Hung-Wei Pan

Overexpression of osteopontin is associated with intrahepatic metastasis, early recurrence, and poorer prognosis of surgically resected hepatocellular carcinoma

Intrahepatic metastasis via portal vein spread is an important feature and a crucial unfavorable prognostic factor of hepatocellular carcinoma (HCC). To identify the molecular factors for tumor progression, the authors used differential display (DD) to analyze aberrant gene expression in HCC. The goal of the current study was to elucidate the clinicopathologic and prognostic significance of osteopontin (OPN) in HCC progression.

Stathmin overexpression cooperates with p53 mutation and osteopontin overexpression, and is associated with tumour progression, early recurrence, and poor prognosis in hepatocellular carcinoma.

Stathmin, a major microtubule‐depolymerizing protein, is involved in cell cycle progression and cell motility. This study aimed to elucidate its role in the progression, early tumour recurrence (ETR), and prognosis of hepatocellular carcinoma (HCC). Stathmin mRNA was overexpressed in 88/156 (56%) resected, unifocal, primary HCCs, while p53 mutation was present in 72 (46%) and osteopontin mRNA overexpression in 79 (51%). Stathmin mRNA expression exhibited high concordance (93%) with protein expression in 107 cases examined by immunohistochemistry. Stathmin overexpression correlated with high α‐fetoprotein (>200 ng/ml, p = 0.02), larger tumour size (>5 cm, p = 0.012), high tumour grade (p < 0.0002), high tumour stage (stage IIIA–IV) with vascular invasion and various degrees of intrahepatic metastasis (p < 1 × 10−8), ETR (p = 0.003), and lower 5‐year survival (p = 0.0007). Stathmin protein expression was often more intense in the peripheral regions of tumour trabeculae, tumour borders, and portal vein tumour thrombi. Stathmin overexpression correlated with p53 mutation (p = 0.017) and osteopontin overexpression (p = 1 × 10−8), both of which were associated with vascular invasion (both p < 0.0001) and poorer prognosis (p < 0.0004 and p = 0.0004, respectively). Regardless of the status of p53 mutation or osteopontin expression, stathmin overexpression was associated with higher vascular invasion (all p < 0.0001). Approximately 90% of HCCs harbouring stathmin overexpression with concomitant p53 mutation or osteopontin overexpression exhibited vascular invasion, and hence the lowest 5‐year survival, p = 0.00018 and p = 0.0009, respectively. However, we did not find that stathmin overexpression exerted prognostic impact independent of tumour stage. In conclusion, stathmin expression correlates with metastatic potential, is an important prognostic factor for HCC, and may serve as a useful marker to predict ETR. Copyright

Lin-28B expression promotes transformation and invasion in human hepatocellular carcinoma

MicroRNAs (miRNAs) play critical roles in embryonic development and are frequently deregulated in human cancers. The let-7 family members are tumor-suppressing miRNAs and are frequently downregulated in cancer cells. Lin-28 and Lin-28B are RNA-binding proteins highly expressed in embryonic tissues. Lin-28 proteins block let-7 precursors from being processed to mature miRNAs by inducing terminal uridylation and degradation of let-7 precursors. Here, we report that Lin-28B, but not Lin-28, is highly expressed in hepatocellular carcinoma (HCC). Lin-28B expression was more frequently noted in high-grade HCCs with high alpha-fetoprotein levels. Knockdown of Lin-28B by RNA interference in the HCC cell line HCC36 suppressed proliferation in vitro and reduced in vivo tumor growth in NOD/SCID mice. In contrast, overexpression of Lin-28B in the HCC cell line HA22T enhanced tumorigenicity. Overexpression of Lin-28B also induced epithelial-mesenchymal transition in HA22T cells and hence, invasion capacity. Large-scale real-time PCR array analysis revealed that, among 380 miRNAs, only let-7/mir-98 family members were regulated by Lin-28B. Lin-28B overexpression enhanced the expression of the known let-7 targets c-myc and HMGA2. It was also found that Lin-28B enhanced the expression of type 1 insulin-like growth factor receptor in a let-7-dependent manner. These results indicate that Lin-28B regulates tumor formation and invasion in HCC through coordinated repression of the let-7/mir-98 family and induction of multiple oncogenic pathways.

