Ming Yang Wang

CTGF enhances the motility of breast cancer cells via an integrin-αvβ3–ERK1/2-dependent S100A4-upregulated pathway

Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin αvβ3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-αvβ3–ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-αvβ3–ERK1/2–S100A4 pathway.

miR-107 promotes tumor progression by targeting the let-7 microRNA in mice and humans

MicroRNAs (miRNAs) influence many biological processes, including cancer. They do so by posttranscriptionally repressing target mRNAs to which they have sequence complementarity. Although it has been postulated that miRNAs can regulate other miRNAs, this has never been shown experimentally to our knowledge. Here, we demonstrate that miR-107 negatively regulates the tumor suppressor miRNA let-7 via a direct interaction. miR-107 was found to be highly expressed in malignant tissue from patients with advanced breast cancer, and its expression was inversely correlated with let-7 expression in tumors and in cancer cell lines. Ectopic expression of miR-107 in human cancer cell lines led to destabilization of mature let-7, increased expression of let-7 targets, and increased malignant phenotypes. In contrast, depletion of endogenous miR-107 dramatically increased the stability of mature let-7 and led to downregulation of let-7 targets. Accordingly, miR-107 expression increased the tumorigenic and metastatic potential of a human breast cancer cell line in mice via inhibition of let-7 and upregulation of let-7 targets. By mutating individual sites within miR-107 and let-7, we found that miR-107 directly interacts with let-7 and that the internal loop of the let-7/miR-107 duplex is critical for repression of let-7 expression. Altogether, we have identified an oncogenic role for miR-107 and provide evidence of a transregulational interaction among miRNAs in human cancer development.

Connective Tissue Growth Factor Confers Drug Resistance in Breast Cancer through Concomitant Up-regulation of Bcl-xL and cIAP1

Connective tissue growth factor (CTGF) expression is elevated in advanced breast cancer and promotes metastasis. Chemotherapy response is only transient in most metastatic diseases. In the present study, we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression was inversely associated with chemotherapy response. Overexpression of CTGF in MCF7 cells (MCF7/CTGF) enhanced clonogenic ability, cell viability, and resistance to apoptosis on exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) mitigated this drug resistance capacity. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cellular inhibitor of apoptosis protein 1 (cIAP1). Knockdown of Bcl-xL or cIAP1 with specific small interfering RNAs abolished the CTGF-mediated resistance to apoptosis induced by the chemotherapeutic agents in MCF7/CTGF cells. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF7/CTGF cells. A neutralizing antibody against integrin alpha(v)beta(3) significantly attenuated CTGF-mediated ERK1/2 activation and up-regulation of Bcl-xL and cIAP1, indicating that the integrin alpha(v)beta(3)/ERK1/2 signaling pathway is essential for CTGF functions. The Bcl-xL level also correlated with the CTGF level in breast cancer patients. We also found that a COOH-terminal domain peptide from CTGF could exert activities similar to full-length CTGF, in activation of ERK1/2, up-regulation of Bcl-xL/cIAP1, and resistance to apoptosis. We conclude that CTGF expression could confer resistance to chemotherapeutic agents through augmenting a survival pathway through ERK1/2-dependent Bcl-xL/cIAP1 up-regulation.

Knockdown of Contactin-1 Expression Suppresses Invasion and Metastasis of Lung Adenocarcinoma

Numerous genetic changes are associated with cancer cell metastasis and invasion. In search for key regulators of invasion and metastasis, a panel of lung cancer cell lines with different invasive ability was screened. The gene for contactin-1 was found to play an essential role in tumor invasion and metastasis. Suppression of contactin-1 expression abolished the ability of lung adenocarcinoma cells to invade Matrigel in vitro as well as the polymerization of filamentous-actin and the formation of focal adhesion structures. Furthermore, knockdown of contactin-1 resulted in extensive inhibition of tumor metastasis and in increased survival in an animal model. RhoA but not Cdc42 or Rac1 was found to serve a critical role in contactin-1-mediated invasion and metastasis. Contactin-1-specific RNA interference resulted in loss of metastatic and invasive capacity in both in vitro and in vivo models. This loss was overturned by constitutive expression of the active form of RhoA. Contactin-1 was differentially expressed in tumor tissues, and its expression correlated with tumor stage, lymph node metastasis, and patient survival. Contactin-1 is proposed to function importantly in the invasion and metastasis of lung adenocarcinoma cells via RhoA-mediated mechanisms.

