3091

HIF1A

Here, we show that NF-kappaB-HIF-1 interaction contributed to breast cancer metastatic capacity by means of an incomplete epithelial/mesenchymal transition and influencing migration, as shown in 1833 (human) and 4T1 (mouse) metastatic cells after different stimuli. The 1833 and the transforming growth factor-beta1-exposed 4T1 cells showed both epithelial (E-cadherins) and mesenchymal (N-cadherins and vimentin) markers, and common mechanisms contributed to the retention of certain epithelial characteristics and the control of migration. The complex NF-kappaB-HIF-1 reciprocal regulation and the enhanced c-Jun expression played a functional role in exacerbating the invasiveness of 1833 cells after p50/p65 transfection and of 4T1 cells exposed to transforming growth factor-beta1. Twist expression seemed to exert a permissive role also regulating epithelial/mesenchymal transition markers. After c-Src wild-type (Srcwt) transfection, c-Src-signal transducer overexpression in 1833 cells increased HIF-1 transactivating activity and invasiveness, and changed E-cadherin/N-cadherin ratio versus mesenchymal phenotype. The transcription factor pattern and the motile phenotype of metastatic 1833 cells were influenced by p65-lysine acetylation and HDAC-dependent epigenetic mechanisms, which positively regulated basal NF-kappaB and HIF-1 activities. However, HDAC3 acted as a corepressor of NF-kappaB activity in parental MDA-MB231 cells, thus explaining many differences from the derived 1833 clone, including reduced HIF-1alpha and c-Jun expression. Invasiveness was differently affected by HDAC knockdown in 1833 and MDA-MB231 cells. We suggest that acetylation/deacetylation are critical in establishing the bone-metastatic gene signature of 1833 cells by regulating the activity of NF-kappaB and HIF-1, and further clarify the epigenetic control of transcription factor network in the motile phenotype of 1833 cells.

Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of alpha-smooth muscle actin (alpha-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of alpha-SMA and vimentin was prevented in mitochondria-deficient rho(0) cells, which are incapable of ROS production during hypoxia. CoCl(2) and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)-alpha under normoxia, failed to induce alpha-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1alpha did not induce alpha-SMA. However, loss of HIF-1alpha or HIF-2alpha abolished induction of alpha-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-beta1, and preincubation of AEC with SB431542, an inhibitor of the TGF-beta1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-beta1 dependent. Furthermore, both ROS and HIF-alpha were necessary for hypoxia-induced TGF-beta1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-beta1 signaling.

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

OBJECTIVE: Wnt/beta-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in epithelial-mesenchymal transition (EMT), however, there was no evidence that Wnt/beta-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1alpha (HIF-1alpha) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of beta-catenin protein increased in LNCaP/HIF-1alpha cells, but not in PC-3/HIF-1alpha. This study mainly aimed at exploring the essentiality and importance of Wnt/beta-catenin signaling pathway in HIF-1alpha-induced EMT. METHODS: Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1alpha. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1alpha was transiently transfected with beta-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/beta-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay. RESULT: We successfully established prostate cancer cell line LNCaP/HIF-1alpha and LNCaP/HIF-1alpha/beta-catenin(-). LNCaP/HIF-1alpha displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1alpha, the epithelial marker E-cadherin was increased in LNCaP/HIF-1alpha/beta-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through beta-catenin shRNA cause a reversal of EMT induced by HIF-1alpha in human prostate cancer. CONCLUSION: Overexpression of HIF-1alpha stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/beta-catenin signaling pathway played a vital role in this process. Wnt/beta-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1alpha.

BACKGROUND: epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-kappaB is closely involved in the progression of EMT. Compared with HIF-1alpha, the correlation between NF-kappaB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-kappaB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1alpha into pancreatic cancer cells under normoxic conditions heightened NF-kappaB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-kappaB activity in this setting attenuated the EMT phenotype. CONCLUSIONS/SIGNIFICANCE: These results suggest that hypoxia or overexpression of HIF-1alpha induces the EMT that is largely dependent on NF-kappaB in pancreatic cancer cells.

Hypoxia is known to promote malignant progression and to induce chemoresistance in cancer. However, the exact mechanisms driving hypoxia induced malignance remain elusive. We found that with exposure to hypoxic condition, hepatocellular carcinoma (HCC) cells experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion, and exhibited high resistance to chemotherapy. We demonstrated that hypoxia-induced EMT and chemoresistance were accompanied by increased HIF-1alpha expression and activation of Akt. HIF-1alpha could be blocked by PI3K inhibitor LY294002, indicating HIF-1alpha activation was regulated by PI3K/Akt pathway. Furthermore, we showed that inhibition of PI3K/Akt and HIF-1alpha enhanced the therapeutic efficacy of hypoxic chemotherapy in the HCC xenograft model. Our findings indicate that the activation of PI3K/Akt/HIF-1alpha pathway plays a critical role in mediating hypoxia-induced EMT and drug resistance leading to unfavorable treatment outcome. Our study provides novel insights into the malignant progression triggered by hypoxic microenvironment in HCC cells.

