Yung-Ming Jeng

ELASTIC MODULUS MEASUREMENTS OF HUMAN LIVER AND CORRELATION WITH PATHOLOGY

Viral hepatitis causes fibrosis in the liver and may change mechanical properties of the liver. To evaluate the impact of fibrosis on elastic properties of human liver and to investigate potential benefits of ultrasonic elasticity imaging, 19 fresh human liver samples and 1 hepatic tumor (focal nodular hyperplasia) sample obtained during operations were studied. Simple 1-D estimates based on the cyclic compression-relaxation method were performed. Elastic modulus values were derived from the predetermined strain (controlled by a step motor system) and the stress values (measured by an electronic balance). Each specimen subsequently received histologic examination and a grade of liver fibrosis was scored from 0 to 5. Results show that the elastic modulus values were on the order of several hundreds to thousands of Pascals. The elastic modulus generally increased with the fibrosis grade, although some discrepancies existed at the middle grades of fibrosis (scores 1 to 3). The correlation between the fibrosis score and the elastic modulus was significant (p < 0.01) based on the statistical analysis using the Pearson correlation method. In addition, the relation between the elastic modulus and the fibrosis grade generally exhibited a quadratic trend. It was concluded that severity of fibrosis had a good correlation with stiffness of the liver. Results also indicated that the elasticity imaging of the liver may provide significant clinical values if the elastic modulus can be accurately measured.

β-Catenin Mutations Are Associated with a Subset of Low-Stage Hepatocellular Carcinoma Negative for Hepatitis B Virus and with Favorable Prognosis

To better understand the role of beta-catenin mutation in hepatocellular carcinoma (HCC), we correlated the gene mutation with hepatitis virus B (HBV) and hepatitis virus C (HCV) status and the clinicopathological features in 366 patients with resected primary unifocal HCC. beta-Catenin mutations were also analyzed in 55 patients with multifocal HCC (68 tumors). Of the whole series, 57 (13.1%) of 434 tumors examined had beta-catenin mutations, 34 occurred at the serine/threonine residues of the GSK-3beta region of beta-catenin. Outside the GSK-3beta phosphorylation site, codons 32 and 34 were two mutational hot spots (17 tumors). The non-HBV-related HCC that was predominantly HCV related had a higher frequency of mutation (P: < 0.00001) and more frequent mutations at codon 45 than HBV-related HCC. HBV-related HCC had a younger mean age (P: < 0.00001), and higher male-to-female ratio (P: < 0.003) and positive familial history of HCC (P: < 0.014). Among 366 unifocal HCCs selected for clinicopathological analysis, beta-catenin mutations were associated with grade I (P: = 0.005) and stage I and II HCC (P: < 0.0001), and a better 5-year survival rate (P: = 0. 00003). These findings suggest mechanisms for beta-catenin mutations differ between HBV-related and non-HBV-related HCCs, and that beta-catenin mutation is a favorable prognostic factor related to low stage. beta-Catenin mutation was associated with nuclear expression of the protein (P: < 0.00001), but we failed to detect point or large fragment deletion mutation in 39 HCCs with nuclear beta-catenin expression, presumably wild-type protein. HCCs expressing mutant nuclear beta-catenin had a better 5-year survival rate (P: < 0.007), suggesting that mutant and wild-type nuclear beta-catenin proteins are not functionally equivalent and deserve more studies for further clarification.

Overexpression and Amplification of Aurora-A in Hepatocellular Carcinoma

Purpose: Aurora-A/STK15/BTAK, a centrosome-associated serine/threonine kinase, has been shown to induce chromosomal instability, leading to aneuploidy and cell transformation. The purpose of this study was to investigate the expression and amplification of Aurora-A in hepatocellular carcinoma (HCC). Experimental Design: Aurora-A mRNA levels were measured in 224 HCCs and 199 paired nontumorous liver tissues by reverse transcription-PCR. Aurora-A mRNA and protein levels of 8 were also measured by reverse transcription-PCR and Western blot hybridization in 8 liver cancer cell lines. Amplification of Aurora-A was determined by Southern blot hybridization in 99 cases. Results: Aurora-A was overexpressed in 137 of 224 (61%) HCCs and all 8 of the cell lines. Overexpression of Aurora-A was associated with high-grade (grade II-IV), and high-stage (stage IIIB-IV) tumors, p53 mutation, infrequent β-catenin mutation, and poor outcome. Aurora-A overexpression and p53 mutation acted synergistically toward poor prognosis. Amplification of Aurora-A was detected only in 3 HCCs. Conclusion: The results show that Aurora-A is overexpressed frequently in HCC, and correlated with high grade and high stage, indicating that overexpression of Aurora-A plays a role in the development and progression of HCC.

