Qiulin Tang

miR-137 targets Cdc42 expression, induces cell cycle G1 arrest and inhibits invasion in colorectal cancer cells.

miRNAs have emerged as post‐transcriptional regulators that are critically involved in the pathogenesis of a number of human cancers. Cdc42, one of the best characterized members of the Rho GTPase family, is found to be up‐regulated in several types of human tumors and has been implicated in cancer initiation and progression. In the present study, we have identified miR‐137 as a potential regulator of Cdc42 expression. A bioinformatics search revealed a putative target‐site for miR‐137 within the Cdc42 3′ UTR at nt 792–798, which is highly conserved across different species. Expression of miR‐137 in colorectal cancer cell lines was found inversely correlated with Cdc42 expression. miR‐137 could significantly suppress Cdc42 3′ UTR luciferase‐reporter activity, and this effect was not detectable when the putative 3′ UTR target‐site was mutated. Consistent with the results of the reporter assay, ectopic expression of miR‐137 reduced both mRNA and protein expression levels of Cdc42 and mimicked the effect of Cdc42 knockdown in inhibiting proliferation, inducing G1 cell cycle arrest, and blocking invasion of the colorectal cancer cells, whereas anti‐miR‐137 expression led to the opposite effect. Furthermore, expression of miR‐137 suppressed the immediate downstream effector of Cdc42, PAK signaling. Our results suggest that miR‐137 may have a tumor suppressor function by directly targeting Cdc42 to inhibit the proliferation and invasion activities of colorectal cancer cells. They raise an interesting possibility that Cdc42 activity and function can be controlled by miRNAs in addition to the classic regulators such as guanine nucleotide exchange factors and GTPase‐activating proteins.

RhoA Regulates G1-S Progression of Gastric Cancer Cells by Modulation of Multiple INK4 Family Tumor Suppressors

RhoA, a member of the Rho GTPase family, has been extensively studied in the regulation of cytoskeletal dynamics, gene transcription, cell cycle progression, and cell transformation. Overexpression of RhoA is found in many malignancies and elevated RhoA activity is associated with proliferation phenotypes of cancer cells. We reported previously that RhoA was hyperactivated in gastric cancer tissues and suppression of RhoA activity could partially reverse the proliferation phenotype of gastric cancer cells, but the underlying mechanism has yet to be elucidated. It has been reported that RhoA activation is crucial for the cell cycle G1-S procession through the regulation of Cip/Kip family tumor suppressors in benign cell lines. In this study, we found that selective suppression of RhoA or its effectors mammalian Diaphanous 1 and Rho kinase (ROCK) by small interfering RNA and a pharmacologic inhibitor effectively inhibited proliferation and cell cycle G1-S transition in gastric cancer lines. Down-regulation of RhoA-mammalian Diaphanous 1 pathway, but not RhoA-ROCK pathway, caused an increase in the expression of p21Waf1/Cip1 and p27Kip1, which are coupled with reduced expression and activity of CDK2 and a cytoplasmic mislocalization of p27Kip1. Suppression of RhoA-ROCK pathway, on the other hand, resulted in an accumulation of p15INK4b, p16INK4a, p18INK4c, and p19INK4d, leading to reduced expression and activities of CDK4 and CDK6. Thus, RhoA may use two distinct effector pathways in regulating the G1-S progression of gastric cancer cells.(Mol Cancer Res 2009;7(4):570–80)

A distinct role of RhoB in gastric cancer suppression.

Although Rho family GTPases RhoA, RhoB and RhoC share more than 85% amino acid sequence identity, they may play distinct roles in tumor progression. RhoA and RhoC have been suggested to have positive effects on tumor progression, but the role of RhoB in cancer, particularly in gastric cancer, remains unclear. In our study, we have examined the expression levels of these three Rho GTPases in a large panel of specimens from gastric cancer patients by immunohistochemistry. We found that RhoA and RhoC expression were significantly elevated, while RhoB was reduced or absent, in surgically removed gastric cancer tissues when compared to normal gastric tissues. The significant reduction of RhoB expression was confirmed in another group of gastric cancer samples in comparison to the adjacent non‐neoplastic tissues. Then we transfected the plasmids containing RhoA, RhoB or RhoC cDNA into two gastric cancer cell lines, SGC7901 and AGS cells, respectively. By overexpression experiments, we found that RhoA promoted the gastric cancer cell proliferation and RhoC stimulated migration and invasion of the cancer cell. RhoB expression, however, significantly inhibited the proliferation, migration and invasion of the gastric cancer cells and also enhanced the chemosensitivity of these cells to anticancer drugs. It appears that RhoB plays an opposing role from that of RhoA and/or RhoC in gastric cancer cells. Our work suggests that RhoB may play a tumor suppressor role and subsequently may have potential implications in future targeted therapy.

The lncRNA MALAT1 is a novel biomarker for gastric cancer metastasis.