RATIONALE: Nasal polyposis implies a refractory clinical course in case of chronic rhinosinusitis (CRS). Although hypoxia is believed to be associated with nasal polyposis, little is known about the mechanism underlying polypogenesis. OBJECTIVES: To determine if hypoxia drives nasal polyposis by epithelial-to-mesenchymal transition (EMT). METHODS: Immunoblotting, immunofluorescence, flow cytometry, and real-time polymerase chain reaction were performed to evaluate EMT and hypoxic markers in human nasal epithelial cells (hNECs) and in sinonasal tissues from patients with CRS with or without polyps. In addition, the effects of hypoxia-inducible factor (HIF)-1alpha inhibitors on nasal polypogenesis were investigated in a murine model. MEASUREMENTS AND MAIN RESULTS: E-cadherin and alpha-smooth muscle actin (alpha-SMA) were down-regulated and up-regulated, respectively, in patients with polyps as compared with patients without polyps. Under hypoxia, hNECs transformed to a mesenchymal shape, and demonstrated representative changes in EMT markers; that is, mesenchymal markers (alpha-SMA, vimentin, and twist) increased but epithelial markers (E-cadherin and beta-catenin) decreased. Mechanistically, E-cadherin level was recovered in hypoxia by silencing HIF-1alpha and decreased in normoxia by expressing HIF-1alpha. Furthermore, hypoxia was found to down-regulate PP2Ac phosphatase and up-regulate pSmad3, which led to alpha-SMA induction. In CRS sinonasal specimens, HIF-1alpha expression was found to correlate with E-cadherin loss and alpha-SMA expression. Finally, HIF-1alpha inhibitors suppressed nasal polypogenesis in a murine model. CONCLUSIONS: hNECs undergo EMT during hypoxia and this process is critically mediated by HIF-1alpha and pSmad3. This study shows that hypoxia-induced EMT is likely to contribute to nasal polyposis in CRS, and suggests that HIF-1alpha be viewed as a therapeutic target for nasal polyposis.

cancer stem cells (CSCs) are predicted to be critical drivers of tumor progression due to their "stemness", but the molecular mechanism of CSCs in regulating metastasis remains to be elucidated. epithelial-mesenchymal transition (EMT), hypoxia-inducible factor (HIF)-1alpha, and miR-21, all of which contribute to cell migration for metastasis, are interrelated with CSCs. In the present study, third-sphere forming (3-S) CSC-like cells, which showed elevated CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and sphereforming capacity as well as migration and invasion capacities, were cultured and isolated from breast cancer MCF-7 parental cells, to evaluate the role of miR-21 in regulating the CSC-like cell biological features, especially EMT. EMT, which was assessed by overexpression of mesenchymal cell markers (N-cadherin, Vimentin, alpha-smooth muscle actin [alpha-SMA]) and suppression of epithelial cell marker (E-cadherin), was induced in 3-S CSC-like cells. Moreover, both of HIF-1alpha and miR-21 were upregulated in the CSC-like cells. Interestingly, antagonism of miR-21 by antagomir led to reversal of EMT, downexpression of HIF-1alpha, as well as suppression of invasion and migration, which indicates a key role of miR-21 involved in regulate CSC-associated features. In conclusion, we demonstrated that the formation of CSC-like cells undergoing process of EMT-like associated with overexpression of HIF-1alpha, both of which are regulated by miR-21.CI - (c) 2012 Japanese cancer Association.

BACKGROUND: Late kidney allograft dysfunction is becoming a significant problem and tubular atrophy and interstitial fibrosis are main causes. It was reported that hypoxia could induce epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells (TECs), and hypoxia-inducible factor-1 (HIF-1) is one of the important regulators of cellular adaptive to hypoxia. METHODS: In this study, we used an HIF-1alphaDeltaODD-expressing adenovirus, which could stably and functionally express HIF-1alpha under normoxia conditions, and used a hypoxia/reoxygenation cell model to simulate ischemia/reperfusion (I/R) injury in vitro, to investigate the effect of HIF-1alpha on EMT-related gene expressions. RESULTS: Our results demonstrated that HIF-1alpha could significantly upregulate alpha-smooth muscle actin expression, and reduced the E-cadherin expression in HK-2 cells during I/R injury. Moreover, miR-21 expression had a positive correlation with HIF-1alpha in this process. CONCLUSION: These results suggest that HIF-1alpha may promote the EMT development through regulating fibrotic gene expression during I/R injury in human renal TECs, and miR-21 could be among the important regulatory pathways in the process.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

Pancreatic cancer is one of the highly aggressive malignant diseases worldwide. To achieve better treatment outcome of pancreatic cancer, in the current study we explore the underlying molecular mechanism of drug resistance in pancreatic cancer cells. We found that resistance to gemcitabine is associated with epithelial-mesenchymal transition (EMT) phenotype in a panel of pancreatic cancer cell lines. Notably, gemcitabine-resistant pancreatic cancer cells acquire EMT phenotype. Moreover, gemcitabine-resistant cells have increased migration and invasion activities. Furthermore, we observed the high expression of HIF-1alpha in gemcitabine-resistant cells. More importantly, inhibition of HIF-1alpha in gemcitabine-resistant cells caused partial reversal of EMT phenotype, suggesting that HIF-1alpha was critically involved in gemcitabine-resistant-mediated EMT. Therefore, targeting HIF-1alpha could be an effective strategy for the treatment of pancreatic cancer.

The aim of the present study was to investigate the effects of tanshinone IIA (Tan IIA), an active constituent of Salvia miltiorrhiza Bunge, on epithelial-mesenchymal transition (EMT) and hypoxia-induced chemoresistance in breast cancer cells. To induce hypoxia, MCF-7 and HCC1973 cells were treated with 100 microM deferoxamine followed by doxorubicin (DOX). Cell viability and proliferation were examined using the CCK-8 and EdU assays, respectively. Western blot and immunofluorescence analyses of the expression of two EMT markers, E-cadherin and vimentin, were also carried out. The role of HIF-1alpha and TWIST in mediating the effects of Tan IIA was determined through siRNA. Based on the results, hypoxia-induced DOX resistance was observed in both MCF-7 and HCC1973 cells (both P=0.001), which was reversed with Tan IIA. Specifically, in hypoxic conditions, Tan IIA significantly decreased cell viability and proliferation (all Pexpression abrogated the effects of Tan IIA on cell viability. Taken together, Tan IIA ameliorated hypoxia-induced DOX resistance and EMT in breast cancer cell lines, which may be attributed to the downregulation of HIF-1alpha expression. Further in vivo studies, however, are required to fully elucidate the therapeutic potential of Tan IIA in increasing the sensitivity of breast cancer cells to chemotherapy.