H3K9 histone methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell adhesion molecule Ep-CAM

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression.

Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the progression of human lung adenocarcinoma. However, the mechanism underlying COX-2’s effect on tumor progression remains largely unknown. Lymphangiogenesis, the formation of new lymphatic vessels, has recently received considerable attention and become a new frontier of tumor metastasis research. Here, we study the interaction between COX-2 and the lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, in human lung cancer cells and their implication in patient outcomes. We developed an isopropyl-β-d-thiogalactopyranoside-inducible COX-2 gene expression system in human lung adenocarcinoma CL1.0 cells. We found that VEGF-C gene expression but not VEGF-D was significantly elevated in cells overexpressing COX-2. COX-2-mediated VEGF-C up-regulation was commonly observed in a broad array of non-small cell lung cancer cell lines. The use of pharmacological inhibitors or activators and genetic inhibition by EP receptor-antisense oligonucleotides revealed that prostaglandin EP1 receptor but not other prostaglandin receptors is involved in COX-2-mediated VEGF-C up-regulation. At the mechanistic level, we found that COX-2 expression or prostaglandin E2 (PGE2) treatment could activate the HER-2/Neu tyrosine kinase receptor through the EP1 receptor-dependent pathway and that this activation was essential for VEGF-C induction. The transactivation of HER-2/Neu by PGE2 was inhibited by way of blocking the Src kinase signaling using the specific Src family inhibitor, PP1, or transfection with the mutant dominant negative src plasmid. Src kinase was involved in not only the HER-2/Neu transactivation but also the following VEGF-C up-regulation by PGE2 treatment. In addition, immunohistochemical staining of 59 lung adenocarcinoma specimens showed that COX-2 level was highly correlated with VEGF-C, lymphatic vessels density, and other clinicopathological parameters. Taken together, our results provided evidence that COX-2 up-regulated VEGF-C and promotes lymphangiogenesis in human lung adenocarcinoma via the EP1/Src/HER-2/Neu signaling pathway.

Expression of the c-kit protein is associated with certain subtypes of salivary gland carcinoma

The c-Kit protein, a receptor type tyrosine kinase that plays an important role in the development of hematopoietic cells, melanocytes, and germ cells, is expressed in mastocytosis, gastrointestinal stromal cell tumors (GISTs), germ cell tumors, and several other tumors. Gain-of-function mutations in exon 11 and exon 17 have been shown as a mechanism of c-kit activation in some tumors. To study the role of c-kit in salivary gland carcinomas, we analyzed the c-kit protein expression in 79 carcinomas of major and minor salivary glands by immunohistochemistry. Although varying in intensity of staining, c-kit expression was identified very often in adenoid cystic carcinomas (20/25), lymphoepithelioma-like carcinomas (6/6) and myoepithelial carcinomas (2/2), but not in other types of salivary gland carcinoma (0/46), P<0.00001. By DNA sequencing, genetic alteration of c-kit juxtamembrane domain (exon 11) and tyrosine kinase domain (exon 17) was not found in all the three types of salivary carcinoma that had c-kit protein expression. In conclusion, c-kit protein overexpression is involved in the pathogenesis of certain types of salivary gland carcinoma, but mutation of the gene is not the mechanism of c-kit activation.