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is frequently over-expressed and serves as a prognostic marker in human cancers. However, little is known about the role of MALAT1 in gastric cancer. Here, we reported that the tissue and plasma MALAT1 levels were significantly higher in gastric cancer patients with distant metastasis (P<0.01) than patients without distant metastasis and the healthy controls. In addition, high levels of plasma MALAT1 independently correlated to a poor prognosis for gastric cancer patients (hazard ratio, 0.242; 95% CI, 0.154-0.836; P=0.036; Cox regression analysis). Functional studies revealed that knockdown of MALAT1 could inhibit cell proliferation, cell cycle progression, migration and invasion, and promote apoptosis in gastric cancer cells. Furthermore, the miR-122-IGF-1R signaling correlated with the dysregulated MALAT1 expression in gastric cancer. These data suggest that MALAT1 could function as an oncogene in gastric cancer, and high MALAT1 level could serve as a potential biomarker for the distant metastasis of gastric cancer.

P-Akt/miR‑200 signaling regulates epithelial-mesenchymal transition, migration and invasion in circulating gastric tumor cells

Both circulating tumor cells (CTCs) and epithelial-mesenchymal transition (EMT) play an important role in invasion, migration and chemoresistant in tumor development. This study aimed to detect whether EMT occurred in human gastric CTCs and to explore the mechanism of EMT in human gastric CTCs. We analysed epithelial markers (pan-CK, E-cadherin), mesenchymal markers (N-cadherin, vimentin) EMT related miR‑200s, and Akt in gastric CTCs. The impact of miR‑200s on EMT, migration and invasion in CTCs was tested. We found that epithelial markers pan-CK, E-cadherin were decreased, and mesenchymal markers N-cadherin, vimentin were overexpressed in gastric CTCs. Expression of EMT related transcriptors, snail1, zeb1, twist1, were reversely correlated with miR‑200s, and were positively correlated with phospho-Akt. Upregulated of miR‑200s downregulated twist1 and zeb1 mRNA expression, and resulted in the supression of EMT, and impaired migration and invasion in gastric CTCs. Inhibition of p-Akt led to upregulation of miR‑200s. In conclusion, gastric CTCs exhibited remarkable EMT process, and p-Akt/miR‑200s signaling regulates EMT, migration and invasion in gastric CTCs.

Chemotherapy and Radiotherapy Downregulate the Activity and Expression of DNA Methyltransferase and Enhance Bcl-2/E1B-19-kDa Interacting Protein-3-Induced Apoptosis in Human Colorectal Cancer Cells

Bcl-2/E1B 19-kDa interacting protein 3 (BNIP3) is a proapoptotic protein whose expression level is often low in colorectal cancer (CRC) cells due to the BNIP3 gene promoter DNA methylation by DNA methyltransferase (DNMT). It is known that chemotherapy and radiotherapy suppress CRC through inducing tumor apoptosis. However, the molecular mechanisms underlying chemotherapy and radiotherapy-induced apoptosis of CRC cells are not well defined. In this study, we observed that the expression level of BNIP3 in colon cancer cells was significantly increased by treatment with therapeutic agents and radiation in vitro. The BNIP3 protein level in CRC tissues from patients who received preoperative concurrent chemotherapy was significantly higher than in those who received surgery alone. Furthermore, treatment with chemotherapeutic agents and radiation significantly decreased the DNMT1 expression level and enzymatic activity. Both expression level and activity of DNMT1 were inversely correlated with the expression level of BNIP3 in colon carcinoma cells after treatment with chemotherapeutic agents and radiation. Consistent with increased BNIP3 expression, chemotherapeutic agents and radiation induced colon carcinoma cell apoptosis in a dose-dependent manner. Based on these observations, we conclude that chemotherapy and radiotherapy inhibit DNMT1 expression to upregulate BNIP3 expression to promote CRC cell apoptosis. And, BNIP3 may play a role in the caspase-dependent apoptosis pathways, mainly during treatment with chemotherapy and radiotherapy.

Isolation and characterization of circulating tumor cells from human gastric cancer patients

AbstractPurpose Circulating tumor cells (CTCs) have been proved to be responsible for tumor metastasis and resistant to anticancer therapies. This study aims to isolate and characterize circulating tumor cells from human gastric cancer patients, and investigate characteristic differences between gastric CTCs and gastric cancer cell lines.MethodsWe analyzed 31 cases of gastric cancer patients using anti-CD45 antibody-conjugated magnetic microbeads negative separation, combined with fluorescence activated cell sorter CD44 positive screening. Abilities of tumor formation, metastasis, invasion, migration, irradiation and drug sensitivity of CTCs and gastric cancer cell lines were detected and compared.ResultsOf all the 31 patients, CD44+/CD45−CTCs were isolated in 14 patients, of which 3 cases were stage IIA, 2 cases stage IIB, 2 cases stage IIIC and 7 cases stage IV. The malignant behavior was demonstrated by both clonogenetic assay and tumor xenograft in nude mice. Compared with human gastric cancer cell lines, the migration and invasion abilities of CTCs increased to 3.21–12.6-fold and 2.3–6.7-fold, respectively (all p values <0.05). In addition, the metastatic potential of CTCs is much higher in vivo than that of the control. Furthermore, CTCs were found to be relatively sensitive to FU, cisplatin and paclitaxel, but relatively resistant to irradiation, oxaliplatin, cetuximab and trastuzumab.ConclusionsCD44+/CD45− gastric CTCs were isolated and found to exhibit stronger malignant behavior when compared with human gastric cancer cell lines. Furthermore, CTCs cultured in vitro have potential implications in drug sensitivity screening for the future anticancer treatments.