epithelial-to-mesenchymal transition (EMT) facilitates the escape of pancreatic cancer cells from the primary tumor site, which is a key early event in metastasis. In the present study, we examined if intermittent hypoxia facilitates the invasiveness of human pancreatic cancer cell lines (Panc-1 and BxPC-3) by Transwell assay. We used western blotting and flow cytometry analysis to quantify stem-like cells in the migratory cells during intermittent hypoxia in the human pancreatic cancer cells. Under normoxia or intermittent hypoxia, the expression of autophagy-related proteins (LC3-II and Beclin), hypoxia-inducible factor-1alpha (HIF-1alpha) and EMT-related markers (E-cadherin, Vimentin and N-cadherin) was examined by western blotting. siRNA and the autophagic inhibitor were used to access the role of HIF-1alpha and autophagy in promoting metastasis and EMT. Under intermittent hypoxia, pancreatic cancer cells demonstrated enhanced invasive ability and enriched stem-like cells. The migratory cells displayed stem-like cell characteristics and elevated the expression of LC3-II and Beclin-1, HIF-1alpha, E-cadherin, Vimentin and N-cadherin under intermittent hypoxia conditions. Moreover, enhanced autophagy was induced by the elevated level of HIF-1alpha. The metastatic ability and EMT of pancreatic cancer stem cells was associated with HIF-1alpha and autophagy. This novel finding may indicate the specific role of HIF-1alpha and autophagy in promoting the metastatic ability of pancreatic cancer stem cells. Additionally, it emphasizes the importance of developing therapeutic strategies targeting cancer stem cells and autophagy to reduce metastasis.

In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-beta and HIF1alpha signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-beta signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1alpha knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1alpha signaling appears to play an important role in this process.

This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1alpha and HIF-2alpha are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1alpha or HIF-2alpha expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1alpha depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on beta-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2alpha knockdown did not affect beta-catenin levels, increasing the transcriptional activity of beta-catenin by inducing its nuclear accumulation, whereas HIF-1alpha silencing negatively affected the stability and transcriptional activity of beta-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1alpha depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2alpha silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1alpha silencing. Remarkably, HIF-2alpha knockdown also enhanced beta-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells.

It is well recognized that hypoxia-inducible factor 1 alpha (HIF-1alpha) 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-1alpha contributing to EMT and cancer metastasis through binding to ZEB1. In this study, we showed that overexpression of HIF-1alpha with adenovirus infection promoted EMT, cell invasion and migration in vitro and in vivo. On a molecular level, HIF-1alpha 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-1alpha-induced EMT and cell invasion. HIF-1alpha expression was highly correlated with the expression of ZEB1 in normal colorectal epithelium, primary and metastatic CRC tissues. Interestingly, both HIF-1alpha 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-1alpha enhances EMT and cancer metastasis by binding to ZEB1 promoter in CRC. HIF-1alpha and ZEB1 are both widely considered as tumor-initiating factors, but our results demonstrate that ZEB1 is a direct downstream of HIF-1alpha, suggesting a novel molecular mechanism for HIF-1alpha-inducing EMT and cancer metastasis.

Choriocarcinoma (CC) is the highest malignant gestational trophoblastic tumor which causes high mortality without timely treatment. HIF-1alpha is a very important molecule promoting neoplasm aggressiveness, metastasis through the induction of the epithelial to mesenchymal transition (EMT). Several researches have shown that Notch signaling is necessary for coupling hypoxia to the EMT. However, the pathway in choriocarcinoma remains undetermined. In this study, overexpression of HIF-1alpha inhibited epithelial index E-cadherin, cytokeratin 18 (CK18) and cytokeratin 19 (CK19) in two choriocarcinoma cell lines (JAR and JEG-3, respectively). The reciprocal changes associated with the EMT phenotypic transformation. The migration and invasive capability was significantly enhanced. HIF-1alpha overexpression was positively correlated with Notch1 and Hes1 (P<0.01; respectively). Using DAPT (Notch1 inhibitor) to knock down Notch1 expression, morphological and molecular typical changes of the EMT were detected in vitro. Knockdown of Notch1 prominently reduced MMP2 and MMP9 activities, and increased E-cadherin, CK18 and CK19 expression in JAR and JEG-3 cells (P<0.01). In vivo, serum-free suspension containing 5x106 JAR cells (lv-HIF-1alpha and lv-NC, respecticely) were implanted subcutaneously or injected intravenously into female NOD SCID mice, which were injected with DAPT or DMSO six times at 3-day intervals. Mice with subcutaneous nodules were injected i.v. fluorine-18 fluorodeoxyglucose (18F-FDG) and then scanned in small animal PET/CT (SA-PET/CT), and mice with lung metastasis were monitored by using the in vivo imaging system (IVIS). The in vivo data showed that inhibition of Notch signaling by DAPT treatment effectively suppressed metastatic tumor growth, metabolic activity and invasion. In summary, overexpression of HIF-1alpha promotes choriocarcinoma cell aggressiveness and metastasis through the regulation of the EMT dependenting on Notch signaling pathway.

