PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells

Background

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.

Methodology/Principal Findings

In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.

Conclusions/Significance

Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.     

HIF-1α is necessary for activation and tumour-promotion effect of cancer-associated fibroblasts in lung cancer

Cancer-associated fibroblasts (CAFs) activation is crucial for the establishment of a tumour promoting microenvironment, but our understanding of CAFs activation is still limited. In this study, we found that hypoxia-inducible factor-1α (HIF-1α) was highly expressed in CAFs of human lung cancer tissues and mouse spontaneous lung tumour. Accordingly, enhancing the expression of HIF-1α in fibroblasts via hypoxia induced the conversion of normal fibroblasts into CAFs. HIF-1α-specific inhibitor or HIF-1α knockout (KO) significantly attenuated CAFs activation, which was manifested by the decreased expression of COL1A2 and α-SMA. In vivo, during tumour formation, the expression of Ki-67 and proliferating cell nuclear antigen (PCNA) in the tumour tissue with HIF-1α KO fibroblasts was significantly lower than that of normal fibroblasts. Moreover, HIF-1α in fibroblasts could activate the NF-κB signalling pathway and enhance a subsequent secretion of CCL5, thus promoting the tumour growth. In conclusion, our results suggest that HIF-1α is essential for the activation and tumour-promotion function of CAFs in lung cancer (LC). And targeting HIF-1α expression on CAFs may be a promising strategy for LC therapy.     

HIF-1α and iNOS levels in crucian carp gills during hypoxia-induced transformation

Hypoxia inducible factor 1 alpha (HIF-1α) initiates expression of a wide variety of genes, some of which are involved in apoptosis and cell cycle arrest. We have previously shown that crucian carp increases its respiratory surface area 7.5-fold in response to hypoxia. This change is due to apoptosis and cell cycle arrest in specific parts of its gills. Here we have characterized crucian carp HIF-1α, and measured mRNA, protein and DNA binding levels during hypoxia exposure in crucian carp gills. We have also measured an HIF-1α-induced gene, the inducible nitric oxide synthase (iNOS), which has the ability to initiate apoptosis and cell cycle arrest. Crucian carp HIF-1α was found to have all critical domains known to be important for function. Comparison of the peptide sequence with other species indicated high similarity with other cyprinid fish, but a pronounced variation compared to the salmonid, rainbow trout. Further, we found HIF-1α protein to be stabilized during hypoxia. Further, HIF-1α was often present in normoxia, and showed marked individual weight-dependent variation. We found no alteration of iNOS mRNA levels during hypoxia exposure. These findings suggest HIF-1α involvement in hypoxia-induced change of respiratory surface area in crucian carp gills. However, its activity does not seem to be mediated through iNOS.     

HIF-1α acts downstream of TNF-α to inhibit vasodilator-stimulated phosphoprotein expression and modulates the adhesion and proliferation of breast cancer cells

Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α.     

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.     

HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells

Background

Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins.

Findings

Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.

Conclusions

Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.     

7-Ketocholesterol and cholestane-triol increase expression of SMO and LXRα signaling pathways in a human breast cancer cell line

Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of 7-ketocholesterol (7-KC), cholestane-3β-5α-6β-triol (triol), and a mixture of 5α-cholestane-3β,6β-diol and 5α-cholestane-3β,6α-diol (diol) to promote cell death in a human breast cancer cell line (MDA-MB-231). We determined cell viability, after 24-h incubation with oxysterols. These oxysterols promoted apoptosis. At least part of the observed effects promoted by 7-KC and triol arose from an increase in the expression of the sonic hedgehog pathway mediator, smoothened. However, this increased expression was apparently independent of sonic hedgehog expression, which did not change. Moreover, these oxysterols led to increased expression of LXRα, which is involved in cellular cholesterol efflux, and the ATP-binding cassette transporters, ABCA1 and ABCG1. Diols did not affect these pathways. These results suggested that the sonic hedgehog and LXRα pathways might be involved in the apoptotic process promoted by 7-KC and triol.     

