Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC‐associated factor X

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine‐producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC‐associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX‐mediated mechanism and opposing influences of hypoxia‐inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene‐manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max‐mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re‐expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX‐related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.     

Bisphosphonates suppress insulin‐like growth factor 1‐induced angiogenesis via the HIF‐1α/VEGF signaling pathways in human breast cancer cells

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the potential molecular mechanisms underlying the antiangiogenic effect of non–nitrogen‐containing and nitrogen‐containing bisphosphonates, clodronate and pamidronate, respectively, in insulin‐like growth factor (IGF)‐1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on hypoxia‐inducible factor (HIF)‐1α/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF‐1‐induced HIF‐1α protein accumulation and VEGF expression in MCF‐7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF‐1α, but they apparently promoted the degradation of IGF‐1‐induced HIF‐1α protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose‐dependent decease in the newly‐synthesized HIF‐1α protein induced by IGF‐1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF‐1α/VEGF axis are associated with the inhibition of the phosphoinositide 3‐kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF‐1‐stimulated MCF‐7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.     

Mutational activation of the epidermal growth factor receptor down‐regulates major histocompatibility complex class I expression via the extracellular signal‐regulated kinase in non–small cell lung cancer

The efficacy of programmed cell death–1 (PD‐1) blockade in patients with non–small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC‐I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC‐I expression in NSCLC cell lines. Appropriate EGFR‐TKIs increased MHC‐I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal–regulated kinase (ERK) kinase MEK, also increased MHC‐I expression, whereas the phosphatidylinositol 3‐kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK‐ERK pathway mediates the down‐regulation of MHC‐I expression in response to EGFR activation. Immunohistochemical analysis of EGFR‐mutated NSCLC specimens obtained before and after EGFR‐TKI treatment also revealed down‐regulation of phosphorylated forms of EGFR and ERK in association with up‐regulation of MHC‐I, an increased number of infiltrating CD8⁺ T cells, and increased PD‐1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC‐I expression through the MEK‐ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR‐MEK‐ERK signaling in and the immune response to EGFR‐mutated NSCLC.     

HIF‐1α is an unfavorable determinant of relapse in gastric cancer patients who underwent curative surgery followed by adjuvant 5‐FU chemotherapy

Among several chemotherapeutic agents, 5‐fluorouracil (5‐FU) has been widely used as a key drug in adjuvant chemotherapy for gastric cancer. However, no reliable marker, which predicts the response to 5‐FU in an adjuvant setting, has been identified. Hypoxia‐induced drug resistance, via upregulation of HIF‐1α, is a major obstacle in the development of effective cancer therapy. However, few clinical studies have so far assessed the relationship between the HIF‐1α expression and the chemo‐resistance of gastric cancer patients in an adjuvant setting. We established 2 HIF‐1α knockdown gastric cancer cell lines in order to clarify the role of HIF‐1α in chemo‐resistance against 5‐FU. Furthermore, expression of HIF‐1α was immunohistochemically assessed in 91 resected specimens. Sixty‐four of 91 patients received 5‐FU adjuvant chemotherapy after surgery. HIF‐1α expression was associated with the significantly shorter relapse‐free survival and disease‐specific survival in the 64 patients of adjuvant group (p = 0.026, 0.014, respectively), but not in the 27 of surgery group. Multivariate analysis showed that HIF‐1α was an independent risk factor for relapse in 64 patients in the adjuvant group (p = 0.029). In conclusion, the current study confirmed, for the first time that HIF‐1α expression is an independent risk factor for relapse in high‐risk gastric cancer patients who underwent curative surgery followed by adjuvant 5‐FU chemotherapy. A favorable effect of 5‐FU might therefore be expected in patients that do not express HIF‐1α, whereas, other types of chemotherapy or additional treatments, such as HIF‐1α inhibitors, should be considered in patients that do express HIF‐1α.     

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1. © 2009 Wiley‐Liss, Inc.     