UNLABELLED: The transcription factor sex determining region (Y SRY)-box 2 (SOX2) is known to play a crucial role in the maintenance of self renewal or pluripotency of undifferentiated embryonic and neuronal stem cells. An elevated expression of SOX2 has been correlated with poor prognosis of esophageal squamous cell carcinoma (ESCC). We sought to investigate the mechanism(s) by which SOX2 modulates the ESCC metastasis. The SOX2 coding DNA sequence was inserted into pCMV vector and stably transfected in ESCC cells (Eca-109). The effect of SOX2 over expression was evaluated on cell migration, invasion and epithelial to mesenchymal transition (EMT). We also measured the expression of Slug to explore if this transcription factor is involved in SOX2-mediated regulation of cell migration/invasion and EMT. In addition, we determined the role of STAT3/HIF-1alpha to further probe the mechanism of SOX2-mediated metastasis via Slug. Our results demonstrated that SOX2 over expressing Eca-109 cells showed an enhanced cell migration/invasion. Moreover, these cells exhibited the EMT characteristics, that is, a significantly suppressed expression of the epithelial cells marker with a concomitant enhancement of those of the mesenchymal markers. An increased expression of Slug in SOX2 over expressing cells suggested the involvement of this transcription factor in SOX2-regulated metastasis. Whereas the expressions of STAT3/HIF-1alpha were found to be up-regulated in SOX2 expressing cells, blockade of these transcription factors resulted in the inhibition of Slug expression at both protein and mRNA levels. CONCLUSION: These results suggest that SOX2 promoted the metastasis of ESCC, at least in part, by modulating Slug expression through the activation of STAT3/HIF-1alpha signaling.

PURPOSE: The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, alpha-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. METHODS: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3beta (GSK-3beta) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of beta-catenin, as well as the total lysate content of fibronectin and alpha-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1alpha (HIF-1alpha) translation inhibitor and an HIF-2alpha translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear beta-catenin and its downstream effect on EMT marker proteins and VEGF expression. RESULTS: SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3beta activity prompting a significant increase in the translocation of cytoplasmic beta-catenin to the nucleus. The enhancement of nuclear beta-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and alpha-SMA. In conjunction with SB216763, coadministration of an HIF-1alpha translation inhibitor, but not an HIF-2alpha translation inhibitor, markedly suppressed the expression of fibronectin and alpha-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear beta-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. CONCLUSIONS: Recently, we reported that nuclear beta-catenin, but not HIF-2alpha, regulates the expression of fibronectin and alpha-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear beta-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1alpha (but not HIF-2alpha) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.

BACKGROUND: Leucine-rich-alpha-2-glycoprotein 1 (LRG1) has been reported to be involved in several tumors, whether it participates in colorectal cancer (CRC) progression remains unclear. Here, we investigated the biological function and underlying molecular mechanisms of LRG1 in CRC. METHODS: The mRNA and protein levels of LRG1 were assessed in CRC tissues through RT-PCR and immunohistochemistry, respectively. HCT116 and SW480 cells were treated with LRG1 siRNA, control siRNA, or recombinant LRG1. Transwell invasion assays and wound healing assays were performed to evaluate the invasion and migration of CRC cells. epithelial-to-mesenchymal transition (EMT) markers of E-cadherin, VDR, N-cadherin, alpha-SMA, Vimentin and Twist1 were detected by RT-PCR and western blot. Enzyme-linked immunosorbent assay was used to measure the secretion level of VEGF-A. Conditioned medium from CRC cells was collected for endothelial cell migration, tube formation and aortic ring sprouting assays. RESULTS: LRG1 was overexpressed in CRC tissues and associated with cancer aggressiveness. LRG1 was further found to induce the EMT process, as well as CRC cell migration and invasion capacity. In addition, LRG1 promoted VEGF-A expression in CRC cells and contributed to tumor angiogenesis. Furthermore, HIF-1alpha could be induced by LRG1 in a concentration- and time-dependent manner, which was responsible for LRG1-induced VEGF-A expression and EMT. CONCLUSIONS: The present study suggests that LRG1 plays a crucial role in the progression of CRC by regulating HIF-1alpha expression, thereby may be a promising therapeutic target of CRC.