Silencing of miR-1-1 and miR-133a-2 cluster expression by DNA hypermethylation in colorectal cancer

MicroRNAs are small non-coding RNA molecules that play important roles in the multistep process of colorectal carcinoma (CRC) development. The present study evaluated the relationship between miR-1-1 and miR-133a-2 expression and DNA methylation, and its putative biological role in CRC. The results indicated that DNA methylation regulated the expression of the miR-1-1 and miR-133a-2 cluster in CRC cell lines. Expression of miR-1 and miR-133a was further evaluated in 64 paired tissue samples (CRC tumor and adjacent normal mucosa) using the stem-loop real-time polymerase chain reaction. The miR-1-133a cluster displayed significantly lower expression in CRC tissue compared to adjacent normal mucosa (P<0.001). The results also indicated frequent hypermethylation of the CpG islands upstream of miR-1-133a (54.6%). Liver metastatic tissues exhibited significantly lower miR-1 (P<0.001) and miR-133a (P<0.001) expression compared to adjacent normal mucosa. Expression of the miR-1-133a cluster inversely correlated with TAGLN2 in the tumor specimens. In conclusion, epigenetic repression of the miR-1-133a cluster may play a critical role in colorectal cancer metastasis by silencing TAGLN2.

Significance of Aurora B overexpression in hepatocellular carcinoma. Aurora B Overexpression in HCC

BackgroundTo investigate the significance of Aurora B expression in hepatocellular carcinoma (HCC).MethodsThe Aurora B and Aurora A mRNA level was measured in 160 HCCs and the paired nontumorous liver tissues by reverse transcription-polymerase chain reaction. Mutations of the p53 and β-catenin genes were analyzed in 134 and 150 tumors, respectively, by direct sequencing of exon 2 to exon 11 of p53 and exon 3 of β-catenin. Anticancer effects of AZD1152-HQPA, an Aurora B kinase selective inhibitor, were examined in Huh-7 and Hep3B cell lines.ResultsAurora B was overexpressed in 98 (61%) of 160 HCCs and in all 7 HCC cell lines examined. The overexpression of Aurora B was associated with Aurora A overexpression (P = 0.0003) and p53 mutation (P = 0.002) and was inversely associated with β-catenin mutation (P = 0.002). Aurora B overexpression correlated with worse clinicopathologic characteristics. Multivariate analysis confirmed that Aurora B overexpression was an independent poor prognostic factor, despite its interaction with Aurora A overexpression and mutations of p53 and β-catenin. In Huh-7 and Hep3B cells, AZD1152-HQPA induced proliferation blockade, histone H3 (Ser10) dephosphorylation, cell cycle disturbance, and apoptosis.ConclusionAurora B overexpression is an independent molecular marker predicting tumor invasiveness and poor prognosis of HCC. Aurora B kinase selective inhibitors are potential therapeutic agents for HCC treatment.

ASPM Is a Novel Marker for Vascular Invasion, Early Recurrence, and Poor Prognosis of Hepatocellular Carcinoma

