Hwudaurw Chen

Cx37 and Cx43 Localize to Zona Pellucida in Mouse Ovarian Follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.

Transforming growth factor Beta2 is required for valve remodeling during heart development

Although the function of transforming growth factor beta2 (TGFβ2) in epithelial mesenchymal transition (EMT) is well studied, its role in valve remodeling remains to be fully explored. Here, we used histological, morphometric, immunohistochemical and molecular approaches and showed that significant dysregulation of major extracellular matrix (ECM) components contributed to valve remodeling defects in Tgfb2−/− embryos. The data indicated that cushion mesenchymal cell differentiation was impaired in Tgfb2−/− embryos. Hyaluronan and cartilage link protein‐1 (CRTL1) were increased in hyperplastic valves of Tgfb2−/− embryos, indicating increased expansion and diversification of cushion mesenchyme into the cartilage cell lineage during heart development. Finally, Western blot and immunohistochemistry analyses indicate that the activation of SMAD2/3 was decreased in Tgfb2−/− embryos during valve remodeling. Collectively, the data indicate that TGFβ2 promotes valve remodeling and differentiation by inducing matrix organization and suppressing cushion mesenchyme differentiation into cartilage cell lineage during heart development. Developmental Dynamics 240:2127–2141, 2011.

Gene Targeted Ablation of High Molecular Weight Fibroblast Growth Factor-2

Fibroblast growth factor‐2 (FGF2) is produced as high molecular weight isoforms (HMW) and a low molecular weight isoform (LMW) by means of alternative usage of translation start sites in a single Fgf2 mRNA. Although the physiological function of FGF2 and FGF2 LMW has been investigated in myocardial capillarogenesis during normal cardiac growth, the role of FGF2 HMW has not been determined. Here, we report the generation of FGF2 HMW‐deficient mice in which FGF2 HMW isoforms are ablated by the Tag‐and‐Exchange gene targeting technique. These mice are normal and fertile with normal fecundity, and have a normal life span. Histological, immunohistochemical, and morphometric analyses indicate normal myocardial architecture, blood vessel, and cardiac capillary density in young adult FGF2 HMW‐deficient mice. These mice along with the FGF2‐ and FGF2 LMW‐deficient mice that we have generated previously will be very useful for elucidating the differential functions of FGF2 isoforms in pathophysiology of cardiovascular diseases. Developmental Dynamics 238:351–357, 2009.

Altered tissue behavior of a non-aneurysmal descending thoracic aorta in the mouse model of Marfan syndrome

Aortic aneurysm is predominantly found in the ascending aorta in patients with Marfan syndrome (MFS). However, descending aortic disease has emerged as a problem since people are living longer because of improved medical and surgical management of the ascending aorta. Diagnostic procedures before disease onset and the mechanisms involved in the transition of normal aortic tissue to aneurysm remain unclear. We determined signs of descending aortic disease before disease onset in mice with a mutation in the fibrillin 1 gene (Fbn1+/C1039G), a validated mouse model of disease susceptibility and progression of aortic aneurysm of MFS. We analyzed a tubular unfixed non-aneurysmal descending thoracic aorta from 8-month-old wild-type and Fbn1+/C1039G mice by a tubular biaxial tester that works in conjunction with a two-photon nonlinear microscope. Fbn1+/C1039G mouse aorta was more compliant in the circumferential direction. Two-photon imaging showed defective organization of adventitial collagen fibers in the pressurized aortas of Fbn1+/C1039G mice. Moreover, disruption in the elastic lamina was noted in the absence of aneurysms in pressurized aortas but not unpressurized aortas of Fbn1+/C1039G mice. At the molecular level, this altered tissue behavior in non-aneurysmal descending aortas of Fbn1+/C1039G mice was accompanied by an increasing trend of canonical but not noncanonical, transforming growth factor-β (TGFβ) signaling. Finally, assays of in vitro collagen lattice formation in mouse wild-type and TGFβ1-deficient embryonic fibroblasts indicate that TGFβ1 can regulate collagen organization. The ability to reveal the presence of altered biomechanics and microstructure coupled with subtle changes in TGFβ signaling provides a novel surrogate measure of tissue susceptibility to aneurysm before disease onset.

The Na +/H + exchanger controls deoxycholic acid-induced apoptosis by a H +-activated, Na +-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na+/H+ exchanger (NHE) and Na+ influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM -0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na+, subsequent loss of intracellular K+, an increase of Ca2+ and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na+, K+ and Ca2+ changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na+ levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na+ concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na+ influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV‐B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV‐B irradiation were banks of 6 Westinghouse FS‐40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30‐min dorsal UV‐B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 105 J m‐2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16‐ to 35‐fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.

Specific microRNA–mRNA Regulatory Network of Colon Cancer Invasion Mediated by Tissue Kallikrein–Related Peptidase 6

Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P = .005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer. Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.

Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis.

Su1163 The Loss of Beclin-1 Expression Correlates With Progression to Esophageal Adenocarcinoma and Autophagy Resistance: the Role of Bile Acids

Background and Aims: Autophagy is a normal physiological mechanism for the degradation of cellular proteins and organelles. Excessive autophagy induced by cellular stress leads to cell death. However, the exact role of autophagy in cancer is not completely clear. Beclin1 plays a central role in the regulation of autophagy. The major goal of our study was to evaluate the role of Beclin-1 and autophagy and the effect of bile acids in the esophageal carcinogenesis. Barretts esophagus (BE) is a premalignant lesion of distal esophagus associated with an increased risk for the development of esophageal adenocarcinoma (EAC). Methods: Beclin-1 expression was evaluated using immunohistochemistry, immunoblotting, or RTPCR in (1) biopsies obtained from 62 patients with BE or EAC, (2) tissues from a rat model of BE and EAC, and (3) esophageal cell lines. Since reflux of bile acids is important in esophageal carcinogenesis, the effect of acute and chronic exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression was evaluated. Autophagy was assessed by electron microscopy or by transfection with GFP-LC3 plasmid and calculating the percentage of GFP-LC3 positive cells with punctuate pattern. Results: Our studies using human biopsies and rat tissues showed that Beclin-1 expression was high in squamous epithelium, while its expression was low in BE and EAC. A similar pattern was observed in esophageal cell lines. HET-1A cells (derived from normal squamous epithelium) showed high levels of Beclin-1, but lower levels of Beclin-1 were found in BE cells (CP-A, CP-C) and EAC cells (OE33 cells). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy in CP-A cells. In contrast, chronic exposure to DCA did not result in any alteration of Beclin-1 or autophagy. To demonstrate that Beclin-1 plays central role in autophagy we used siRNA to decrease Beclin-1 expression and then autophagy was evaluated after treatment with DCA. No increase in autophagy was detected compared to untreated control CP-A cells. Conclusions: In summary, our data suggests that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance. We propose that autophagy resistance in combination with apoptosis resistance induced by chronic exposure to bile acids contributes to EAC development.

Abstract 3793: Bile acids and autophagy resistance in Barrett's esophagus

Background: Barrett9s esophagus (BE) is a premalignant lesion of the distal esophagus that is associated with increased risk of esophageal cancer (EAC). Bile acids are implicated in BE development. Autophagy is a tightly-regulated process involving degradation and recycling of cellular proteins and organelles. The genetic links between autophagy defects and cancer suggest that autophagy is involved in tumor suppression. We have previously shown that acute exposure to bile acids induces autophagy. In this study we tested the hypothesis that chronic exposure to bile acids leads to loss of capability to undergo autophagy and that BE is resistant to autophagy. Methods: Tissue biopsies from sixty patients with different grades of BE dysplasia, EAC and control tissues were evaluated for the expression of Beclin, p-mTOR, p-AKT by immunohistochemistry. Transmission electron microscopy (TEM) was used to evaluate autophagy in tissues starved for 4 hours in Hank9s balanced salt solution (HBSS, autophagy inducer). To study chronic effects of DCA in vitro we developed CP-A cells resistant to DCA. These new CP-AR cells are able to survive and proliferate in medium containing 0.2mM DCA. Both control CP-A cells and CP-AR cells were treated with DCA and then evaluated for autophagic markers, Beclin 1 and p62. Autophagy was also assessed by quantification of cells with the punctuate pattern of GFP-LC3. Results: Our data indicate that tissue biopsies from patients with BE dysplasia and esophageal adenocarcinoma: (1) are resistant to autophagy induced by HBSS, (2) express increased levels of proteins associated with autophagy resistance [phosphorylated Akt (p-Akt) and p-mTOR] and (3) express decreased levels of the key pro-autophagic protein Beclin-1. In addition, the levels of Beclin-1 in non-dysplastic tissue or control tissues (duodenum, squamous epithelium) were high, while p-mTOR and p-Akt were low. CP-AR cells treated with DCA expressed same levels of p62 and Beclin 1 as untreated cells. In contrast, the levels of these proteins were increased in parental CP-A cells exposed to DCA indicating an increase in autophagy. Furthermore, there was no significant difference in number of GFP-LC3 positive cells with punctuate pattern between untreated and DCA treated resistant CP-AR cells. However, 103% increase in GFP-LC3 positive cells with punctuate pattern was observed after DCA treatment in sensitive CP-A cells compared to untreated CP-A cells. Conclusion: Dysplastic BE and EAC have decreased capability to undergo autophagy. In vitro studies show that chronic exposure to bile acids may be crucial factor in the development of autophagy resistance in BE esophagus. These alterations may cause a consequent increase in DNA damage leading to cancer progression. 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 3793. doi:10.1158/1538-7445.AM2011-3793