The critical role of mast cell‐derived hypoxia‐inducible factor‐1α in human and mice melanoma growth

Mast cells play an important role in tumorigenesis. Histamine released from mast cells stimulates new vessel formation by acting through the histamine1 (H1) receptor. Despite the evidence of the role of mast cells in tumor growth and angiogenesis, the potential mechanism remains to be elucidated. Therefore, we investigated the role of mast cell‐derived HIF‐1α in melanoma growth. Here, we identify that the most positive cells for HIF‐1α staining are seen in mast cells of human and animal melanoma tissue. The number of the stromal cell types (fibroblasts, macrophages and endothelial cells) was also increased in melanoma tissues. In activated bone marrow‐derived mast cells (BMMCs), expressions of HIF‐1α and VEGF were increased. Histamine also induced the expressions of HIF‐1α and VEGF in BMMCs. H1 receptor antagonists significantly improved overall survival rates and substantially suppressed tumor growth as well as the infiltration of mast cells and levels of VEGF through the inhibition of HIF‐1α expression in B16F10 melanoma‐bearing mice. Furthermore, the injection of HIF‐1α depleted BMMCs markedly inhibited the growth of tumors and migration of mast cells and increased the survival rate of the mice. These findings emphasize that the growth of melanoma can actually be exacerbated by mast cell‐derived HIF‐1α. In aggregate, our results reveal a novel role for mast cell‐derived HIF‐1α in the melanoma microenvironment and have important implications for the design of therapeutic strategies.     

Hypoxia induces gefitinib resistance in non‐small‐cell lung cancer with both mutant and wild‐type epidermal growth factor receptors

Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non‐small‐cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild‐type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor‐α (TGFα) was dramatically upregulated under hypoxia, and the knockdown of TGFα or hypoxia‐inducible factor‐1α (HIF1α) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild‐type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGFα or HIF1α. Our results indicate that hypoxia causes gefitinib resistance in EGFR‐mutant NSCLC through the activation of wild‐type EGFR mediated by the upregulation of TGFα. The presence of wild‐type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.     

Overexpression of hypoxia‐inducible factor 1 alpha impacts FoxP3 levels in mycosis fungoides—Cutaneous T‐cell lymphoma: Clinical implications

Mycosis fungoides (MF) is the most common variant of primary cutaneous T‐cell lymphoma, and decreased forkhead box P3 (FoxP3) expression has been reported in MF late stages. Hypoxia‐inducible factor 1 alpha (HIF‐1α) may regulate FoxP3 expression; however, it is unknown whether HIF‐1α is expressed in the CD4⁺ T cells of MF patients and how it could affect the expression of FoxP3. Therefore, we evaluated the expression of HIF‐1α and FoxP3 in CD4⁺ T cells obtained from the skin lesions of MF patients. We found increased cell proliferation and an increase in CD4⁺ T cells with an aberrant phenotype among early stage MF patients. HIF‐1α was overexpressed in these CD4⁺ T cells. In addition, we found a decrease in the percentage of FoxP3⁺ cells both in the skin of MF patients, when compared with control skin samples, and with disease progression. In addition, a negative correlation was established between HIF‐1α and FoxP3 expression. Skin HIF‐1α expression in MF patients correlated with the extent of the affected area and increased with the disease progression. Finally, we showed that ex vivo inhibition of HIF‐1α degradation increases the percentage of FoxP3⁺ T cells in skin lesions. Our results suggest that overexpression of HIF‐1α affects the levels of FoxP3 in MF patients, which could have relevant implications in terms of disease outcome.     

Extracellular ATP promotes breast cancer invasion and epithelial‐mesenchymal transition via hypoxia‐inducible factor 2α signaling

Extracellular ATP has been shown to play an important role in invasion and the epithelial‐mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia‐inducible factor (HIF) signaling and upregulate hypoxia‐inducible factor 1/2α (HIF‐1/2α) expression. After knocking down HIF‐1/2α using siRNA, we found that ATP‐driven invasion and EMT were significantly attenuated via HIF2A‐siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF‐2α direct targets, among which lysyl oxidase‐like 2 (LOXL2) and matrix metalloproteinase‐9 (MMP‐9) mediated ATP‐driven invasion, and E‐cadherin and Snail mediated ATP‐driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF‐2α and mediate ATP‐driven HIF‐2α upregulation. Furthermore, we demonstrated that expressions of HIF‐2α and its target proteins could be regulated via ATP by AKT‐PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF‐2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP‐HIF‐2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF‐2α signaling, which may be a potential target for future anti–metastasis therapy.     

Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia‐inducible factor 1α

Increased levels of hypoxia and hypoxia‐inducible factor 1α (HIF‐1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF‐1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF‐A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category “Response to hypoxia” was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia‐responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF‐1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF‐1α shRNA or Dox at low concentrations blocked HIF‐1α induction of VEGF‐A by 84–97% and carbonic anhydrase 9 by 83–93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF‐1α activity with increasing tumor size and with anti‐VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF‐1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF‐1α target genes that may promote resistance to antiangiogenic and other therapies. HIF‐1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.     