Purpose:Abnormal spindle-like microcephaly associated (ASPM) plays an important role in neurogenesis and cell proliferation. This study is to elucidate its role in hepatocelllular carcinoma (HCC), particularly early tumor recurrence (ETR) and prognosis. Experimental Design: We used reverse transcription-PCR assays to measure the ASPM mRNA levels in 247 HCC and correlated with clinicopathologic and molecular features. Results:ASPM mRNA levels were high in fetal tissues but very low in most adult tissues. ASPM mRNA was overexpressed in 162 HCC (66%) but not in benign liver tumors. ASPM overexpression correlated with high α-fetoprotein (P = 1 × 10-8), high-grade (grade II-IV) HCC (P = 2 × 10-6), high-stage (stage IIIA-IV) HCC (P = 1 × 10-8), and importantly ETR (P = 1 × 10-8). ETR is the most critical unfavorable clinical prognostic factor. Among the various independent histopathologic (tumor size, tumor grade and tumor stage) and molecular factors (p53 mutation, high α-fetoprotein, and ASPM overexpression), tumor stage was the most crucial histologic factor (odds ratio, 14.7; 95% confidence interval, 6.65-33.0; P = 1 × 10-8), whereas ASPM overexpression (odds ratio, 6.49; P = 1 × 10-8) is the most important molecular factor associated with ETR. ASPM overexpression was associated with vascular invasion and ETR in both p53-mutated (all P values = 1 × 10-8) and non-p53-mutated HCC (P = 1 × 10-8 and 0.00088, respectively). Hence, patients with APSM-overexpressing HCC had lower 5-year survival (P = 0.000001) in both p53-mutated (P = 0.00008) and non-p53-mutated HCC (P = 0.0027). In low-stage (stage II) HCC, ASPM overexpression also correlated with higher ETR (P = 0.008). Conclusion:ASPM overexpression is a molecular marker predicting enhanced invasive/metastatic potential of HCC, higher risk of ETR regardless of p53 mutation status and tumor stage, and hence poor prognosis.

Overexpression of KIAA0101 Predicts High Stage, Early Tumor Recurrence, and Poor Prognosis of Hepatocellular Carcinoma

Purpose: KIAA0101 is a proliferating cell nuclear antigen–associated factor and involved in cell proliferation. This study is to elucidate its role in the progression, early tumor recurrence (ETR), and prognosis of hepatocellular carcinoma (HCC). Experimental Design: KIAA0101 mRNA was measured by reverse transcription-PCR in 216 resected, unifocal, primary HCCs and its protein in 164 cases by immunohistochemistry. Results: KIAA0101 mRNA was overexpressed in 131 (61%) HCCs, and protein was detected in 105 (64%). KIAA0101 mRNA overexpression correlated with higher tumor grade (P = 0.0001), higher tumor stage with vascular invasion and various extents of intrahepatic spread (P = 1 × 10−8), ETR (P = 1.8 × 10−6), and lower 5-year survival (P = 0.0026). Multivariate analysis confirmed that KIAA0101 overexpression was an independent risk factor associated with high-grade tumor (P = 0.0001), high-stage tumor (P < 0.0001), and ETR (P = 0.0052) and thus contributed to poor prognosis. KIAA0101 protein–positive tumor cells accumulated at the borders of tumor macrotrabeculae and were more abundant in tumor thrombi than in the main tumors. Hence, KIAA0101 may contribute to growth advantage and resistance to hypoxic insult. In this series, p53 mutation was detected in 93 of 184 (51%) HCCs. In both p53-mutated and non–p53-mutated HCCs, KIAA0101 overexpression correlated with higher vascular invasion (stages IIIA to IV; all Ps < 0.0001) and, accordingly, led to lower 5-year survival rates (P = 0.011 and 0.029, respectively). Conclusion: KIAA0101 correlates with enhanced metastatic potential and is a significant prognostic factor of HCC.

MicroRNA Dysregulation in Gastric Cancer

Gastric carcinogenesis is a complex multistep process involving genetic dysregulation of proto-oncogenes and tumorsuppressor genes, and has recently entered the era of microRNAs (miRNAs), a class of small non-coding RNAs that posttranscriptionally regulate gene expression and control various cellular functions. MicroRNAs are small (approximately 22 nucleotides) non-coding RNAs that play fundamental roles in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis. MicroRNAs have been revealed to be involved in various stages of cancer development, showing that abnormal miRNA expressions play critical roles in modulating expression of known oncogenes or tumor suppressor genes during cancer progression. Therefore, microRNAs can perform the function of onco-miRs or tumor-suppressor-miRs in gastric carcinogenesis. This review summarizes a current understanding of the connection between miRNAs and gastric cancer. Additionally, this paper outlines the applications of miRNAs in clinical practice, such as diagnosis, prognosis, detection, and therapy of gastric cancer.