STUDY QUESTION: How does hypoxia promote growth of lesions in the development of endometriosis? SUMMARY ANSWER: Hypoxia induces the epithelial-mesenchymal transition (EMT) of endometrial cells, resulting in changes in cellular characteristics, which may be a prerequisite for the establishment of endometriotic lesions. WHAT IS KNOWN ALREADY: Up-regulated hypoxia-inducible factor 1alpha (HIF-1alpha) has recently been found in ectopic endometrial lesions. There is increasing evidence that EMT, in which epithelial cells acquire mesenchymal and migratory properties, may also play a role in the establishment of the disease. EMT induced by HIF-1alpha has been reported to play a role in the development of many tumor types. STUDY DESIGN, SIZE, DURATION: We investigated expression changes of N-cadherin, E-cadherin, beta-catenin, HIF-1alpha and vimentin using immunohistochemistry in normal, eutopic and ectopic endometria. Endometrial tissues from 96 additional females without related pathology were collected, and these tissues were subjected to subsequent primary cell culture for further experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression of N-cadherin, E-cadherin, beta-catenin, HIF-1alpha and vimentin in 20 normal, 21 eutopic and 21 ectopic endometrial samples was assessed by immunohistochemistry. Human primary endometrial epithelial cells were isolated from 96 normal endometrial tissues. Times for hypoxia treatment for western blot analysis were 1, 2, 4 and 8 h, for transwell experiments was 48 h. Ishikawa cells were used instead of primary endometrial epithelial cells for transfection and part of the transwell experiments. The impact of hypoxia on invasion was evaluated by transwell assays. Overexpression and inhibition of HIF-1alpha were achieved by transfection of pG/CMV/HIF-1alpha/IRES/EGFP and MCS-shHIF-1alpha-EGFP-IRES plasmids separately. The effect of hypoxia, overexpression and knockdown of HIF-1alpha on hypoxia-induced changes of EMT markers and beta-catenin were analyzed by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). MAIN RESULTS AND THE ROLE OF CHANCE: Overexpression of HIF-1alpha and changes associated with EMT were observed in normal, eutopic and ectopic endometrial tissues. Primary cultured human endometrial epithelial cells responded to hypoxia with classic EMT changes (fibroblastoid phenotype, increased expression of snail family zinc finger 1, beta-catenin and mesenchymal markers, and decreased expression of E-cadherin) and increased invasiveness. The decreased invasiveness of Ishikawa cells by knockdown of HIF-1alpha was observed under hypoxic conditions. While up-regulation of HIF-1alpha induced changes characteristic of EMT, down-regulation of HIF-1alpha had the opposite effect. Statistical significance was defined as P < 0.05. LIMITATIONS, REASONS FOR CAUTION: A weakness of this study is the relatively small sample size for immunohistochemistry. Ishikawa cells were used instead of primary endometrial epithelial cells for transfection and part of the transwell experiments. WIDER IMPLICATIONS OF THE FINDINGS: Hypoxia-stabilized HIF-1alpha may play an important role in the invasion of endometrial cells in ectopic endometrial lesions, and it may induce EMT in the development of endometriosis. HIF-1alpha may be a new and important target for the endometriosis treatment. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the National Nature Science Foundation of China (grant number 81170545 and 81471439). The authors declare no competing interests.CI - (c) The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Recently, increasing numbers of long noncoding RNAs (lncRNAs), with both oncogenic and tumor-suppressive potential, have been found to be aberrantly expressed in various human cancers. However, the function of lncRNAs in hepatocellular carcinoma (HCC) progression remains largely unknown. In this study, we performed a comprehensive microarray analysis of lncRNA expression using human HCC specimens. After validation in 119 human HCC tissues, we identified a novel tumor suppressor lncRNA, CPS1 intronic transcript 1 (CPS1-IT1). To elucidate the clinical significance of CPS1-IT1 in HCC, correlations between CPS1-IT1 levels, clinical parameters, and survival outcomes were analyzed. In vitro and in vivo functional assays were also performed to dissect the potential underlying mechanisms. expression of CPS1-IT1 was significantly decreased in 73% of HCC tissues, and patients with low CPS1-IT1 expression had poor survival outcomes. Furthermore, in vitro functional assays indicated that CPS1-IT1 significantly reduced cell proliferation, migration and invasion capacities through reduced Hsp90 binding to and activation of HIF-1alpha, thereby suppressing the epithelial-mesenchymal transition (EMT). An in vivo animal model also demonstrated the tumor suppressor role of CPS1- IT1 via decreased tumor growth and metastasis. In conclusion, lncRNA CPS1-IT1 acts as a tumor suppressor in HCC by reducing HIF-1alpha activation and suppressing EMT. The findings of this study establish a function for CPS1-IT1 in HCC progression and suggest its potential as a new prognostic biomarker and target for HCC therapy.

Choroidal neovascularization (CNV) is a major cause of vision loss in many retinal diseases. Hypoxia is determined to be a key inducer of CNV and hypoxia-inducible factor-1 (HIF-1) is an important transcription factor. epithelial-mesenchymal transition (EMT) and the synthesis of proangiogenic cytokines make great contributions to the development of CNV. In the present study, the role of HIF-1alpha signaling in the regulation of angiogenin (ANG) expression and EMT in hypoxic retinal pigment epithelial cells was investigated. A significant elevation expression of ANG expression level in a mouse model of laser-induced CNV was demonstrated. In a hypoxic model of ARPE-19, an increased expression level of ANG and induction of EMT accompanied with stabilization and nucleus translocation of HIF-1alpha. Blockage of HIF-1alpha signaling resulted in inhibition of high expression of ANG and EMT features. The direct interaction between HIF-1alpha and ANG promoter region was identified by ChIP-qPCR. The association of RNase 4 mRNA level with HIF-1alpha signaling was also clarified in APRE-19. Moreover, the exogenous ANG translocated into the nucleus, enhanced 45S rRNA transcription, promoted cell proliferation and tube formation in human retinal microvascular endothelial cells. In conclusion, the hypoxic conditions regulate the expression of ANG and EMT via an activation of HIF-1alpha signaling. It provides molecular evidence for potential therapy strategies of treating CNV.CI - Copyright (c) 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Prospero-related homeobox 1 (PROX1) is an essential regulator in lymphangiogenesis and has been implicated in both oncogenic and tumor-suppressive functions in many types of human cancers. However, the role of PROX1 in prostate cancer (PCa) remains poorly understood. In this study, based on different PCa cell lines and knockout mice, we showed that PROX1 could be suppressed by DAB2IP, a novel member of the Ras GTPase-activating protein family and a critical player in control of epithelial-mesenchymal transition (EMT) and PCa metastasis. Mechanistically, PROX1 overexpression in DAB2IP-deficient PCa cells could enhance the accumulation of HIF1alpha protein by inhibiting ubiquitin pathway and then consequently induce an EMT response, which is characterized by repression of E-cadherin, up-regulation of vimentin and matrix metallopeptidases (MMPs) and enhancement of cell migration. Together, our data provides a new insight into mechanism that DAB2IP regulates EMT and PCa metastasis, especially points out the potential roles of its downstream PROX1/HIF1alpha signaling in a unique non-skeletal metastasis of PCa.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Metastasis is the major cause of treatment failure in anaplastic thyroid carcinoma (ATC) patients. In the preliminary study, we demonstrated that interleukin (IL)-11 expression is positively correlated with distant metastasis in ATC. However, the mechanisms underlying remain largely unknown. Here, we found that cobalt chloride (a hypoxia mimetic) promoted IL-11 expression via HIF-1alpha activation. Furthermore, the resultant increase in IL-11 expression significantly induced epithelial-mesenchymal transition (EMT) in ATC cells, accompanied by Akt/GSK3beta pathway activation and increased invasive and migratory abilities. Conversely, HIF-1alpha or IL-11 knockdown, or treating cells with a neutralizing antibody against IL-11, a PI3K inhibitor, or Akt inhibitor V, significantly suppressed the induction of EMT and counteracted the enhancements in invasive and migratory abilities. These results indicate that hypoxia increases IL-11 secretion in ATC cells via HIF-1alpha induction and that IL-11 then induces EMT in these cells via the PI3K/Akt/GSK3beta pathway, ultimately improving their invasive and migratory potential. This study elucidates the prometastatic role played by IL-11 in ATC metastasis and indicates it as a potential target for the treatment of cancer metastasis. However, many questions remain to be explored.

