Yao Ching Hung

Androgen Receptor Promotes Hepatitis B Virus–Induced Hepatocarcinogenesis Through Modulation of Hepatitis B Virus RNA Transcription

Targeting androgen receptor may prevent hepatitis B virus–induced liver cancer. Destroying the Source of Liver Cancer Hepatocellular carcinoma represents the majority of cases of primary liver cancer; it is the fifth most common cancer and the third leading cause of cancer death worldwide. Several risk factors for hepatocellular carcinoma have been identified, but gender and hepatitis B virus (HBV) infection have proven to be uniquely associated with the disease by undetermined mechanisms. HBV infection alone, which is endemic in many Asian countries including China, accounts for approximately 53% of hepatocellular carcinoma cases worldwide. Decades of research have examined risk factor exposures such as age, history of hepatitis, occupation, regular alcohol drinking, regular cigarette smoking, and family history of liver cancer to explain the gender disparities that are so prevalent among HBV-induced liver cancers, but none of these were fully accountable, suggesting that other unknown susceptibilities are lurking. Liver cancer arises most frequently in the setting of chronic liver inflammation. After infection by the hepatitis virus, the host’s inflammatory immune response to the viral antigens induces hepatocyte damage and is followed by the pathogenesis of liver cancer. Although it has been postulated that sex hormones may interact with HBV infection in the process and lead to a dominant sex disparity in liver cancer risk, this has never been decisively shown. Now, Wu et al. use a genetically modified mouse model of HBV-induced liver cancer to explore how sex hormones, and specifically their receptors, play a role in promoting the disease. They find that targeting of the androgen receptor—and not androgens, as is frequently done in the clinic—hampers tumor formation at the gross and mechanistic levels. These results shed new light on a previously unexplored pathway and provide an intervention modality that is translatable in the clinic, and may explain why men are more susceptible to liver cancer than women. Hepatitis B virus (HBV)–induced hepatitis and carcinogen-induced hepatocellular carcinoma (HCC) are associated with serum androgen concentration. However, how androgen or the androgen receptor (AR) contributes to HBV-induced hepatocarcinogenesis remains unclear. We found that hepatic AR promotes HBV-induced hepatocarcinogenesis in HBV transgenic mice that lack AR only in the liver hepatocytes (HBV-L-AR−/y). HBV-L-AR−/y mice that received a low dose of the carcinogen N′-N′-diethylnitrosamine (DEN) have a lower incidence of HCC and present with smaller tumor sizes, fewer foci formations, and less α-fetoprotein HCC marker than do their wild-type HBV-AR+/y littermates. We found that hepatic AR increases the HBV viral titer by enhancing HBV RNA transcription through direct binding to the androgen response element near the viral core promoter. This activity forms a positive feedback mechanism with cooperation with its downstream target gene HBx protein to promote hepatocarcinogenesis. Administration of a chemical compound that selectively degrades AR, ASC-J9, was able to suppress HCC tumor size in DEN-HBV-AR+/y mice. These results demonstrate that targeting the AR, rather than the androgen, could be developed as a new therapy to battle HBV-induced HCC.

Correlation between vascular endothelial growth factor-C expression and invasion phenotype in cervical carcinomas

The correlation between vascular endothelial growth factor (VEGF)‐C gene expression and in vitro invasive activity and matrix metalloproteinase (MMP)‐2 or 9 gene expression and proteolytic activity in 11 cervical carcinoma cell lines, was investigated. Immunohistochemical expression of VEGF‐C in 52 cervical carcinoma tissues was also correlated with tumor aggressiveness with respect to clinicopathologic features, tumor vascularity, MMP‐2 expression and patient outcome. Expression of VEGF‐C mRNA differed remarkably among the cell lines and there was a statistical correlation between VEGF‐C gene expression and the number of invaded tumor cells (p = 0.0009) and MMP‐2 gene expression and activity (p < 0.05). Anti‐VEGF‐C antibody inhibited the invasive and proteolytic activity of tumor cells in a concentration‐dependent manner. VEGF‐C or MMP‐2 expression in clinical tissue samples was well correlated with depth of myometrial invasion, endometrial invasion, pelvic lymphnode metastasis and tumor vascularity (p < 0.05) and there was a close relation between VEGF‐C and MMP‐2 expression (p < 0.0001) in cervical carcinomas. Overall survival rates for 14 patients with strong VEGF‐C staining tumors were lower than those for 38 patients with weak VEGF‐C staining tumors (p = 0.0132) and VEGF‐C tissue status emerged as an independent prognostic parameter (p = 0.0232). These results suggest that VEGF‐C expression is closely related to invasion phenotype and affects the patients survival in cervical carcinomas.