Lymphotoxin‐β receptor activation by lymphotoxin‐α1β2 and LIGHT promotes tumor growth in an NFκB‐dependent manner

Lymphotoxin beta receptor (LTβR) activation on mouse fibrosarcoma cells (BFS‐1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro‐angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTβR, namely LTα₁β₂ and LIGHT, are involved in the activation of LTβR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTβR in these fibrosarcoma tumors, we used conditional LTβ‐deficient mice that specifically lack LTβ expression either on T cells (T‐LTβ^?/?) or on B cells (B‐LTβ^?/?). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild‐type mice, indicating the participation of both T and B host lymphocytes in the activation of LTβR in these tumors. Tumor growth was also reduced in LIGHT‐deficient mice, suggesting a contribution of this ligand to the activation of LTβR in BFS‐1 fibrosarcomas. LTβR signaling can involve IκBα and/or NFκB‐inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro‐angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro‐angiogenic signaling after LTβR activation. Our data support the idea that the ablation of LTβR signaling should be considered for cancer treatment.     

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells

Activation of transforming growth factor β (TGF‐β) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial‐mesenchymal transition (EMT). Hypoxia‐inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF‐1α expression by protein phosphatase activity on dissociation of the E‐cadherin/β‐catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF‐β. By using lung cancer cells treated with HIF‐1α stabilizers or carrying doxycycline‐dependent HIF‐1α deletion or point mutants, we investigated the role of stabilized HIF‐1α expression on TGF‐β‐induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF‐β and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF‐1α expression was inhibited. Stabilized HIF‐1α protein expression inhibited the TGF‐β‐stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF‐1α‐induced repression of the TGF‐β‐stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF‐1α expression on inhibition of TGF‐β‐induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.     

Deletions of chromosomes 3p and 14q molecularly subclassify clear cell renal cell carcinoma

BACKGROUND:

The short arm of chromosome 3 (3p) harbors the von Hippel‐Lindau (VHL) tumor suppressor gene, and the long arm of chromosome 14 (14q) harbors the hypoxia‐inducible factor 1α (HIF‐1α) gene. The objective of this study was to evaluate the significance of 3p loss (loss VHL gene) and 14q loss (loss HIF‐1α gene) in clear cell renal cell carcinoma (ccRCC).

METHODS:

In total, 288 ccRCC tumors underwent a prospective cytogenetic analysis for alterations in chromosomes 3p and 14q. Tumors were assigned to 1 of 4 possible chromosomal alterations: VHL +3p/+14q (VHL wild type [VHL‐WT]), VHL +3p/?14q (VHL‐WT plus HIF2α [WT/H2]), ?3p/+14q (HIF1α and HIF2α [H1H2]), and ?3p/?14q (HIF2α [H2]).

RESULTS:

Among patients who had loss of 3p, tumors with ?3p/?14q (H2) alterations were larger (P = .002), had higher grade (P = .002) and stage (P = .001), and more often were metastatic (P = .029) than tumors that retained 14q (H1H2). All patients who had tumors with ?3p/?14q (H2) had worse cancer‐specific survival (P = .014), and patients who had localized disease (P = .012) and primary T1 (pT1) tumors (P = .008) had worse recurrence‐free survival. In patients who had pT1 tumors, combined 3p/14q loss was an independent predictor of recurrence‐free survival (hazard ratio, 11.19; 95% confidence interval, 1.91‐65.63) and cancer‐specific survival (hazard ratio, 15.93; 95% confidence interval, 3.09‐82.16). The current investigation was limited by its retrospective design, single‐center experience, and a lack of confirmatory protein analyses.

CONCLUSIONS:

Loss of chromosome 3p (the VHL gene) was associated with improved survival in patients with ccRCC, whereas loss of chromosome 14q (the HIF‐1α gene) was associated with worse outcomes. The results of the current study support the hypothesis that HIF‐1α functions as an important tumor suppressor gene in ccRCC. Cancer 2013. © 2013 American Cancer Society.     

