Been-Ren Lin

Connective Tissue Growth Factor Activates Pluripotency Genes and Mesenchymal–Epithelial Transition in Head and Neck Cancer Cells

The epithelial-mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal-epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through αvβ3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These findings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classification system and therapeutic strategies for HNSCC.

Epithelial–Mesenchymal Transition Induced by TNF-α Requires NF-κB–Mediated Transcriptional Upregulation of Twist1

Proinflammatory cytokines produced in the tumor microenvironment facilitate tumor development and metastatic progression. In particular, TNF-α promotes cancer invasion and angiogenesis associated with epithelial-mesenchymal transition (EMT); however, the mechanisms underlying its induction of EMT in cancer cells remain unclear. Here we show that EMT and cancer stemness properties induced by chronic treatment with TNF-α are mediated by the upregulation of the transcriptional repressor Twist1. Exposure to TNF-α rapidly induced Twist1 mRNA and protein expression in normal breast epithelial and breast cancer cells. Both IKK-β and NF-κB p65 were required for TNF-α-induced expression of Twist1, suggesting the involvement of canonical NF-κB signaling. In support of this likelihood, we defined a functional NF-κB-binding site in the Twist1 promoter, and overexpression of p65 was sufficient to induce transcriptional upregulation of Twist1 along with EMT in mammary epithelial cells. Conversely, suppressing Twist1 expression abrogated p65-induced cell migration, invasion, EMT, and stemness properties, establishing that Twist1 is required for NF-κB to induce these aggressive phenotypes in breast cancer cells. Taken together, our results establish a signaling axis through which the tumor microenvironment elicits Twist1 expression to promote cancer metastasis. We suggest that targeting NF-κB-mediated Twist1 upregulation may offer an effective a therapeutic strategy for breast cancer treatment.

IL-6 induces AGS gastric cancer cell invasion via activation of the c-Src/RhoA/ROCK signaling pathway

Interleukin‐6 (IL‐6) is a multifunctional cytokine that is associated with the disease status and outcomes of gastric cancer. Nonetheless, the underlying mechanism of how IL‐6 promotes the spread of gastric cancer is still unclear. In this study, we used a modified Boyden chamber assay to test the invasion ability of different gastric cancer cell lines. Liposome‐mediated transfection was used to introduce an IL‐6 expression vector into AGS cells, and the transfectants were further examined for the expression of active RhoA and phosphorylated Src using a pull‐down assay and coimmunoprecipitation/Western blot analysis. Furthermore, RhoA expression in gastric adenocarcinoma specimens was investigated immunohistochemically. We documented that IL‐6 could promote AGS cell motility and invasiveness, and inhibition of RhoA expression by dominant negative RhoA, C3 transferase, or dominant negative Src expressing plasmids could effectively decrease the invasiveness of IL‐6 transfectants. We also documented an interaction between active RhoA and phosphorylated‐Src following IL‐6 treatment. Gastric cancers displaying high expression of RhoA are highly correlated with aggressive lymph node metastasis, more advanced tumor stage, histologically diffuse type and poorer survival. In conclusion, IL‐6 induces AGS gastric cancer cell invasion via activation of the c‐Src/RhoA/ROCK signaling pathway and RhoA expression could be used as a prognostic factor in patients with gastric adenocarcinoma.

Cyr61Induces Gastric Cancer Cell Motility/Invasion via Activation of the Integrin/Nuclear Factor-KB/Cyclooxygenase-2 Signaling Pathway

Purpose: Cysteine-rich 61 (Cyr61/CCN1) is involved in many different types of tumor development and progression. Nonetheless, the role of Cyr61 in human gastric cancer has not yet been fully characterized. Experimental design: We addressed the issue by immunohistochemical staining of 81 gastric adenocarcinoma specimens. Liposome-mediated transfection was used to introduce a Cyr61 expression vector into gastric cancer AGS cell lines. Transfectants were tested in invasion assay by a Boyden chamber. Furthermore, a cyclooxygenase-2 (COX-2) reporter assay and gel mobility shift assay were done to investigate the potential signal pathway of Cyr61. Results: Patients with gastric adenocarcinoma whose tumor displayed high expression of Cyr61 correlated well with aggressive lymph node metastasis, more advanced tumor stage, histologic diffuse type, and early recurrence. Stable transfection of Cyr61 into the AGS cell line strongly enhanced its invasive activity. The overexpression of Cyr61 into AGS cells significantly increased the expression of COX-2 mRNA, protein, and enzymatic activity. Gel mobility shift assays further showed that the nuclear factor-κB (NF-κB) pathway was evidently activated in Cyr61-expressing AGS cells. Function-neutralizing antibody to αvβ3 but not αvβ5 effectively suppressed Cyr61-mediated NF-κB activation, COX-2 gene expression, and cell invasiveness. Conclusions: Cyr61 may contribute to the malignant progression of gastric cancer by promoting tumor cell motility/invasion through up-regulation of the functional COX-2 via an integrin αvβ3/NF-κB-dependent pathway.

CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway

Laryngeal and hypopharyngeal squamous cell carcinomas (LHSCCs) are common head and neck cancers with a high propensity for lymph node (LN) and lung metastasis. Here, we report that LHSCCs express high levels of functional CXCR4 receptors, native for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). Primary tumor immunohistochemistry from LHSCC patients has revealed significant expression of CXCR4 and CXCL12. Greater expression of CXCR4 but not that of CXCL12 is correlated with LN and distant metastasis. Reverse transcription-polymerase chain reaction and western blots have demonstrated that CXCR4 messenger RNA (mRNA) and protein were expressed in LHSCC cell lines as well, but failed to detect CXCL12 mRNA expression. CXCL12 treatment enhanced extracellular signal-regulated kinase (ERK) pathway activation and the motility/invasiveness of LHSCC cell lines, which were blocked by treatment with a CXCR4 antagonist (AMD3100) and a specific MEK inhibitor (U0126). Results show that the mRNA and protein levels of matrix metalloproteinase (MMP)-13, but not MMP-2 or MMP-9, were elevated in HEp-2 cells in response to CXCL12. Again, U0126 almost inhibited the induction of MMP-13 in HEp-2 cells by stimulating CXCL12. The transcriptional factor, c-Jun, a downstream factor of ERK pathway, was found to be readily phosphorylated and translocated to the nucleus after 10 min of exposure to CXCL12. Blockage of c-Jun activity by transfection with c-jun antisense oligodeoxynucleotide significantly decreased CXCL12-induced MMP-13 expression and cell invasion. CXCL12 seems to enhance LHSCC cell invasion through paracrine-activated CXCR4, which triggers ERK/c-Jun-dependent MMP-13 upregulation.

Involvement of Hypoxia-inducing Factor-1α-dependent Plasminogen Activator Inhibitor-1 Up-regulation in Cyr61/CCN1-induced Gastric Cancer Cell Invasion

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1α (HIF-1α) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1α protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1α protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl2 treatment, both well known for induction of HIF-1α. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1α up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1α protein accumulation. Blockage of HIF-1α activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1α strongly inhibited their invasion ability, suggesting that elevation in HIF-1α protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1α-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1α to block HIF-1α activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1α-dependent up-regulation of PAI-1.

Elevated Expression of Cyr61 Enhances Peritoneal Dissemination of Gastric Cancer Cells through Integrin α2β1

Cysteine-rich 61 (Cyr61/CCN1) is involved in human gastric cancer development and progression. Nonetheless, the role of Cyr61 as regards peritoneal dissemination of such cancers has not yet been completely characterized. We used liposome-mediated transfection to establish Cyr61, or antisense Cyr61, expression vectors into gastric cancer AGS or MKN45 cell lines. Transfectants were tested by means of a cancer-cell adhesion assay in vitro and ex vivo. Furthermore, a functional integrin fluorescence-activated cell sorting assay, reverse transcription-PCR, and an AP-1 reporter assay were performed to investigate the potential signaling pathway of Cyr61. It was shown that stable transfection of Cyr61 into the AGS cell line strongly enhanced its adhesion ability. The overexpression of Cyr61 within AGS cells significantly increased the functional expression of integrin α2β1. Function-neutralizing antibody to integrin α2β1 effectively suppressed the Cyr61-mediated enhanced adhesion of AGS cells to peritoneal tissue. Promoter assays of integrin α2 gene further revealed that the AP-1 pathway was evidently activated within Cyr61-expressing AGS cells. Animal studies have revealed that mice injected with Cyr61-overexpressed AGS cells featured a greater number of peritoneal seeding nodules and a lower survival rate than the Neo control cell lines, and when such cells were treated with functional blocking antibody to integrin α2β1, they were able to elicit a decline in the peritoneal dissemination. The data suggest that Cyr61 may contribute to the peritoneal dissemination of gastric cancer by promoting tumor-cell adhesion ability through the up-regulation of the functional integrin α2β1 via an AP-1-dependent pathway.

Cysteine-Rich 61 (CCN1) Enhances Chemotactic Migration, Transendothelial Cell Migration, and Intravasation by Concomitantly Up-Regulating Chemokine Receptor 1 and 2