Hepatic androgen receptor suppresses hepatocellular carcinoma metastasis through modulation of cell migration and anoikis

Early reports suggested androgen/androgen receptor (AR) signals promote hepatocarcinogenesis. However, all antiandrogen clinical trials failed in advanced hepatocellular carcinoma (HCC) without reasonable explanations. We examined AR functions in HCC cancer metastasis in this study. We examined hepatic AR roles in HCC metastasis by comparing liver hepatocyte AR knockout and wildtype in a carcinogen‐induced HCC mouse model. We examined tumor histology, cancer metastatic risks, and cancer survival in vivo, as well as cell anoikis and migration using primary hepatic tumor culture in vitro. We also examined therapeutic potentials of AR expression combined with the molecular targeting agent sorafenib in an HCC metastasis mouse model. We found a novel cancer phenotype in which mice lacking hepatic AR developed more undifferentiated tumors and larger tumor size at the metastatic stage. These mice also died earlier with increased lung metastasis, suggesting that hepatic AR may play dual yet opposite roles to promote HCC initiation but suppress HCC metastasis. Mechanistic dissection found that hepatic AR could enhance anoikis and suppress migration of HCC cells by way of suppression of p38 phosphorylation/activation and the nuclear factor kappa B (NF‐κB)/matrix metallopeptidase 9 (MMP9) pathway, respectively. In addition, the in vivo preclinical trials concluded that a combination therapy of increased AR expression and reduced multiple‐kinase inhibitor (sorafenib) exhibited better therapeutic efficacy. Conclusion: Our study demonstrates that AR could orchestrate intrahepatic signaling hierarchies and cellular behaviors, consequently affect HCC progression. Results from combination therapy shed light on developing new therapeutic paradigms for battling HCC at later metastatic stages. (HEPATOLOGY 2012;56:176–185)

Vascular endothelial growth factor-C expression and invasive phenotype in ovarian carcinomas

Purpose: To investigate the biological correlation between vascular endothelial growth factor (VEGF)-C expression and invasive phenotype in ovarian carcinomas. Experimental Design: Gene and protein expression levels of VEGF-C in 10 ovarian carcinoma cell lines were correlated with invasive activity of the cells. The correlation between immunohistochemical expression of VEGF-C and tumor aggressiveness in 73 ovarian carcinomas was also examined with respect to clinicopathologic features and patient outcome. Results:VEGF-C gene and protein expression differed remarkably among the cell lines, and there was a statistical correlation among VEGF-C expression, in vitro invasive activity, and matrix metalloproteinase-2 (MMP-2) gene expression and its activity. Anti-VEGF-C and anti-MMP-2 antibodies inhibited the invasive activity of tumor cells. VEGF-C expression in clinical tissue samples was well correlated with clinical stages, retroperitoneal lymph node metastasis, MMP-2 expression, angiogenesis, lymphangiogenesis, and low apoptotic index (AI). The patients whose tumors had strong VEGF-C expression and low AI underwent a poorer prognosis than did those with weak VEGF-C expression and high AI. Conclusion: VEGF-C expression is closely related to invasive phenotype and affects the patients survival in ovarian carcinomas.

