Abstract Hepatocellular carcinoma (HCC) is one the most fatal cancers worldwide. Early diagnosis is crucial for curing the disease. However, patients are often diagnosis at late-stage with distant metastasis, and the prognosis of metastatic HCC is poor. Molecular pathological studies confirm that gene expression alteration plays a critical role in disease progression. Recently it has been discovered that microRNA (miRNA) can alter the gene expression at post-transcription level. This study aimed to elucidate the role of miRNA in development of metastatic HCC using an orthotopic animal model and the molecular pathway involved. Orthotopic metastasis animal model was established by implanting HCC cell lines onto the liver of the SCID mice. Tumor growth was monitored by in vivo imaging system. Primary tumor and lung metastasis were observed after 12 weeks of inoculation. Tumor were then excised and established into primary tumor cell lines (PT) and lung metastatic cell lines (LM). Functional studies comparing the PT and LM cell lines derived from HCC cell line PLC demonstrated higher cell invasion and migration ability in the metastatic cell line compared with the primary tumor cell line. In addition, the LM cell line possessed more stress fiber than the PT cell line. The PLC-derived cell lines and another metastatic HCC cell line, MHCC97H-derived cell lines were subjected to the miRNA microarray analysis. Fifteen human miRNAs were found to be differentially expressed in the LM cell lines when compared to the PT cell lines. miR-106b was one of the miRNAs which was over-expressed in LM cell lines. We further confirmed our findings in HCC clinical sample and found that miR-106b was over-expressed in HCC tumor compared with the adjacent non-tumor tissue, and its expression was significantly associated with the tumor grade (p=0.018). MicroRNA knock-down study was performed by using the miR-106b LNA knock-down probe. The miR-106b knock-down reverted the cell migration phenotype, and fluorescent staining of stress fiber formation showed that the miR-106b knock-down cells have less stress fiber formation than the parental cell lines and the scramble control. We further studied the expression of E-cadherin in PLC-PT, PLC-LM, the scramble control and the miR-106b knock-down cell lines. Our result demonstrated that miR-106b expression inversely correlated with E-cadherin level. Since decrease of E-cadherin expression is the hallmark for epithelia-mesenchymal transition (EMT), further studies will focus on the role of miR-106b in EMT of HCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2024.
Aims We previously demonstrated Proline rich tyrosine kinase 2 (Pyk2) plays important roles in regulating tumor progression, migration and invasion in hepatocellular carcinoma (HCC). In this study, we aimed to examine the role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC and to explore its underlying molecular mechanism. Methodology/Principal Findings Stable transfectants either overexpressing or suppressing Pyk2 were established in different HCC cell lines. MTT, colony formation and Annexin-V assays were employed to examine their in vitro responses to cisplatin. Xenograft ectopic and orthotopic nude mice models were generated to investigate the in vivo responses of them to cisplatin treatment. cDNA microarray was performed to identify Pyk2-induced genes which were further validated by quantitative real-time RT-PCR using clinical HCC samples. In vitro functional study demonstrated that Pyk2-overexpressing HCC transfectants exhibited relatively lower cytotoxicity, higher colony-forming ability and lower apoptosis to cisplatin compared with the control transfectants. Moreover, Pyk2 overexpressing HCC transfectants had a higher survival rate under cisplatin treatment by up-regulation of AKT phosphorylation. In vivo xenograft nude mice model demonstrated that Pyk2-overexpressing transfectants developed higher tolerance to cisplatin treatment together with less tumor necrosis and apoptosis. cDNA microarray analysis revealed that there were more than 4,000 genes differentially expressed upon overexpression of Pyk2. Several upregulated genes were found to be involved in drug resistance and invasion in cancers. Among them, the expression profiles of MDR1, GAGE1, STAT1 and MAP7 were significantly associated with the expression of Pyk2 in clinical HCC samples. Conclusions Our results may suggest a new evidence of Pyk2 on promoting cisplatin resistance of HCC cells through preventing cell apoptosis, activation of AKT pathway and upregulation of drug resistant genes.
Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.
Abstract Functional studies to identify the potential role of a chromosome 3p14-21 gene, protein tyrosine phosphatase receptor type G (PTPRG), were performed. PTPRG was identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by differential gene profiling of tumorigenic and nontumorigenic NPC chromosome 3 microcell hybrids (MCH). Down-regulation of this gene was found in tumor segregants when compared with their corresponding tumor-suppressive MCHs, as well as in NPC cell lines and tumor biopsies. Promoter hypermethylation and loss of heterozygosity were found to be important mechanisms contributing to PTPRG silencing. PTPRG overexpression in NPC cell lines induces growth suppression and reduced anchorage-independent growth in vitro. This is the first study to use a tetracycline-responsive vector expression system to study PTPRG stable transfectants. Results indicate its ability to induce significant tumor growth suppression in nude mice under conditions activating transgene expression. These studies now provide functional evidence indicating critical interactions of PTPRG in the extracellular matrix milieu induce cell arrest and changes in cell cycle status. This is associated with inhibition of pRB phosphorylation through down-regulation of cyclin D1. These novel findings enhance our current understanding of how PTPRG may contribute to tumorigenesis. [Cancer Res 2008;68(19):8137–45]
<div>Abstract<p>Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including <i>RB1</i>, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to <i>RB1</i>, but instead likely involves <i>thrombospondin type I domain-containing 1</i> (<i>THSD1</i>), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of <i>THSD1</i> expression in 100% of ESCC and other cancer cell lines. Mechanisms for <i>THSD1</i> silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type <i>THSD1</i> into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that <i>THSD1</i> is a good candidate TSG. (Mol Cancer Res 2008;6(4):592–603)</p></div>
Abstract Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG. (Mol Cancer Res 2008;6(4):592–603)
Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.
Abstract Chromosome 3p plays an important role in tumorigenesis in many cancers, including nasopharyngeal carcinoma (NPC). We have previously shown chromosome 3p can suppress tumor growth in vivo by using the monochromosome transfer approach, which indicated the chromosome 3p21.3 region was critical for tumor suppression. BLU/ZMYND10 is one of the candidate tumor suppressor genes mapping in the 3p21.3 critical region and is a candidate TSG for NPC. By quantitative RT‐PCR, it is frequently downregulated in NPC cell lines (83%) and NPC biopsies (80%). However, no functional studies have yet verified the functional role of BLU/ZMYND10 as a tumor suppressor gene. In the current study, a gene inactivation test (GIT) utilizing a tetracycline regulation system was used to study the functional role of BLU/ZMYND10 . When BLU/ZMYND10 is expressed in the absence of doxycycline, the stable transfectants were able to induce tumor suppression in nude mice. In contrast, downregulation of BLU/ZMYND10 in these tumor suppressive clones by doxycycline treatment restored the tumor formation ability. This study provides the first significant evidence to demonstrate BLU/ZMYND10 can functionally suppress tumor formation in vivo and is, therefore, likely to be one of the candidate tumor suppressor genes involved in NPC. Published 2006 Wiley‐Liss, Inc.
Supplementary Figures S1-S3 from Monochromosome Transfer and Microarray Analysis Identify a Critical Tumor-Suppressive Region Mapping to Chromosome 13q14 and <i>THSD1</i> in Esophageal Carcinoma
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