Blocking OLFM4/HIF-1α axis alleviates hypoxia-induced invasion,epithelial–mesenchymal transition,and chemotherapy resistance in non-small-cell lung cancer

Hypoxia is a common biological hallmark of solid cancers, which has been proposed to be associated with oncogenesis and chemotherapy resistance. The purpose of the present study was to investigate the role and underlying mechanisms of olfactomedin 4 (OLFM4) in the hypoxia-induced invasion, epithelial–mesenchymal transition (EMT), and chemotherapy resistance of non-small-cell lung cancer (NSCLC). We observed dramatically upregulated expression of OLFM4 in several NSCLC cell lines, and this effect was more pronounced in A549 and H1299 cells. In addition, our data revealed that OLFM4 expression was remarkably increased in both A549 and H1299 cells under hypoxic microenvironment, accompanied by enhanced levels of hypoxia-inducible factor (HIF)-1α protein. The HIF-1α level was elevated in response to hypoxia, resulting in the regulation of OLFM4. Interestingly, OLFM4 was a positive regulator of hypoxia-driven HIF-1α production. Moreover, depletion of OLFM4 modulated multiple EMT-associated proteins, as evidenced by the enhanced E-cadherin levels along with the diminished expression of N-cadherin and vimentin in response to hypoxia, and thus blocked invasion ability of A549 and H1299 cells following exposure to hypoxia. Furthermore, ablation of OLFM4 accelerated the sensitivity of A549 cells to cisplatin under hypoxic conditions, implying that OLFM4 serves as a key regulator in chemotherapeutic resistance under hypoxia. In conclusion, OLFM4/HIF-1α axis might be a potential therapeutic strategy for NSCLC.     

Prognostic value of HIF-1α expression in patients with gastric cancer

The role of hypoxia-inducible factors-1 alpha (HIF-1α) expression in gastric cancer remains controversial. We performed a systematic review of the literature with meta-analysis. Electronic databases were used to identify published studies before December 1, 2012. Pooled hazard ratio (HR) or odds ratio (OR) with 95 % confidence interval (95 % CI) was used to estimate the strength of the association between HIF-1α expression and survival of gastric cancer patients. Heterogeneity and publication bias were also assessed. Final analysis of 1,268 patients from 9 eligible studies was performed. High HIF-1α expression was significantly correlated with poor overall survival (OS) of gastric cancer patients (HR = 2.14, 95 % CI = 1.32–3.48). Subgroup analysis indicated that HIF-1α over-expression had an unfavorable impact on OS in Asian patients (HR = 2.35, 95 % CI = 1.41–3.92). Moreover, up-regulation of HIF-1α was significantly associated with the depth of invasion (OR = 2.49, 95 % CI = 1.28–4.83), lymph node metastasis (OR = 2.15, 95 % CI = 1.27–3.66), and vascular invasion (OR = 2.23, 95 % CI = 1.20–4.14). HIF-1α expression might be a predicative factor of poor prognosis for gastric cancer particularly in Asia.     

Expression profiling after induction of demethylation in MCF-7 breast cancer cells identifies involvement of TNF-α mediated cancer pathways

Epigenetic methylation change is a major process that occurs during cancer development. Even though many tumor-related genes have been identified based on their relationship between methylation and expression, few studies have been conducted to investigate the relevant biological pathways involved in these changes. To identify essential pathways likely to be affected by methylation in breast cancer, we examined a pool of genes in which expression was upregulated after induction of demethylation by 5-Aza-2′-deoxycytidine (Aza) in the MCF-7 breast cancer cell line. Genome-wide demethylation was confirmed by monitoring the demethylation of a previously known gene, SULT1A1. Overall, 210 and 213 genes were found to be upregulated and downregulated (fold change ≥ 2), respectively, in common in cells treated with 5 and 10 μM of Aza. Network analysis of these 423 genes with altered expression patterns identified the involvement of a cancer related network of genes that were heavily regulated by TNF-α in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis may be intimately involved in tumorigenesis in MCF-7 cells.     

Differential effects of calcium on PI3K-Akt and HIF-1α survival pathways

Calcium signaling participates in the regulation of numberless cellular functions including cell cycle progression and cellular migration, important processes for cancer expansion. Cancer cell growth, migration, and invasion are typically supported by PI3K/Akt activation, while a hypoxic environment is critical in cancer development. Accordingly, in the present study, we aimed at investigating whether perturbations in calcium homeostasis induce alterations of HIF-1α and activate Akt levels in epithelial A549 and A431 cells. Survival was drastically reduced in the presence of calcium chelator BAPTA-AM and thapsigargin, a SERCA inhibitor inducing store-operated calcium entry, to a lesser extent. Calcium chelation provoked a transient but strong upregulation of HIF-1α protein levels and accumulation in the nucleus, whereas in the presence of thapsigargin, HIF-1α levels were rapidly abolished before reaching and exceeding control levels. Despite cell death, calcium chelation merely inhibited Akt, which was significantly activated in the presence of thapsigargin. Moreover, when store-operated calcium entry was simulated by reintroducing calcium ions in cell suspensions, Akt was rapidly activated in the absence of any growth factor. These data further underscore the growing importance of calcium entry and directly link this elementary event of calcium homeostasis to the Akt pathway, which is commonly deregulated in cancer.