A long-term follow-up study of 176 cases with adult-type ovarian granulosa cell tumors

OBJECTIVE Because of rarity, indolent clinical course, and of most importance, small sample size studies of previous ovarian granulosa cell tumors (GCTs), this study was conducted to report the clinical characteristics and long-term outcomes of 176 pathologically confirmed GCTs. METHODS Between 1984 and 2010, we retrospectively evaluated 176 patients from multiple medical centers in Taiwan. RESULTS The mean age at the diagnosis was 46 years and nearly half of the patients (45.7%) were in their fourth or fifth decades of life. The most common symptoms included abdominal pain (28.5%), followed by irregular menstruation (16.7%). The mean tumor size was 10.4 cm. The stage distribution at diagnosis was stage I in 77.8% of patients, stage II in 5.1%, stages III-V in 6.1%, and unknown in 11% of patients. The median follow-up period was 60.7 months. The recurrence rate was 21%. The overall 5- and 10-year survival rates were 96.5% and 94.1%, respectively. In univariate analysis, initial stage, presence of residual tumor after initial surgery, need for adjuvant chemotherapy, and tumor size were associated with disease recurrence. In the multivariate analysis, only the presence of residual tumor after initial surgery and tumor size were significantly associated with recurrence. CONCLUSIONS The outcomes of patients with GCTs were good, with nearly to 95% of patients surviving 5 and 10 years. The prognosis was related to initial stage, presence of residual tumor after initial surgery, and tumor size (>13.5 cm). Different surgical methods and/or adjuvant therapy appear not to affect the outcome.

Caffeic acid induces apoptosis in human cervical cancer cells through the mitochondrial pathway.

OBJECTIVE The anti-proliferation effect of caffeic acid (3,4-dihydroxycinnamic acid), isolated from Ocimum gratissimum Linn, on human cervical cancer cells (HeLa cells) was examined to elucidate the associated mechanism and death mode. MATERIALS AND METHODS Flow cytometry showed that caffeic acid treatment results in dramatically increased apoptosis of HeLa cells. Western blot analysis revealed that caffeic acid activates various processed caspases. RESULTS Caffeic acid significantly reduced proliferation of HeLa cells in a concentration-dependent manner. Morphological evidence of apoptosis, including nuclei fragmentation was clearly observed 24 and 48 hours after exposure to caffeic acid (1 mM and 10 mM) by flow cytometry. Time-dependent inhibition was also observed. Caffeic acid decreased levels of uncleaved caspase-3 and Bcl-2, and induced cleaved caspase-3 and p53. CONCLUSION Caffeic acid induces apoptosis by inhibiting Bcl-2 activity, leading to release of cytochrome c and subsequent activation of caspase-3, indicating that caffeic acid induces apoptosis via the mitochondrial apoptotic pathway. This also suggests that caffeic acid has a strong anti-tumor effect and may be a promising chemopreventive or chemotherapeutic agent.

Homeobox gene expression and mutation in cervical carcinoma cells.

An association between deregulation of homeobox (HOX) gene expression and oncogenic transformation has been recently reported in human tumors. In this study, we investigated HOX gene expression and mutation in cervical carcinoma cells. Using reverse transcription‐PCR, 11 human cervical carcinoma cell lines and 14 normal cervical tissue samples were examined for mRNA expression of the 39 class I HOX genes. DNA samples from 11 cell lines were tested for mutations in exons 1 and 2 of the HOXA10 and A13 genes using overlapping primer pairs which also cover intron 1 of these genes. HOXA1, B2, B4, C5, C10 and D13 genes were expressed in 8, 7, 9, 9, 9 and 11 of 11 cervical carcinoma cell lines, respectively, but not in any of the normal cervical tissues. HOXA9, A11, A13, B5, C4, D3 and D9 genes were expressed in all cell lines and normal tissues. In contrast, 13 of 39 HOX genes were silent in all materials examined. Single‐strand conforma‐tional polymorphism and sequence analysis revealed a C insertion after base 1042 and/or a G to C substitution at base 1113 in intron 1 of the HOXA13 gene in 4 of 11 cell lines, however, neither deletions nor mutations were detected in exons 1 and 2 of the HOXA10 and A13 genes. Our data suggest that the expression of HOXA1, B2, B4, C5, C10 and D13 genes might be involved in the process leading to the transformation of normal cervical cells. (Cancer Sci 2003; 94: 437–441)

Glutathione S-transferase GSTM1, GSTT1 and p53 codon 72 polymorphisms in human tumor cells

