Cheng-Chi Chang

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.

Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the progression of human lung adenocarcinoma. However, the mechanism underlying COX-2’s effect on tumor progression remains largely unknown. Lymphangiogenesis, the formation of new lymphatic vessels, has recently received considerable attention and become a new frontier of tumor metastasis research. Here, we study the interaction between COX-2 and the lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, in human lung cancer cells and their implication in patient outcomes. We developed an isopropyl-β-d-thiogalactopyranoside-inducible COX-2 gene expression system in human lung adenocarcinoma CL1.0 cells. We found that VEGF-C gene expression but not VEGF-D was significantly elevated in cells overexpressing COX-2. COX-2-mediated VEGF-C up-regulation was commonly observed in a broad array of non-small cell lung cancer cell lines. The use of pharmacological inhibitors or activators and genetic inhibition by EP receptor-antisense oligonucleotides revealed that prostaglandin EP1 receptor but not other prostaglandin receptors is involved in COX-2-mediated VEGF-C up-regulation. At the mechanistic level, we found that COX-2 expression or prostaglandin E2 (PGE2) treatment could activate the HER-2/Neu tyrosine kinase receptor through the EP1 receptor-dependent pathway and that this activation was essential for VEGF-C induction. The transactivation of HER-2/Neu by PGE2 was inhibited by way of blocking the Src kinase signaling using the specific Src family inhibitor, PP1, or transfection with the mutant dominant negative src plasmid. Src kinase was involved in not only the HER-2/Neu transactivation but also the following VEGF-C up-regulation by PGE2 treatment. In addition, immunohistochemical staining of 59 lung adenocarcinoma specimens showed that COX-2 level was highly correlated with VEGF-C, lymphatic vessels density, and other clinicopathological parameters. Taken together, our results provided evidence that COX-2 up-regulated VEGF-C and promotes lymphangiogenesis in human lung adenocarcinoma via the EP1/Src/HER-2/Neu signaling pathway.

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.

RNA‐binding protein insulin‐like growth factor II mRNA‐binding protein 3 expression promotes tumor invasion and predicts early recurrence and poor prognosis in hepatocellular carcinoma

Insulin‐like growth factor II mRNA‐binding protein 3 (IMP3) is an RNA‐binding protein expressed in embryonic tissues and multiple cancers. To investigate the role of IMP3 in hepatocellular carcinoma (HCC), its protein expression in the surgically resected unifocal tumors of 377 HCC patients (296 men and 81 women) with ages ranging from 7 to 88 years (mean, 55.49 years) was analyzed by immunohistochemistry. IMP3 was expressed in 255 (67.6%) of 377 resected unifocal primary HCCs. IMP3 protein was predominantly expressed in tumor border and invasive front, and it was more abundant in the satellite nodules and tumor thrombi than in the main tumors. The expression correlated with high α‐fetoprotein (>200 ng/mL, P < 1 × 10−7), larger tumor size (>5 cm, P = 0.006), high tumor grade (P < 1 × 10−7), and high tumor stage with vascular invasion and various degrees of intrahepatic metastasis (P < 1 × 10−7). IMP3 expression predicted early tumor recurrence (P < 1 × 10−7) and was a strong indicator of poor prognosis (P < 0.0001). Depletion of IMP3 with RNA interference in HCC cell line HA22T caused a decrease in cell motility, invasion, and transendothelial migration. Microarray analysis revealed that IMP3 depletion was associated with downregulation of multiple genes involved in tumor invasion. Conclusion: Our results indicate that IMP3 plays an important role in tumor invasion and metastasis and is a strong prognostic factor for patients with HCC. (HEPATOLOGY 2008.)