Vasculogenic mimicry (VM) plays an important role in colorectal cancer (CRC) metastasis, and both hypoxia and the epithelial-mesenchymal transition (EMT) are necessary for VM. In this study, HIF-1alpha expression was upregulated in the VM-positive CRC cell line HCT-116 and thereby affected the expression of the EMT-related markers Claudin-4, E-cadherin (E-cd) and Vimentin(VIM). SB431542 and U0126EtOH, which can inhibit of EMT were used to treat HCT-116 and HCT-8 in these experiments. Both of the inhibitors had significant effect on EMT markers and the formations of VM in CRC cells. In addition, knockdown of HIF-1alpha in the HCT-116 cells inhibited their capacity for VM. Our study reveals a regulatory role for HIF-1alpha in VM and suggests that targeting either HIF-1alpha or EMT may be a valuable strategy for the elimination of CRC metastasis.

Hypoxia is a major driving force of cancer invasion and metastasis. Here we show that death domain-associated protein (Daxx) acts to negatively regulate hypoxia-induced cell dissemination and invasion by inhibiting the HIF-1alpha/HDAC1/Slug pathway. Daxx directly binds to the DNA-binding domain of Slug, impeding histone deacetylase 1 (HDAC1) recruitment and antagonizing Slug E-box binding. This, in turn, stimulates E-cadherin and occludin expression and suppresses Slug-mediated epithelial-mesenchymal transition (EMT) and cell invasiveness. Under hypoxic conditions, stabilized hypoxia-inducible factor (HIF)-1alpha downregulates Daxx expression and promotes cancer invasion, whereas re-expression of Daxx represses hypoxia-induced cancer invasion. Daxx also suppresses Slug-mediated lung cancer metastasis in an orthotopic lung metastasis mouse model. Using clinical tumour samples, we confirmed that the HIF-1alpha/Daxx/Slug pathway is an outcome predictor. Our results support that Daxx can act as a repressor in controlling HIF-1alpha/HDAC1/Slug-mediated cancer cell invasion and is a potential therapeutic target for inhibition of cancer metastasis.

Substantial evidence suggests that the epithelial-mesenchymal transition (EMT) phenotype is associated with the invasive characteristics of cancer stem cells (CSCs),which possess an EMT phenotype that may predominate in tumor invasion and metastasis. However, the mechanisms for the generation and regulation of these CSCs have not been clearly defined. As hypoxia and EMT-related factors may have important functions in EMT-like CSCs, the aim of this study was to investigate the effects of hypoxia on these cells. CSCs were established from the gastric cancer cell lines MGC-803 and SGC7901, and the relationship between hypoxia and EMT-like CSCs was investigated in gastric cancer. After hypoxia treatment, some gastric CSCs exhibited a marked increase in hypoxia-inducible factor-1alpha (HIF-1alpha)expression and increased migration and invasion capabilities compared with the normoxic control. These CSCs were defined by activation of the mesenchymal cell marker Vimentin and by inhibition of the epithelial cell marker E-cadherin. Our analyses also show that HIF-1alpha was responsible for activating EMT via increased expression of the transcription factor Snail in gastric CSCs. Moreover, inhibition of Snail by shRNA reduced HIF-1alpha-induced EMT in gastric CSCs. The results demonstrated that hypoxia-induced EMT-like CSCs rely on HIF-1alphato activate Snail, which may result in recurrence and metastasis of gastric cancer.

Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown.Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial-mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs.Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1alpha (HIF1alpha), HIF2alpha, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration.Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin cancer Res; 23(14); 3896-905. (c)2017 AACR.CI - (c)2017 American Association for cancer Research.

BACKGROUND: The incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in recent years. A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis, epithelial-mesenchymal transition and angiogenesis. epithelial-mesenchymal transition and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression. HIF-1alpha functions as a master transcriptional regulator in the adaptive response to hypoxia. Lysyl oxidases like 2 (LOXL2) is a member of the lysyl oxidase family, which main function is to catalyze the covalent cross-linkages of collagen and elastin in the extracellular matrix. Recent work has demonstrated that HIF-1alpha promotes the expression of LOXL2, which is believed to amplify tumor aggressiveness. LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma. The purpose of our study is to explore the role of HIF-1alpha in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism. METHODS: HIF-1alpha, LOXL2 expression and CD31/periodic acid-Schiff double staining in HCC patient samples were examined by immunohistochemical staining. shRNA plasmids against HIF-1alpha was used to determine whether LOXL2 been increased by HIF-1alpha. We monitored a series of rescue assays to demonstrate our hypothesis that LOXL2 is required and sufficient for HIF-1alpha induced EMT and VM formation, which mediates cellular transformation and takes effect in cellular invasion. Then we performed GeneChip(R) Human Transcriptome Array (HTA) 2.0 in HepG2 cells, HepG2 cells overexpressed LOXL2 and HepG2 cells treated with CoCl2. RESULTS: In clinical HCC tissues, it confirmed a positive relationship between HIF-1alpha and LOXL2 protein. Importantly, HIF-1alpha and LOXL2 high expression and the presence of vasculogenic mimicry were correlated to poor prognosis. HIF-1alpha was found to induce EMT, HCC cell migration, invasion and VM formation by regulating LOXL2. The results of microarray assays were analyzed. CONCLUSION: HIF-1alpha plays an important role in the development of HCC by promoting HCC metastasis, EMT and VM through up-regulating LOXL2. This study highlights the potential therapeutic value of targeting LOXL2 for suppression of HCC metastasis and progression.