The genes of the glutathione S-transferase (GST) family encode enzymes that appear to be critical in cellular protection against the cytotoxic effects, whereas p53 is a tumor suppressor gene. Despite a large number of studies on germline polymorphisms of GSTM1, GSTT1 and p53 genes, there have been very few reports on genotyping of these genes in human malignant tumor cells. In this study, we investigated GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human tumor cell lines originating from different organs to clarify tissuespedic polymorphic frequency of these genes in human solid tumors. The GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex PCR techniques and PCR-RFLP analysis was conducted to identify p53 codon 72 genotypes. Gene expression of GSTM1 or GSTT1 was detected by RT-PCR in the cells with respective present genotype for each. Polymorphisms of p53 codon 72 detected by PCR-RFLP were also confirmed using SSCP and sequence analyses. GSTM1 and GSTT1 genotypes were various in 104 cell lines examined. Null GSTM1 genotype was dominant in small cell lung, kidney and ovarian carcinoma cells, whereas null GSTT1 genotype was dominant in cervical and endometrial Carcinoma cells. GSTM1 and GSTT1 genotypes in ovarian carcinoma cells were quite similar to those in small cell lung carcinoma cells. Polymorphic frequency of p53 codon 72 was also various among the cells, however, the Pro allele was found in only 1 of 6 kidney, 14 cervical and 4 endometrial carcinoma cell lines. There was a significant difference in GSTM1 and p53 genotypes between 34 small cell and 24 non small cell lung carcinoma cells (P < 0.01). Combined study on the distribution of GSTM1, GSTT1 and p53 genotypes revealed that null GSTM1 genotype was associated with the Arg allele of p53 codon 72 in 58 lung carcinoma cells and null GSTT1 genotype was associated with the Pro/Pro homozygote in 104 tumor cell lines examined. This is the first study examining GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human solid tumor cells and suggesting that polymorphic frequency of these genes may be tissue- and organ-specific. The molecular interaction between GST gene defects and p53 codon 72 genotype in the development of human malignant tumors should be further investigated.

MicroRNA-21 promotes the ovarian teratocarcinoma PA1 cell line by sustaining cancer stem/progenitor populations in vitro

IntroductionResistance of cancer stem/progenitor cells (CSPCs) to chemotherapy can lead to cancer relapse. Ovarian teratocarcinoma (OVTC) arises from germ cells and comprises pluripotent cells that can be used to study cancer cell stemness. In this study, we evaluated whether microRNA-21 (miR-21) promotes ovarian teratocarcinoma by maintaining cancer stem/progenitor populations.MethodsThe lentiviral delivery system was used to upregulate or to suppress the expression of miR-21 in the human ovarian teratocarcinoma cell line PA1 and cell growth assays were used to monitor the expression of miR-21 at different time points. Antibodies directed toward CD133, a stem cell marker, were used to identify CSPCs in the PA1 cell population, and the level of miR-21 expression was determined in enriched CSPCs. Stem cell functional assays (sphere assay and assays for CD133 expression) were used to assess the effects of miR-21 on progression of the CD133+ population.ResultsKnockdown of miR-21 in PA1 cells attenuated growth of PA1 cells whereas overexpression of miR-21 promoted cell growth. Moreover, knockdown of miR-21 resulted in a marked reduction in the CD133+ population and sphere formation of CSPCs. In contrast, overexpression of miR-21 resulted in a marked increase in the population of CD133+ cells as well as sphere formation of CSPCs.ConclusionsMicroRNA-21 plays a significant role in cancer growth by regulating stemness in cancer cells.

Successful treatment of uterine arteriovenous malformation with percutaneous embolization.

OBJECTIVE Uterine arteriovenous malformation (AVM) is a rare condition and can be life-threatening if not managed properly. We report a case that was diagnosed by typical ultrasound imaging and treated successfully with uterine arterial embolization. CASE REPORT A 28-year-old female, gravida 4, para 3, abortus 1, presented with massive vaginal bleeding 19 days after a termination of pregnancy due to fetal anomaly. After a dilatation and curettage 3 years previously, typical ultrasound image findings and a declining pattern of serum beta-hCG (human chorionic gonadotrophin), acquired AVM was highly suspected. The patient underwent bilateral uterine arterial embolization. Four weeks later, there was nearly complete resolution of the AVM and the patients menstrual cycle was restored 6 weeks after embolization. CONCLUSION AVM can be diagnosed at an early stage with the aid of history taking and ultrasound. Percutaneous embolotherapy is a safe and effective treatment for AVM, especially when fertility preservation is desired.