HIF-1α Promotes Epithelial-Mesenchymal Transition and Metastasis through Direct Regulation of ZEB1 in Colorectal Cancer

It is well recognized that hypoxia-inducible factor 1 alpha (HIF-1α) is involved in cancer metastasis, chemotherapy and poor prognosis. We previously found that deferoxamine, a hypoxia-mimetic agent, induces epithelial-mesenchymal transition (EMT) in colorectal cancer. Therefore, here we explored a new molecular mechanism for HIF-1α contributing to EMT and cancer metastasis through binding to ZEB1. In this study, we showed that overexpression of HIF-1α with adenovirus infection promoted EMT, cell invasion and migration in vitro and in vivo. On a molecular level, HIF-1α directly binding to the proximal promoter of ZEB1 via hypoxia response element (HRE) sites thus increasing the transactivity and expression of ZEB1. In addition, inhibition of ZEB1 was able to abrogate the HIF-1α-induced EMT and cell invasion. HIF-1α expression was highly correlated with the expression of ZEB1 in normal colorectal epithelium, primary and metastatic CRC tissues. Interestingly, both HIF-1α and ZEB1 were positively associated with Vimentin, an important mesenchymal marker of EMT, whereas negatively associated with E-cadherin expression. These findings suggest that HIF-1α enhances EMT and cancer metastasis by binding to ZEB1 promoter in CRC. HIF-1α and ZEB1 are both widely considered as tumor-initiating factors, but our results demonstrate that ZEB1 is a direct downstream of HIF-1α, suggesting a novel molecular mechanism for HIF-1α-inducing EMT and cancer metastasis.     

miR-221 Promotes Epithelial-Mesenchymal Transition through Targeting PTEN and Forms a Positive Feedback Loop with β-catenin/c-Jun Signaling Pathway in Extra-Hepatic Cholangiocarcinoma

Extrahepatic cholangiocarcinoma (EHCC) is a refractory malignancy with poor prognosis due to its early invasion, metastasis and recurrence after operation. Therefore, understanding the mechanisms of invasion and metastasis is the key to the development of new and effective therapeutic strategies for EHCC. In the present study we demonstrated that miR-221 promoted EHCC invasion and metastasis through targeting PTEN and formed a positive feedback loop with β-catenin/c-Jun signaling pathway. We found miR-221 was upregulated in EHCC specimens and CC cell lines. Moreover, miR-221 was found strongly associated with the metastasis and prognosis of EHCC patients. The expression of PTEN was downregulated in EHCC patients and CC cell lines, and was further demonstrated as one of the downstream targets of miR-221. In addition, our data indicated that β-catenin activated miR-221 through c-jun, while miR-221 enhanced β-catenin signaling induced-epithelial-mesenchymal transition (EMT) by targeting PTEN, hence forming a positive feedback loop in EHCC cell lines. In conclusion, our results suggested that miR-221 promotes EMT through targeting PTEN and forms a positive feedback loop with β-catenin/c-Jun signaling pathway in EHCC.     

Honokiol Suppresses Renal Cancer Cells’ Metastasis via Dual-Blocking Epithelial-Mesenchymal Transition and Cancer Stem Cell Properties through Modulating miR-141/ZEB2 Signaling

Renal cell carcinoma (RCC) is associated with a high frequency of metastasis and only few therapies substantially prolong survival. Honokiol, isolated from Magnolia spp. bark, has been shown to exhibit pleiotropic anticancer effects in many cancer types. However, whether honokiol could suppress RCC metastasis has not been fully elucidated. In the present study, we found that honokiol suppressed renal cancer cells’ metastasis via dual-blocking epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. In addition, honokiol inhibited tumor growth in vivo. It was found that honokiol could up-regulate miR-141, which targeted ZEB2 and modulated ZEB2 expression. Honokiol reversed EMT and suppressed CSC properties partly through the miR-141/ZEB2 axis. Our study suggested that honokiol may be a suitable therapeutic strategy for RCC treatment.     

A 100-kDa Protein Tyrosine Phosphorylation Is Concurrent with β1 Integrin-Mediated Morphological Differentiation in Neuroblastoma and Small Cell Lung Cancer Cells

IMR32, a neuroblastoma cell line, and CADO LC6, a small cell lung cancer (SCLC) cell line, extended neurite-like processes when cultured on fibronectin (FN)-coated surfaces or cultured in a serum-free medium. Monoclonal antibodies against the integrin β1 subunit inhibited this process formation, suggesting that their morphological change is initiated by β1 integrin-dependent signal transduction to the cell interior. Anti-phosphotyrosine immunoblots demonstrated that the phosphorylation level of a 100-kDa protein, but not 125-kDa focal adhesion kindase, correlated well with the morphological change in both cell lines. This 100-kDa protein phosphorylation did not accompany FN-induced morphological changes in NIH 3T3 fibroblasts or A549 adenocarcinoma cells. These findings suggest that neuroblastoma and SCLC may share β1 integrin-mediated signaling events distinct from nonneuronal cells.     