In cancer cells, intracellular Ca(2+) homeostasis is altered, and this is involved in tumor initiation, progression, and metastasis. However, little is known about the underlying mechanisms. Here, we report that transient receptor potential channel 5 (TrpC5), a receptor-activated non-selective Ca(2+) channel, is correlated with tumor metastasis in colon cancer patients. Moreover, in colon cancer cells, overexpression of TrpC5 caused a robust rise in the concentration of ([Ca(2+)]i), decreased E-cadherin, and increased mesenchymal biomarker expression, then promoted cell migration, invasion, and proliferation. Interestingly, we found that TrpC5 mediated hypoxia-inducible factor 1alpha (HIF-1alpha) expression, activating Twist to promote the epithelial-mesenchymal transition (EMT). Notably, patients with high expression of TrpC5 displayed poorer overall and metastasis-free survival. Taken together, our findings demonstrate that TrpC5 induces the EMT through the HIF-1alpha-Twist signaling pathway to promote tumor metastasis in colon cancer.CI - (c) 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

We have reported that Achaete scute-like 2 (Ascl2) transcriptionally repressed miR-200 family members and affected the epithelial-mesenchymal transition (EMT)-mesenchymal-epithelial transition (MET) plasticity in colorectal cancer (CRC) cells. However, little is known about the regulation of the Ascl2/miR-200 axis. Here, we found that hypoxia inducible factor-1alpha (HIF-1alpha) mRNA levels were positively correlated with Ascl2 mRNA levels and inversely correlated with miR-200b in CRC samples. Mechanistically, we showed that Ascl2 was a downstream target of HIF-1alpha and had a critical role in the EMT phenotype induced by hypoxia or HIF-1alpha over-expression. Hypoxia or HIF-1alpha over-expression activated Ascl2 expression in CRC cells in a direct transcriptional mechanism via binding with the hypoxia-response element (HRE) at the proximal Ascl2 promoter. HIF-1alpha-induced Ascl2 expression repressed miR-200b expression to induce EMT occurrence. Furthermore, we found HIF-1alpha was a direct target of miR-200b. MiR-200b bound with the 3'-UTR of HIF-1alpha in CRC cells. HIF-1alpha/Ascl2/miR-200b regulatory feedback circuit modulated the EMT-MET plasticity of CRC cells. Our results confirmed a novel HIF-1alpha/Ascl2/miR-200b regulatory feedback circuit in modulating EMT-MET plasticity of CRC cells, which could serve as a possible therapeutic target.CI - Copyright (c) 2017. Published by Elsevier Inc.

OBJECTIVE: Hypoxia is a common phenomenon in solid tumor microenvironment. Thereby, the aim of this study was to investigate the molecular mechanisms of tumor metastasis and epithelial-mesenchymal transition (EMT) regulated by lncRNA CPS1 intronic transcript 1 (CPS1-IT1) under hypoxia in CRC. METHODS: expression of lncRNA CPS1-IT1, hypoxia-inducible factor-1 alpha (HIF-1alpha) and autophagy related protein (LC3) were initially assessed in human CRC tissues and in a series of CRC cell lines. The relationship of CPS1-IT1, HIF-1alpha and autophagy were analyzed in CRC were performed through in vitro and in vivo functional assays. RESULTS: expression of CPS1-IT1 were significantly reduced, while HIF-1alpha and LC3-II were increased in CRC tissues and cell lines. Then, in vitro assays revealed that CPS1-IT1 suppresses EMT and autophagy by inhibiting the activation of HIF-1alpha in CRC. An in vivo animal model also demonstrated the tumor suppressor mechanism of CPS1- IT1. CONCLUSION: In this study, we found that hypoxia induce autophagy, and inhibition of autophagy could suppress tumor metastasis and EMT in CRC. Additionally, lncRNA CPS1-IT might suppresses metastasis and EMT by inhibiting hypoxia-induced autophagy through inactivation of HIF-1alpha in CRC.CI - Copyright (c) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

The development and progression of hepatocellular carcinoma (HCC) are dependent on its local microenvironment. Hypoxia and inflammation are two critical factors that shape the HCC microenvironment; however, the interplay between the two factors and the involvement of cancer cells under such conditions remain poorly understood. We found that tumor-associated macrophages, the primary proinflammatory cells within tumors, secreted more interleukin 1beta (IL-1beta) under moderate hypoxic conditions due to increased stability of hypoxia inducible factor 1alpha (HIF-1alpha). Under persistent and severe hypoxia, we found that the necrotic debris of HCC cells induced potent IL-1beta release by tumor-associated macrophages with an M2 phenotype. We further confirmed that the necrotic debris-induced IL-1beta secretion was mediated through Toll-like receptor 4/TIR domain-containing adapter-inducing interferon-beta/nuclear factor kappa-light-chain-enhancer of activated B cells signaling in a similar, but not identical, fashion to lipopolysaccharide-induced inflammation. Using mass spectrometry, we identified a group of proteins with O-linked glycosylation to be responsible for the necrotic debris-induced IL-1beta secretion. Following the increase of IL-1beta in the local microenvironment, the synthesis of HIF-1alpha was up-regulated by IL-1beta in HCC cells through cyclooxygenase-2. The epithelial-mesenchymal transition of HCC cells was enhanced by overexpression of HIF-1alpha. We further showed that IL-1beta promoted HCC metastasis in mouse models and was predictive of poor prognosis in HCC patients. CONCLUSION: Our findings revealed an HIF-1alpha/IL-1beta signaling loop between cancer cells and tumor-associated macrophages in a hypoxic microenvironment, resulting in cancer cell epithelial-mesenchymal transition and metastasis; more importantly, our results suggest a potential role of an anti-inflammatory strategy in HCC treatment. (Hepatology 2018;67:1872-1889).CI - (c) 2017 by the American Association for the Study of Liver diseases.