Angiopoietin-like protein 1 suppresses SLUG to inhibit cancer cell motility.

Angiopoietin-like protein 1 (ANGPTL1) is a potent regulator of angiogenesis. Growing evidence suggests that ANGPTL family proteins not only target endothelial cells but also affect tumor cell behavior. In a screen of 102 patients with lung cancer, we found that ANGPTL1 expression was inversely correlated with invasion, lymph node metastasis, and poor clinical outcomes. ANGPTL1 suppressed the migratory, invasive, and metastatic capabilities of lung and breast cancer cell lines in vitro and reduced metastasis in mice injected with cancer cell lines overexpressing ANGPTL1. Ectopic expression of ANGPTL1 suppressed the epithelial-to-mesenchymal transition (EMT) by reducing the expression of the zinc-finger protein SLUG. A microRNA screen revealed that ANGPTL1 suppressed SLUG by inducing expression of miR-630 in an integrin α(1)β(1)/FAK/ERK/SP1 pathway-dependent manner. These results demonstrate that ANGPTL1 represses lung cancer cell motility by abrogating the expression of the EMT mediator SLUG.

Multiple gene sequencing for risk assessment in patients with early-onset or familial breast cancer

Since BRCA mutations are only responsible for 10–20% of cases of breast cancer in patients with early-onset or a family history and since next-generation sequencing technology allows the simultaneous sequencing of a large number of target genes, testing for multiple cancer-predisposing genes is now being considered, but its significance in clinical practice remains unclear. We then developed a sequencing panel containing 68 genes that had cancer risk association for patients with early-onset or familial breast cancer. A total of 133 patients were enrolled and 30 (22.6%) were found to carry germline deleterious mutations, 9 in BRCA1, 11 in BRCA2, 2 in RAD50, 2 in TP53 and one each in ATM, BRIP1, FANCI, MSH2, MUTYH, and RAD51C. Triple-negative breast cancer (TNBC) was associated with the highest mutation rate (45.5%, p = 0.025). Seven of the 9 BRCA1 mutations and the single FANCI mutation were in the TNBC group; 9 of the 11 BRCA2, 1 of the 2 RAD50 as well as BRIP1, MSH2, MUTYH, and RAD51C mutations were in the hormone receptor (HR)(+)Her2(−) group, and the other RAD50, ATM, and TP53 mutations were in the HR(+)Her2(+) group. Mutation carriers were considered as high-risk to develop malignancy and advised to receive cancer screening. Screening protocols of non-BRCA genes were based on their biologic functions; for example, patients carrying RAD51C mutation received a screening protocol similar to that for BRCA, since BRCA and RAD51C are both involved in homologous recombination. In conclusion, we consider that multiple gene sequencing in cancer risk assessment is clinically valuable.

Glutamine Supplementation Attenuates Expressions of Adhesion Molecules and Chemokine Receptors on T Cells in a Murine Model of Acute Colitis

Background. Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease. This study investigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cells in mice with dextran sulfate sodium- (DSS-) induced colitis. Methods. C57BL/6 mice were fed either a standard diet or a Gln diet replacing 25% of the total nitrogen. After being fed the diets for 5 days, half of the mice from both groups were given 1.5% DSS in drinking water to induce colitis. Mice were killed after 5 days of DSS exposure. Results. DSS colitis resulted in higher expression levels of P-selectin glycoprotein ligand- (PSGL-) 1, leukocyte function-associated antigen- (LFA-) 1, and C-C chemokine receptor type 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells, and mRNA levels of endothelial adhesion molecules in colons were upregulated. Gln supplementation decreased expressions of PSGL-1, LFA-1, and CCR9 by Th cells. Colonic gene expressions of endothelial adhesion molecules were also lower in Gln-colitis mice. Histological finding showed that colon infiltrating Th cells were less in the DSS group with Gln administration. Conclusions. Gln supplementation may ameliorate the inflammation of colitis possibly via suppression of T cell migration.