Peginterferon Alfa-2a Plus Ribavirin for the Treatment of Dual Chronic Infection With Hepatitis B and C Viruses

BACKGROUND & AIMS Dual chronic infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) is common in areas endemic for either virus. Combination therapy with ribavirin and pegylated interferon (peginterferon) is the standard of care for patients with HCV monoinfection. We investigated the effects of combination therapy in patients infected with both HBV and HCV (genotypes 1, 2, or 3). METHODS The study included 321 Taiwanese patients with active HCV infection; 161 also tested positive for hepatitis B surface antigen (HBsAg) and 160 were HBsAg-negative (controls). Patients with HCV genotype 1 infection received peginterferon alfa-2a (180 mug) weekly for 48 weeks and ribavirin (1000-1200 mg) daily. Patients with HCV genotypes 2 or 3 received peginterferon alfa-2a weekly for 24 weeks and ribavirin (800 mg) daily. At 24 weeks posttreatment, patient samples were examined for a sustained virologic response (SVR) against HCV (serum HCV levels decreased to <25 IU/mL). RESULTS In patients with HCV genotype 1 infection, the SVR was 72.2% in dually infected patients vs 77.3% in monoinfected patients after treatment. For patients with HCV genotype 2/3 infections, the SVR values were 82.8% and 84.0%, respectively, after treatment. Serum HBV DNA eventually appeared in 36.3% of 77 dual-infected patients with undetectable pretreatment levels of HBV DNA; this was not accompanied by significant hepatitis. Posttreatment HBsAg clearance was observed in 11.2% of 161 dual-infected patients. CONCLUSIONS Combination therapy with peginterferon alfa-2a and ribavirin is equally effective in patients with HCV monoinfection and in those with dual chronic HCV/HBV infection.

Mutation and loss of expression of ARID1A in uterine low-grade endometrioid carcinoma.

ARID1A is a recently identified tumor suppressor gene that is mutated in approximately 50% of ovarian clear cell and 30% of ovarian endometrioid carcinomas. The mutation is associated with loss of protein expression as assessed by immunohistochemistry. In this study, we evaluated ARID1A immunoreactivity in a wide variety of carcinomas to determine the prevalence of ARID1A inactivation in carcinomas. Mutational analysis of ARID1A was carried out in selected cases. Immunoreactivity was not detected (corresponding to inactivation or mutation of ARID1A) in 36 (3.6%) of 995 tumors. Uterine low-grade endometrioid carcinomas showed a relatively high-frequency loss of ARID1A expression, as 15 (26%) of 58 cases were negative. The other tumor that had a relatively high-frequency loss of ARID1A expression was gastric carcinoma (11%). Mutational analysis showed 10 (40%) of 25 uterine endometrioid carcinomas; none of 12 uterine serous carcinomas and none of 56 ovarian serous and mucinous carcinomas harbored somatic ARID1A mutations. All mutations in endometrioid carcinomas were nonsense or insertion/deletion mutations, and tumors with ARID1A mutations showed complete loss or clonal loss of ARID1A expression. In conclusion, this study is the first large-scale analysis of a wide variety of carcinomas showing that uterine low-grade endometrioid carcinoma is the predominant tumor type harboring ARID1A mutations and frequent loss of ARID1A expression. These findings suggest that the molecular pathogenesis of low-grade uterine endometrioid carcinoma is similar to that of ovarian low-grade endometrioid and clear cell carcinoma, tumors that have previously been shown to have a high-frequency loss of expression and mutation of ARID1A.

Multiple Lineages of Human Breast Cancer Stem/Progenitor Cells Identified by Profiling with Stem Cell Markers

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44+/CD24-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR+/ESA+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44+/CD24−/low, ESA+, CD133+, CXCR4+ and PROCR+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

TET1 Suppresses Cancer Invasion by Activating the Tissue Inhibitors of Metalloproteinases

Tumor suppressor gene silencing through cytosine methylation contributes to cancer formation. Whether DNA demethylation enzymes counteract this oncogenic effect is unknown. Here, we show that TET1, a dioxygenase involved in cytosine demethylation, is downregulated in prostate and breast cancer tissues. TET1 depletion facilitates cell invasion, tumor growth, and cancer metastasis in prostate xenograft models and correlates with poor survival rates in breast cancer patients. Consistently, enforced expression of TET1 reduces cell invasion and breast xenograft tumor formation. Mechanistically, TET1 suppresses cell invasion through its dioxygenase and DNA binding activities. Furthermore, TET1 maintains the expression of tissue inhibitors of metalloproteinase (TIMP) family proteins 2 and 3 by inhibiting their DNA methylation. Concurrent low expression of TET1 and TIMP2 or TIMP3 correlates with advanced node status in clinical samples. Together, these results illustrate a mechanism by which TET1 suppresses tumor development and invasion partly through downregulation of critical gene methylation.