Inorganic arsenic is widely distributed in the environment, and epidemiologic data show a strong association between arsenic exposure and risk of liver cancer. An understanding of the mechanisms underlying development of liver cancer and metastasis would be useful in reducing the incidence and mortality of liver cancer. MicroRNAs (miRs) act as regulators in liver cancer. Here, we show that acute or chronic exposure of human liver epithelial L-02 cells to arsenite increased expression of miR-191. There were decreased levels of BASP-1 and E-cadherin and increased levels of WT-1 and N-cadherin, indicating that arsenite induced epithelial-mesenchymal transition (EMT). Moreover, arsenite increased EpCAM and CD90 mRNA levels, showing the acquisition of stem cell-like properties by these cells. Suppression of miR-191 resulted in repression of EMT and reduced expression of stem-cell markers. Further, a miR-191 inhibitor blocked spheroid formation and production of side population cells. Luciferase reporter assays indicated that miR-191 was a target of HIF-2alpha, and inhibition of miR-191 decreased the neoplastic and metastatic properties of arsenite-transformed L-02 cells. Thus, in arsenite-transformed liver epithelial cells, transcriptional activation of the miR-191 promoter by HIF-2alpha is involved in EMT and in the acquisition of a stem cell-like phenotype.CI - Copyright (c) 2017. Published by Elsevier Ltd.

Human lens epithelial cells (HLE) undergo mesenchymal transition and become fibrotic during posterior capsule opacification (PCO), which is a frequent complication after cataract surgery. TGF-beta2 has been implicated in this fibrosis. Previous studies have focused on the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in fibrotic diseases, but the role of HIF-1alpha in the TGF-beta2-mediated fibrosis in HLE is not known. TGF-beta2 treatment (10 ng/mL, 48 h) increased the HIF-1alpha levels along with the EMT markers in cultured human lens epithelial cells (FHL124 cells). The increase in HIF-1alpha corresponded to an increase in VEGF-A in the culture medium. However, exogenous addition of VEGF-A (up to 10 ng/mL) did not alter the EMT marker levels in HLE. Addition of a prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, up to 10 microM), enhanced the levels of HIF-1alpha, and secreted VEGF-A but did not alter the EMT marker levels. However, treatment of cells with a HIF-1alpha translational inhibitor, KC7F2, significantly reduced the TGF-beta2-mediated EMT response. This was accompanied by a reduction in the ERK phosphorylation and nuclear translocation of Snail and Slug. Together, these data suggest that HIF-1alpha is important for the TGF-beta2-mediated EMT of human lens epithelial cells.CI - (c) 2018 Wiley Periodicals, Inc.

BACKGROUND/AIMS: During development of liver fibrosis, an important source of myofibroblasts is hepatocytes, which differentiate into myofibroblasts by epithelial to mesenchymal transition (EMT). In epithelial tumours and kidney fibrosis, hypoxia, through activation of hypoxia-inducible factors (HIFs), is an important stimulus of EMT. Our recent studies demonstrated that HIF-1alpha is important for the development of liver fibrosis. Accordingly, the hypothesis was tested that hypoxia stimulates hepatocyte EMT by a HIF-dependent mechanism. METHODS: Primary mouse hepatocytes were exposed to room air or 1% oxygen and EMT evaluated. In addition, bile duct ligations (BDLs) were performed in control and HIF-1alpha-deficient mice and EMT quantified. RESULTS: Exposure of hepatocytes to 1% oxygen increased expression of alpha-smooth muscle actin, vimentin, Snail and fibroblast-specific protein-1 (FSP-1). Levels of E-cadherin and zona occludens-1 were decreased. Upregulation of FSP-1 and Snail by hypoxia was completely prevented in HIF-1beta-deficient hepatocytes and by pretreatment with SB431542, a transforming growth factor-beta (TGF-beta) receptor inhibitor. HIFs promoted TGF-beta-dependent EMT by stimulating activation of latent TGF-beta1. To determine whether HIF-1alpha contributes to EMT in the liver during the development of fibrosis, control and HIF-1alpha-deficient mice were subjected to BDL. FSP-1 was increased to a greater extent in the livers of control mice when compared with HIF-1alpha-deficient mice. CONCLUSIONS: Results from these studies demonstrate that hypoxia stimulates hepatocyte EMT by a HIF and TGF-beta-dependent mechanism. Furthermore, these studies suggest that HIF-1alpha is important for EMT in the liver during the development of fibrosis.

One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1alpha (HIF-1alpha) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury on tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling.CI - Copyright (c) 2015 Elsevier Inc. All rights reserved.

Paraquat (PQ) poisoning-induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia-inducible factor-1alpha (HIF-1alpha) and epithelial-mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and beta-catenin are two other factors involved in promoting EMT. However, the relationship among HIF-1alpha, Snail and beta-catenin in PQ poisoning-induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF-1alpha in EMT occurs via the Snail and beta-catenin pathways in PQ poisoning-induced pulmonary fibrosis. Sixty-six Sprague-Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE-6TN cell lines were transfected with HIF-1alpha siRNA for 48 hrs before being exposed to PQ. Western blotting, real-time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF-1alpha and alpha-SMA were increased at 2 hrs and the level of ZO-1 (Zonula Occluden-1) was reduced at 12 hrs. In vitro, the transient transfection of HIF-1alpha siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and beta-catenin were significantly reduced when HIF-alpha was silenced. These data demonstrate that EMT may be involved in PQ poisoning-induced pulmonary fibrosis and regulated by HIF-1alpha via the Snail and beta-catenin pathways. Hypoxia-inducible factor-1alpha may be a therapeutic target for the treatment of PQ poisoning-induced pulmonary fibrosis.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.