Co-modulated behavior and effects of differentially expressed miRNA in colorectal cancer

BackgroundMicroRNAs (miRNAs) are short noncoding RNAs (approximately 22 nucleotides in length) that play important roles in colorectal cancer (CRC) progression through silencing gene expression. Numerous dysregulated miRNAs simultaneously participate in the process of colon cancer development. However, the detailed mechanisms and biological functions of co-expressed miRNA in colorectal carcinogenesis have yet to be fully elucidated.ResultsThe objective of this study was to identify the dysfunctional miRNAs and their target mRNAs using a wet-lab experimental and dry-lab bioinformatics approach. The differentially expressed miRNA candidates were identified from 2 miRNA profiles, and were confirmed in CRC clinical samples using reported target genes of dysfunctional miRNAs to perform functional pathway enrichment analysis. Potential target gene candidates were predicted by an in silico search, and their expression levels between normal and colorectal tumor tissues were further analyzed using real-time polymerase chain reaction (RT-PCR).We identified 5 miRNAs (miR-18a, miR-31, miR-96, miR-182, and miR-224) and 10 miRNAs (miR-1, miR-9, miR-10b, miR-133a, miR-143, miR-137, miR-147b, miR-196a/b, and miR-342) that were significantly upregulated and downregulated in colon tumors, respectively. Bioinformatics analysis showed that the known targets of these dysregulated miRNAs simultaneously participated in epithelial-to-mesenchymal transition (EMT), cell growth, cell adhesion, and cell cycles. In addition, we identified that several pivotal target gene candidates may be comodulated by dysfunctional miRNAs during colon cancer progression. Finally, 7 candidates were proven to be differentially expressed, and had an anti-correlationship with dysregulated miRNA in 48 CRC samples.ConclusionFifteen dysfunctional miRNAs were engaged in metastasis-associated pathways through comodulating 7 target genes, which were identified by using a multi-step approach. The roles of these candidate genes are worth further exploration in the progression of colon cancer, and could potentially be targets in future therapy.

Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer

Prostate cancer is a complex disease that can be relatively harmless or extremely aggressive. Although androgen-deprivation therapy is a commonly used treatment for men with prostate cancer, the adverse effects can be detrimental to patient health and quality of life. Therefore, identifying new target genes for tumor growth will enable the development of novel therapeutic intervention. TPX2 plays a critical role in chromosome segregation machinery during mitosis. Low rates of chromosome missegregation can promote tumor development, whereas higher levels might promote cell death and suppress tumorigenesis. Hence, the strategy of promoting cell death by inducing massive chromosome missegregation has been a therapeutic application for selectively eliminating highly proliferating tumor cells. RNAi was used for TPX2 protein expression knockdown, and a clonogenic assay, immunostaining, double thymidine block, image-cytometry analysis, and tumor spheroid assay were used to analyze the role of TPX2 in tumor cell growth, cell cycle progression, multinuclearity, ploidy, and tumorigenicity, respectively; finally, Western blotting was used to analyze anticancer mechanisms in TPX2 targeting. We demonstrated that targeting TPX2 reduced cell cycle regulators and chromosome segregation genes, resulting in increased cell micronucleation. Moreover, TPX2 depletion led to prostate cancer cell growth inhibition, increased apoptosis, and reduced tumorigenesis. These results confirmed the therapeutic potential of targeting TPX2 in prostate cancer treatment. Moreover, we found that TPX2 silencing led to deregulation of CDK1, cyclin B, securin, separase, and aurora A proteins; by contrast, p21 mRNA was upregulated. We also determined the molecular mechanisms for TPX2 targeting in prostate cancer cells. In conclusion, our study illustrates the power of TPX2 as a potential novel target gene for prostate cancer treatment.