Connective tissue growth factor (CTGF) and cancer progression

Connective tissue growth factor (CTGF) is a member of the CCN family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth. CCN family proteins share uniform modular structure which mediates various cellular functions such as regulation of cell division, chemotaxis, apoptosis, adhesion, motility, angiogenesis, neoplastic transformation, and ion transport. Recently, CTGF expression has been shown to be associated with tumor development and progression. There is growing body of evidence that CTGF may regulate cancer cell migration, invasion, angiogenesis, and anoikis. In this review, we will highlight the influence of CTGF expression on the biological behavior and progression of various cancer cells, as well as its regulation on various types of protein signals and their mechanisms.

Regulation of Excision Repair Cross-Complementation Group 1 by Snail Contributes to Cisplatin Resistance in Head and Neck Cancer

Purpose: We investigated the mechanism and clinical significance of the epithelial-mesenchymal transition (EMT)-induced chemoresistance in head and neck squamous cell carcinoma (HNSCC). Experimental Design: The correlation between the expression of different EMT regulators and chemoresistance genes, such as excision repair cross complementation group 1 (ERCC1), was evaluated in cancer cell lines from the NCI-60 database and four human HNSCC cell lines. Ectopic expression of Snail or short-interference RNA-mediated repression of Snail or ERCC1 was done in HNSCC cell lines. Cell viability was examined for cells after cisplatin treatment. A luciferase reporter assay and chromatin immunoprecipitation were used to identify the transcriptional regulation of ERCC1 by Snail. Immunohistochemical analysis of Snail, Twist1, ERCC1, hypoxia inducible factor-1 α (HIF-1α), and NBS1 were done in samples from 72 HNSCC patients receiving cisplatin-based chemotherapy. Results: The correlation between the expression of Snail and ERCC1 was confirmed in different cell lines, including HNSCC cells. In HNSCC cell lines, overexpression of Snail in the low endogenous Snail/ERCC1 cell lines FaDu or CAL-27 increased ERCC1 expression, and hypoxia or overexpression of NBS1 also upregulated ERCC1. Knockdown of Snail in the high endogenous Snail/ERCC1 cell line OECM-1 downregulated ERCC1 expression and attenuated cisplatin resistance. Furthermore, suppression of ERCC1 in Snail- or NBS1-overexpressing HNSCC cells enhanced sensitivity to cisplatin. Snail directly regulated ERCC1 transcription. In patients with HNSCC, coexpression of Snail and ERCC1 correlated with cisplatin resistance and a poor prognosis. Conclusions: Activation of ERCC1 by Snail is critical in the generation of cisplatin resistance of HNSCC cells. Clin Cancer Res; 16(18); 4561–71. ©2010 AACR.

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.

Breast Tumor Kinase Phosphorylates p190RhoGAP to Regulate Rho and Ras and Promote Breast Carcinoma Growth, Migration, and Invasion

Breast tumor kinase (Brk), an Src-like nonreceptor tyrosine kinase, is overexpressed in breast cancer and several other cancer types. Our previous study indicates that Brk promotes cell migration and tumor invasion by phosphorylating the focal adhesion protein paxillin. Here, we report the identification of p190RhoGAP-A (p190) as a Brk substrate. Brk phosphorylates p190 at the Y(1105) residue both in vitro and in vivo, thereby promoting the association of p190 with p120RasGAP (p120). As a consequence, Brk stimulates p190 and attenuates p120 functions, leading to RhoA inactivation and Ras activation, respectively. In carcinoma cells expressing high levels of Brk, endogenous Brk functions as a key contributor to epidermal growth factor-induced p190 tyrosine phosphorylation. We present evidence showing that p190 phosphorylation plays essential roles in both migratory and proliferative effects of Brk. Furthermore, disruption of p190 phosphorylation-induced p190/p120 complex in breast cancer cells abolishes not only the abilities of Brk to regulate RhoA and Ras but also the stimulatory effects of Brk on proliferation, migration, invasion, transformation, and tumorigenicity. Together, our findings reveal a previously unknown function of Brk in regulating both RhoA and Ras by phosphorylating p190 and provide evidence for the crucial roles of this Brk-elicited signaling pathway in promoting breast malignancy.

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.