miR-520h is crucial for DAPK2 regulation and breast cancer progression

MicroRNAs (miRNAs) are small RNAs that suppress gene expression by their interaction with 3’untranslated region of specific target mRNAs. Although the dysregulation of miRNAs has been identified in human cancer, only a few of these miRNAs have been functionally documented in breast cancer. Thus, defining the important miRNA and functional target involved in chemoresistance is an urgent need for human breast cancer treatment. In this study, we, for the first time, identified a key role of miRNA 520h (miR-520h) in drug resistance. Through protecting cells from paclitaxel-induced apoptosis, expression of miR-520h promoted the drug resistance of human breast cancer cells. Bioinformatics prediction, compensatory mutation and functional validation further confirmed the essential role of miR-520h-suppressed Death-associated protein kinase 2 (DAPK2) expression, as restoring DAPK2 abolished miR-520h-promoted drug resistance, and knockdown of DAPK2 mitigated cell death caused by the depletion of miR-520h. Furthermore, we observed that higher level of miR-520h is associated with poor prognosis and lymph node metastasis in human breast cancer patients. These results show that miR-520h is not only an independent prognostic factor, but is also a potential functional target for future applications in cancer therapeutics.

Deacetylation of HSPA5 by HDAC6 leads to GP78-mediated HSPA5 ubiquitination at K447 and suppresses metastasis of breast cancer

Heat-shock protein 5 (HSPA5) is a marker for poor prognosis in breast cancer patients and has an important role in cancer progression, including promoting drug resistance and metastasis. In this study, we identify that the specific lysine residue 447 (K447) of HSPA5 could be modified with polyubiquitin for subsequent degradation through the ubiquitin proteasomal system, leading to the suppression of cell migration and invasion of breast cancer. We further found that GP78, an E3 ubiquitin ligase, interacted with the C-terminal region of HSPA5 and mediated HSPA5 ubiquitination and degradation. Knock down of GP78 significantly increased the expression of HSPA5 and enhanced migration/invasive ability of breast cancer cells. Knock down of histone deacetylase-6 (HDAC6) increased the acetylation of HSPA5 at lysine residues 353 (K353) and reduced GP78-mediated ubiquitination of HSPA5 at K447 and then increased cell migration/invasion. In addition, we demonstrate that E3 ubiquitin ligase GP78 preferentially binds to deacetylated HSPA5. Notably, the expression levels of GP78 inversely correlated with HSPA5 levels in breast cancer patients. Patients with low GP78 expression significantly correlated with invasiveness of breast cancer, advanced tumor stages and poor clinical outcome. Taken together, our results provide new mechanistic insights into the understanding that deacetylation of HSPA5 by HDAC6 facilitates GP78-mediated HSPA5 ubiquitination and suggest that post-translational regulation of HSPA5 protein is critical for HSPA5-mediated metastatic properties of breast cancer.

Keap1-Nrf2 Interaction Suppresses Cell Motility in Lung Adenocarcinomas by Targeting the S100P Protein.

Purpose: Kelch-like ECH-associated protein 1 (Keap1) is an E3 ligase participated in the cellular defense response against oxidative stress through nuclear factor erythroid-2–related factor 2 (Nrf2). However, the role of Keap1 in regulating cancer motility is still controversial. We investigated the contribution of the Keap1–Nrf2 axis in the progression of non–small cell lung cancer (NSCLC). Experimental Design: The expression of Keap1 and Nrf2 was examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of NSCLC tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of the Keap1–Nrf2 axis in lung cancer mobility and progression. Results: Keap1 expression was decreased in specimens from NSCLC patients with lymph node metastasis compared with patients without metastasis. Higher Keap1 expression levels were correlated with the survival of NSCLC patients. Moreover, manipulation of Keap1 expression affected cell migration/invasion abilities. Depletion of Nrf2 relieved the migration promotion imposed by Keap1 suppression. Mechanistic investigations found that S100P was downregulated in both Keap1-overexpressing and Nrf2-knockdown NSCLC cells. Overexpression of Keap1 and knockdown of Nrf2 both suppressed S100P expression in NSCLC cells. Knockdown of S100P inhibited cell migration in highly invasive NSCLC cells and also relieved the migration promotion imposed by Keap1 suppression in weakly invasive NSCLC cells. Conclusions: Our findings suggest that Keap1 functions as a suppressor of tumor metastasis by targeting the Nrf2/S100P pathway in NSCLC cells. In addition, overexpression of Keap1 may be a novel NSCLC treatment strategy and/or useful biomarker for predicting NSCLC progression. Clin Cancer Res; 21(20); 4719–32. ©2015 AACR.