Cysteine-rich 61 (Cyr61; CCN1) plays an important role in tumor development and progression in many kinds of human malignancies. Here, we further show the enforced expression of the Cyr61 gene or treatment with recombinant Cyr61 protein enhanced expression of chemokine receptors CXCR1 and CXCR2 in gastric cancer AGS cells. Attenuation of Cyr61 levels in MKN-45 cells by transfecting with antisense Cyr61 significantly reduced the level of CXCR1 and CXCR2. It is suggested that Cyr61 tightly regulates the downstream genes CXCR1 and CXCR2 in gastric cancer cells. Supportively, reverse transcription–PCR and immunohistochemical analysis of human gastric adenocarcinoma showed that there was a high correlation between the expression level of Cyr61 and CXCR1/CXCR2. The up-regulated functionality of CXCR1 andCXCR2 in Cyr61-overexpressing AGS cells could facilitate their chemotactic migration toward interleukin-8, a physiologic ligand of CXCR1 and CXCR2. In addition, the Cyr61-mediated up-regulation of CXCR1/CXCR2 also contributed to transendothelial migration, as well as intravasation in a chick embryo model. Pharmacologic and genetic approaches revealed that phosphoinositide 3-kinase (PI3K)/Akt, but not extracellular signal-regulated kinase 1/2 or p38, signaling pathway is requisite for the up-regulation of CXCR1/CXCR2 mRNA and protein induced by Cyr61. Function-neutralizing antibody to integrin αvβ3, but not α2β1, effectively abolished Cyr61-elicited Src activation and the subsequent PI3K/Akt pathway. Antagonists toward integrin αvβ3, Src kinase, and PI3K/Akt not only suppressed CXCR1/CXCR2 elevation but also blocked chemotactic migration induced by Cyr61. In conclusion, we suggest that Cyr61 promotes interleukin-8–dependent chemotaxis, transendothelial migration, and intravasation by induction of CXCR1/CXCR2 through integrin αvβ3/Src/PI3K/Akt–dependent pathway. (Mol Cancer Res 2007;5(11):1111–23)

HDAC2 promotes cell migration/invasion abilities through HIF-1α stabilization in human oral squamous cell carcinoma

BACKGROUND Histone deacetylase 2 (HDAC2) expressions in oral squamous cell carcinoma (OSCC) had been implicated in advanced stage and poor prognosis. It suggests a possible link between the migration/invasion potential of oral cancer cells and the prevalent expression of HDAC2. METHODS Five head and neck cancer (HNC) cell lines, including Ca9-22, Cal-27, HSC-3, SAS, and TW2.6, were used. Cells stably overexpressing HDAC2 and shRNA against HDAC2 were established to investigate migration/invasion ability in vitro and tumorigenesis and progression in vivo. RESULTS We found that alterations in the HDAC2 level in OSCC cell lines modulated their invasive ability with a positive correlation. Animal model also showed that knockdown of HDAC2 expression in SAS cells, originally containing high endogenous HDAC2 expression, resulted in decrease in tumor initiation and progression. Using high-throughput transcriptome analysis, numerous genes involved in HIF-1α-associated pathways were found. At the mechanism levels, using agents to block de novo protein synthesis or prevent protein degradation by ubiquitination, we found the stability of hypoxia inducible factor 1α (HIF-1α) protein was maintained in OSCC cells with HDAC2 overexpression. In addition, co-immunoprecipitation assay also revealed that HDAC2-mediated HIF-1α protein stability is because of direct interaction of HIF-1α with von Hippel-Lindau (VHL) protein. CONCLUSIONS Our work demonstrates that HDAC2 maintains HIF-1α stability, probably at the level of protein modification, which in turn leads to the increase in cell invasion/migration ability in oral cancer progression. These findings implicate the potential of HDAC inhibitors for oral cancer therapy.

Connective Tissue Growth Factor Acts as a Therapeutic Agent and Predictor for Peritoneal Carcinomatosis of Colorectal Cancer

Purpose: Here, we aimed to investigate the role of connective tissue growth factor (CTGF) in peritoneal carcinomatosis (PC) associated with colorectal cancer (CRC) and to characterize the underlying mechanism of CTGF mediating adhesion. Experimental Design: A cohort of 136 CRC patient specimens was analyzed in this study. CRC cell lines were used for in vitro adhesion assay and in vivo peritoneal dissemination experiment. Recombinant CTGF protein treatment, transfection of CTGF expression plasmids, and knockdown of CTGF expression in CRC cells were utilized to evaluate the integrin α5, which served as a target of CTGF in inhibiting peritoneal seeding. Results: The analysis of CRC tissues revealed an inverse correlation between CTGF expression and prevalence of PC. Lower CTGF level in CRC patients was associated with higher peritoneal recurrence rate after surgery. Inducing CTGF expression in cancer cells resulted in decreased incidence of PC and increased rate of mice survival. The mice received intraperitoneal injection of recombinant CTGF protein simultaneously with cancer cells or following tumor formation; in both cases, peritoneal tumor dissemination was found to be effectively inhibited in the mouse model. Functional assay revealed that CTGF significantly decreased the CRC cell adhesion ability, and integrin α5 was confirmed by reverse transcriptase PCR and functional blocking assay as a downstream effector in the CTGF-mediated inhibition of CRC cell adhesion. Conclusions: CTGF acts as a molecular predictor of PC and could be a potential therapeutic target for the chemoprevention and treatment of PC in CRC patients. Clin Cancer Res; 17(10); 3077–88. ©2011 AACR.