EGFR-PI3K-PDK1 pathway regulates YAP signaling in hepatocellular carcinoma: the mechanism and its implications in targeted therapy

The epidermal growth factor receptor (EGFR) pathway and Hippo signaling play an important role in the carcinogenesis of hepatocellular carcinoma (HCC). However, the crosstalk between these two pathways and its implications in targeted therapy remains unclear. We found that the activated EGFR signaling could bypass RhoA to promote the expression of YAP(Yes-associated protein), the core effector of the Hippo signaling, and its downstream target Cyr61. Further studies indicated that EGFR signaling mainly acted through the PI3K-PDK1 (Phosphoinositide 3-kinase-Phosphoinositide-dependent kinase-1) pathway to activate YAP, but not the AKT and MAPK pathways. While YAP knockdown hardly affected the EGFR signaling. In addition, EGF could promote the proliferation of HCC cells in a YAP-independent manner. Combined targeting of YAP and EGFR signaling by simvastatin and the EGFR signaling inhibitors, including the EGFR tyrosine kinase inhibitor (TKI) gefitinib, the RAF inhibitor sorafenib and the MEK inhibitor trametinib, presented strong synergistic cytotoxicities in HCC cells. Therefore, the EGFR-PI3K-PDK1 pathway could activate the YAP signaling, and the activated EGFR signaling could promote the HCC cell growth in a YAP-independent manner. Combined use of FDA-approved inhibitors to simultaneously target YAP and EGFR signaling presented several promising therapeutic approaches for HCC treatment.

The Rho GTPase RhoE is a p53-regulated candidate tumor suppressor in cancer cells

Previous studies have shown that RhoE, an atypical member of the Rho GTPase family, may play an opposite role to RhoA in regulating cell proliferation and invasion. To explore the relationship between RhoE and the malignant phenotypes of human cancer, we have determined the expression patterns of RhoE in varying grade of human cancer tissues and tested the effects of RhoE expression in several RhoE underexpressing cancer cell lines. Systemic immunocytochemistry analyses of gastric, colorectal, lung and breast carcinomas, respectively, showed that RhoE protein expression was significantly decreased in most cancer cases compared with that of adjacent normal tissues. Enhanced RhoE expression could markedly inhibit proliferation, migration and invasion and induce apoptosis of the cancer cells which have relatively low levels of endogenous RhoE expression. Wild-type p53 (wt-p53) could strongly increase RhoE expression in p53-transfected cells. Furthermore, the luciferase assays indicated that wt-p53 significantly enhanced the activities of RhoE promoter compared with mutant p53 (mt-p53) in PC3 cells (p53 null). Collectively, data are presented showing that RhoE may participate in human cancer progression and act as a candidate target of p53, and these findings also strongly suggest that RhoE may be a new candidate tumor suppressor and could serve as a potential target in the gene therapy of cancer.

Effects of the Proapoptotic Regulator Bcl2/Adenovirus EIB 19 kDa-Interacting Protein 3 on Radiosensitivity of Cervical Cancer

PURPOSE Bcl2/adenovirus EIB 19 kDa-interacting protein 3 (BNIP3) is a proapoptotic member of the Bcl-2 family. To address its potential as a therapeutic target for radiosensitization, this study investigated the effect of Bnip3 expression on radiosensitivity of cervical cancer in vitro and in vivo. MATERIALS AND METHODS In vitro: A plasmid expressing the BNIP3 gene was transfected into human cervical cancer HeLa cells using Lipofectamine(2000), and western blot and immunohistochemistry analysis were performed to evaluate the expression of BNIP3 in transfected cells. The effects on radiation-induced apoptosis were investigated using a clone formation assay and flow cytometry. In vivo: A total of 6 × 10⁶ HeLa cells were subcutaneously inoculated into the dorsal flank of nude mice, and plasmids expressing the BNIP3 gene were injected into the mice via the tail vein. Tumor volume was calculated, and immunohistochemistry was used to detect the expression of BNIP3 in tumor cells. TUNEL assays were performed to determine the apoptosis rates in tumor tissues. RESULTS Transfection with the recombinant BNIP3 plasmid increased expression of the Bnip3 protein in tumor cells. This apoptosis regulator significantly decreased the viability of cells (p < 0.01) and increased the apoptosis rates (p < 0.01) both in vitro and in vivo. The antitumor effect of radiotherapy was enhanced by overexpression of BNIP3, as revealed by tumor growth curve analysis. CONCLUSIONS Radiosensitization in human cervical cancer cells was observed after treatment with the recombinant BNIP3 plasmid in vitro and in vivo. Results suggested that BNIP3 may play a role in enhancement of radiotherapy efficiency, and its expression may have a synergistic effect on radiation treatments.