Helicobacter pylori Promotes Epithelial–Mesenchymal Transition in Gastric Cancer by Downregulating Programmed Cell Death Protein 4 (PDCD4)

Helicobacter pylori, a Gram-negative, microaerophilic bacterium found in the stomach, is assumed to be associated with carcinogenesis, invasion and metastasis in digestive diseases. Cytotoxin-associated gene A (CagA) is an oncogenic protein of H. pylori that is encoded by a Cag pathogenicity island related to the development of gastric cancer. The epithelial–mesenchymal transition (EMT) is the main biological event in invasion or metastasis of epithelial cells. H. pylori may promote EMT in human gastric cancer cell lines, but the specific mechanisms are still obscure. We explored the underlying molecular mechanism of EMT induced by H. pylori CagA in gastric cancer. In our article, we detected gastric cancer specimens and adjacent non-cancerous specimens by immunohistochemistry and found increased expression of the EMT-related regulatory protein TWIST1 and the mesenchymal marker vimentin in cancer tissues, while programmed cell death factor 4 (PDCD4) and the epithelial marker E-cadherin expression decreased in cancer specimens. These changes were associated with degree of tissue malignancy. In addition, PDCD4 and TWIST1 levels were related. In gastric cancer cells cocultured with CagA expression plasmid, CagA activated TWIST1 and vimentin expression, and inhibited E-cadherin expression by downregulating PDCD4. CagA also promoted mobility of gastric cancer cells by regulating PDCD4. Thus, H. pylori CagA induced EMT in gastric cancer cells, which reveals a new signaling pathway of EMT in gastric cancer cell lines.     

Metformin Plays a Dual Role in MIN6 Pancreatic β Cell Function through AMPK-dependent Autophagy

Metformin improves insulin sensitivity in insulin sensitive tissues such as liver, muscle and fat. However, the functional roles and the underlying mechanism of metformin action in pancreatic β cells remain elusive. Here we show that, under normal growth condition, metformin suppresses MIN6 β cell proliferation and promotes apoptosis via an AMPK-dependent and autophagy-mediated mechanism. On the other hand, metformin protects MIN6 cells against palmitic acid (PA)-induced apoptosis. Our findings indicate that metformin plays a dual role in β cell survival and overdose of this anti-diabetic drug itself may lead to potential β cell toxicity.     

Zinc Protoporphyrin Suppresses β-Catenin Protein Expression in Human Cancer Cells: The Potential Involvement of Lysosome-Mediated Degradation

Zinc protoporphyrin (ZnPP) has been found to have anticancer activity both in vitro and in vivo. We have recently demonstrated that ZnPP diminishes β-catenin protein expression in cancer cells. The present study examined the cellular mechanisms that mediate ZnPP’s suppression of β-catenin expression. We demonstrate that ZnPP induces a rapid degradation of the β-catenin protein in cancer cells, which is accompanied by a significant inhibition of proteasome activity, suggesting that proteasome degradation does not directly account for the suppression. The possibility that ZnPP induces β-catenin exportation was rejected by the observation that there was no detectable β-catenin protein in the conditioned medium after ZnPP treatment of cancer cells. Further experimentation demonstrated that ZnPP induces lysosome membrane permeabilization, which was reversed by pretreatment with a protein transportation inhibitor cocktail containing Brefeldin A (BFA) and Monensin. More significantly, pretreatment of cancer cells with BFA and Monensin attenuated the ZnPP-induced suppression of β-catenin expression in a concentration- and time-dependent manner, indicating that the lysosome protein degradation pathway is likely involved in the ZnPP-induced suppression of β-catenin expression. Whether there is cross-talk between the ubiquitin-proteasome system and the lysosome pathway that may account for ZnPP-induced β-catenin protein degradation is currently unknown. These findings provide a novel mechanism of ZnPP’s anticancer action and reveal a potential new strategy for targeting the β-catenin Wnt signaling pathway for cancer therapy.     

Functional Analysis of Novel Candidate Regulators of Insulin Secretion in the MIN6 Mouse Pancreatic β Cell Line

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic β cells is important for understanding and treating diabetes. The pancreatic β cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.     

Inactivation of DNA–Dependent Protein Kinase Promotes Heat–Induced Apoptosis Independently of Heat–Shock Protein Induction in Human Cancer Cell Lines

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.     

Tubulin-dependent secretion of S100A6 and cellular signaling pathways activated by S100A6-integrin β1 interaction

S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin β1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin β1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin–dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and β tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin β1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin β1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways.     