Retinoid X receptor α enhances human cholangiocarcinoma growth through simultaneous activation of Wnt/β‐catenin and nuclear factor‐κB pathways

Retinoid X receptor α (RXRα) plays important roles in the malignancy of several cancers such as human prostate tumor, breast cancer, and thyroid tumor. However, its exact functions and molecular mechanisms in cholangiocarcinoma (CCA), a chemoresistant carcinoma with poor prognosis, remain unclear. In this study we found that RXRα was frequently overexpressed in human CCA tissues and CCA cell lines. Downregulation of RXRα led to decreased expression of mitosis‐promoting factors including cyclin D1and cyclin E, and the proliferating cell nuclear antigen, as well as increased expression of cell cycle inhibitor p21, resulting in inhibition of CCA cell proliferation. Furthermore, RXRα knockdown attenuated the expression of cyclin D1 through suppression of Wnt/β‐catenin signaling. Retinoid X receptor α upregulated proliferating cell nuclear antigen expression through nuclear factor‐κB (NF‐κB) pathways, paralleled with downregulation of p21. Thus, the Wnt/β‐catenin and NF‐κB pathways account for the inhibition of CCA cell growth induced by RXRα downregulation. Retinoid X receptor α plays an important role in proliferation of CCA through simultaneous activation of Wnt/β‐catenin and NF‐κB pathways, indicating that RXRα might serve as a potential molecular target for CCA treatment.     

Epithelial–mesenchymal transition in human gastric cancer cell lines induced by TNF‐α‐inducing protein of Helicobacter pylori

Helicobacter pylori strains produce tumor necrosis factor‐α (TNF‐α)‐inducing protein, Tipα as a carcinogenic factor in the gastric epithelium. Tipα acts as a homodimer with 38‐kDa protein, whereas del‐Tipα is an inactive monomer. H. pylori isolated from gastric cancer patients secreted large amounts of Tipα, which are incorporated into gastric cancer cells by directly binding to nucleolin on the cell surface, which is a receptor of Tipα. The binding complex induces expression of TNF‐α and chemokine genes, and activates NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells). To understand the mechanisms of Tipα in tumor progression, we looked at numerous effects of Tipα on human gastric cancer cell lines. Induction of cell migration and elongation was found to be mediated through the binding to surface nucleolin, which was inhibited by the nucleolin‐targeted siRNAs. Tipα induced formation of filopodia in MKN‐1 cells, suggesting invasive morphological changes. Tipα enhanced the phosphorylation of 11 cancer‐related proteins in serine, threonine and tyrosine, indicating activation of MEK‐ERK signal cascade. Although the downregulation of E‐cadherin was not shown in MKN‐1 cells, Tipα induced the expression of vimentin, a significant marker of the epithelial–mesenchymal transition (EMT). It is of great importance to note that Tipα reduced the Young's modulus of MKN‐1 cells determined by atomic force microscopy: This shows lower cell stiffness and increased cell motility. The morphological changes induced in human gastric cancer cells by Tipα are significant phenotypes of EMT. This is the first report that Tipα is a new inducer of EMT, probably associated with tumor progression in human gastric carcinogenesis.     

Clinicopathological significance of nuclear factor‐kappa B,HIF‐1 alpha,and vascular endothelial growth factor expression in stage III colorectal cancer

Nuclear factor‐κB (NF‐κB), hypoxia‐inducible factor 1α (HIF‐1α), and vascular endothelial growth factor (VEGF) are involved in cell proliferation, invasion, angiogenesis, and metastases. The principal objective of this study was to assess the prognostic significance of NF‐κB, HIF‐1α, and VEGF expression in stage III colorectal cancer. Tumor tissues from 148 patients with stage III colorectal carcinoma, all of whom underwent potentially curative resection, were immunohistochemically evaluated using monoclonal antibodies against NF‐κB, HIF‐1α, and VEGF. Positivity rates of NF‐κB, HIF‐1α, and VEGF were 47.3%, 42.6%, and 61.5%, respectively. NF‐κB expression in tumor tissues was correlated significantly with HIF‐1α expression (P < 0.001), VEGF expression (P = 0.044), and the presence of vascular invasion (P = 0.013). Univariate analysis demonstrated that NF‐κB expression was associated with poor 5‐year overall survival (55.8 months vs 76.9 months, P = 0.012). Multivariate analysis verified that NF‐κB was independently associated with adverse outcomes (relative risk: 1.92, P = 0.049). However, HIF‐1α and VEGF did not appear to be related to clinical outcomes. NF‐κB expression in tumor tissue is associated with angiogenesis and poor 5‐year overall survival in stage III colorectal cancer patients. (Cancer Sci 2010; 00: 000–000)