52.5 and 48.8 months). Lymphatic and capillary invasion (21.4 vs 45.3 months; P<.02) and proximal location of primary tumors (28.5 vs 58.6 months; P<.02) were the only other factors adversely affecting survival. Lauren classification and histologic grade were not significant predictors of patient outcomes. CONCLUSIONS In addition to the American Joint Committee on Cancer stage, microvessel involvement and tumor location are important predictors of DFS and OS in gastric cancer and should be included in risk stratification and selection criteria for patients entering novel adjuvant or neoadjuvant clinical trials.

Magnetic resonance elastography and biomarkers to assess fibrosis from recurrent hepatitis C in liver transplant recipients.

Background. Imaging techniques evaluating liver stiffness (magnetic resonance elastography [MRE]) and biomarkers may be useful indicators of fibrosis stage in hepatitis C virus (HCV)+patients. Our aim was to compare the accuracy of MRE and biomarkers in staging fibrosis because of recurrent HCV in liver transplant (LT) recipients with hepatocellular carcinoma. Methods. Liver magnetic resonance imaging and MRE, FIBROSpectII, aspartate aminotransferase-to-platelet ratio index (aspartate aminotransferase [AST]: platelet index), AST:alanine aminotransferase ratio, and magnetic resonance imaging/MRE-guided biopsies targeting the stiffest regions (right and left lobes) were performed in HCV+LT recipients. Sensitivity, specificity, positive predictive value (PPV)/negative predictive value (NPV), and likelihood ratios were calculated for the best cutoff by receiver operating characteristic analysis. Results. Thirty-two recipients were included: 28 men, age 60 (±6.4) years, and time since LT 3.25 (±1.68) years. Both MRE (P=0.0001) and FIBROSpectII (P=0.009) were significantly different between no fibrosis and more than or equal to stage 1 groups, whereas aspartate aminotransferase-to-platelet ratio index and AST:alanine aminotransferase ratio were not different. Areas under the receiver operating characteristic curve were 0.87 for MRE and 0.84 for FIBROSpectII. MRE cutoff of 3.81 kPa had 87.5% sensitivity, 79.2% specificity, 58.3% PPV, and 95.0% NPV; FIBROSpectII cutoff of 42 had 87.5% sensitivity, 70.0% specificity, 53.8% PPV, and 93.3% NPV for detection of more than or equal to stage 1 fibrosis. Two patients had high MRE values because of unexpected acute rejection and portal vein thrombosis. Conclusions. MRE and FIBROSpectII are highly sensitive in detecting fibrosis due to recurrent HCV. Both are limited by the low specificity/PPV and confounding because of other graft complications. Values below the MRE and FIBROSpectII cutoffs, however, strongly suggest the absence of fibrosis and may avert the need for protocol biopsy staging.

Concurrent PEDF deficiency and Kras mutation induce invasive pancreatic cancer and adipose-rich stroma in mice

Background and aims Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. Methods In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-KrasG12D mouse model of non-invasive cystic papillary neoplasms. Results EL-KrasG12D/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-KrasG12D/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). Conclusions These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

A high omega-3 fatty acid diet mitigates murine pancreatic precancer development.

BACKGROUND Diets containing omega-3 (ω-3) fat have been associated with decreased tumor development in the colon, breast, and prostate. We assessed the effects of a diet rich in ω-3 fat on the development of pancreatic precancer in elastase (EL)-Kras transgenic mice and examined the effect of an ω-3 fatty acid on pancreatic cancer cells in vitro. MATERIALS AND METHODS Two cohorts of EL-Kras mice were fed a high ω-3 fat diet (23% menhaden oil) for 8 and 11 mo and compared with age-matched EL-Kras mice fed standard chow (5% fat). Pancreata from all mice were scored for incidence and frequency of precancerous lesions. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA) to assess proliferative index in lesions of mice fed either a high ω-3 or standard diet. In vitro, the effect of the ω-3 fatty acid, docosahexaenoic acid (DHA), on two pancreatic cancer cell lines was assessed. Cancer cell proliferation was assessed with an MTT assay; cell cycle analysis was performed by flow cytometry; and apoptosis was assessed with annexin/PI staining. RESULTS The incidence, frequency, and proliferative index of pancreatic precancer in EL-Kras mice was reduced in mice fed a high ω-3 fat diet compared with mice fed a standard chow. In vitro, DHA treatment resulted in a concentration-dependent decrease in proliferation through both G1/G0 cell cycle arrest and induction of apoptosis. CONCLUSIONS A high ω-3 fat diet mitigates pancreatic precancer by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.