Accumulation of Phosphorylated β-Catenin Enhances ROS-Induced Cell Death in Presenilin-Deficient Cells

Presenilin (PS) is involved in many cellular events under physiological and pathological conditions. Previous reports have revealed that PS deficiency results in hyperproliferation and resistance to apoptotic cell death. In the present study, we investigated the effects of PS on β-catenin and cell mortality during serum deprivation. Under these conditions, PS1/PS2 double-knockout MEFs showed aberrant accumulation of phospho-β-catenin, higher ROS generation, and notable cell death. Inhibition of β-catenin phosphorylation by LiCl reversed ROS generation and cell death in PS deficient cells. In addition, the K19/49R mutant form of β-catenin, which undergoes normal phosphorylation but not ubiquitination, induced cytotoxicity, while the phosphorylation deficient S37A β-catenin mutant failed to induce cytotoxicity. These results indicate that aberrant accumulation of phospho-β-catenin underlies ROS-mediated cell death in the absence of PS. We propose that the regulation of β-catenin is useful for identifying therapeutic targets of hyperproliferative diseases and other degenerative conditions.     

Modulation of Myoepithelial-Associated α6β4 Integrin in a Breast Cancer Cell Line Alters Invasive Potential

In normal breast, cell–stromal contact is mediated by myoepithelial cells which strongly express α2β1, α3β1, and α6β4 integrins, while epithelial cells exhibit α2β1 and α3β1 integrins at cell–cell borders, but do not express α6β4 integrin. Breast carcinomas consistently show down-regulation of all integrins. We have investigated the modulatory effect of stromal proteins, hormones, and transforming growth factor β (TGF-β) on integrin expression in breast cancer cell lines MCF-7, T47-D, and MDA-MB 231 using indirect immunofluorescence and confocal laser scanning microscopy. MCF-7 and T47-D cells displayed low levels of both α2β1 and α3β1 integrins, and no α6β4 integrin, and this profile remained unchanged by modulatory agents. The MDA-MB 231 cells exhibited stronger staining for α2β1 and α3β1 integrins and focal staining for α6β4 integrin under control conditions, but markedly enhanced reactivity for the α6β4 complex in the presence of TGF-β. This was associated with acquisition of a spread cellular morphology and localization of α6β4 at the cell periphery in a discrete punctate distribution. There was associated enhanced expression of epiligrin, the ligand for α6β4, with similar localization to the cell periphery. Cell invasion assays through a Matrigel barrier revealed significantly reduced invasive potential of TGF-β-treated cells, an effect largely reversed following preincubation of the treated cells with anti-β4 integrin antibody. We conclude that α6β4 integrin can be up-regulated by TGF-β and has an anti-invasive effect on MDA-MB 231 cells. In addition to α6β4, MDA-MB 231 cells exhibit other myoepithelial markers including cytokeratin 14, vimentin, and weak expression of CALLA. These findings support the concept of a subgroup of breast carcinomas displaying features of myoepithelial differentiation.     

Microarray Analysis of Novel Candidate Genes Responsible for Glucose-Stimulated Insulin Secretion in Mouse Pancreatic β Cell Line MIN6

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet β cells is important for understanding and treating diabetes. MIN6 cells, a transformed β-cell line derived from a mouse insulinoma, retain GSIS and are a popular in vitro model for insulin secretion. However, in long-term culture, MIN6 cells'' GSIS capacity is lost. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells, that shows well-regulated insulin secretion in response to glucose, glybenclamide, and KCl, even after prolonged culture. To investigate the molecular mechanisms responsible for preserving GSIS in this subclone, we compared four groups of MIN6 cells: Pr-LP (parental MIN6, low passage number), Pr-HP (parental MIN6, high passage number), C4-LP (MIN6 clone 4, low passage number), and C4-HP (MIN6 clone 4, high passage number). Based on their capacity for GSIS, we designated the Pr-LP, C4-LP, and C4-HP cells as “responder cells.” In a DNA microarray analysis, we identified a group of genes with high expression in responder cells (“responder genes”), but extremely low expression in the Pr-HP cells. Another group of genes (“non-responder genes”) was expressed at high levels in the Pr-HP cells, but at extremely low levels in the responder cells. Some of the responder genes were involved in secretory machinery or glucose metabolism, including Chrebp, Scgn, and Syt7. Among the non-responder genes were Car2, Maf, and Gcg, which are not normally expressed in islet β cells. Interestingly, we found a disproportionate number of known imprinted genes among the responder genes. Our findings suggest that the global expression profiling of GSIS-competent and GSIS-incompetent MIN6 cells will help delineate the gene regulatory networks for insulin secretion.