3091

HIF1A

hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor);HIF1A;hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)

ARNT interacting protein|ARNT-interacting protein|HIF-1-alpha|PAS domain-containing protein 8|basic-helix-loop-helix-PAS protein MOP1|class E basic helix-loop-helix protein 78|hypoxia-inducible factor 1 alpha isoform I.3|hypoxia-inducible factor 1, alpha

14q23.2

protein-coding

Although metastatic disease is lethal in the majority of bladder cancer cases, study on the molecular mechanism(s) of this process suffers from the limited source of distant metastatic tumor tissues and very few suitable animal models. To address this need, we generated an orthotopic animal model by instilling human bladder cancer T24-tumorigenic (T24-t) cells into mouse bladder, and sublines were subsequently derived as primary (T24-parental, T24-P) and lung metastatic (T24-L) sites. Data from invasion, migration, and adhesion assays suggested higher metastatic potential of T24-L cells than T24-P cells in vitro. Using two metastatic models to assess the metastatic ability in vivo, T24-L cells exhibited higher incidence of tumor metastasis. Mechanistically, the up-regulation of MMP-1 and HIF-1alpha was observed in T24-L cells. Knocking down HIF-1alpha can significantly down-regulate the expression of MMP-1, accompanied by the decreased invasion ability in vitro. Using immunohistochemical staining, we further observed HIF-1alpha elevation in the metastatic lymphomatic tissues compared with the primary bladder cancer tissues from the same patients. Taken together, our study provides the evident of the function of HIF-1alpha/MMP-1 in regulating metastasis of bladder cancer and HIF-1alpha as a potential target for controlling bladder cancer metastasis.

Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While beta-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of beta-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of beta-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with beta-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following beta-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered beta-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following beta-carotene treatment. Level of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following beta-carotene treatment. In conclusion, the present study provides the first evidence that beta-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1alpha.

BACKGROUND: Hypoxia-inducible factor 1 alpha (hif-1alpha) furnishes tumor cells with the means of adapting to stress parameters like tumor hypoxia and promotes critical steps in tumor progression and aggressiveness. We investigated the role of hif-1alpha expression in patients with node-positive breast cancer. METHODS: tumor samples from 77 patients were available for immunohistochemistry. The impact of hif-1alpha immunoreactivity on survival endpoints was determined by univariate and multivariate analyses, and correlations to clinicopathological characteristics were determined by cross-tabulations. RESULTS: hif-1alpha was expressed in 56% (n = 43/77) of the patients. Its expression correlated with progesterone receptor negativity (P = 0.002). The Kaplan-Meier curves revealed significantly shorter distant metastasis-free survival (DMFS) (P = 0.04, log-rank) and disease-free survival (DFS) (P = 0.04, log-rank) in patients with increased hif-1alpha expression. The difference in overall survival (OS) did not attain statistical significance (5-year OS, 66% without hif-1alpha expression and 55% with hif-1alpha expression; P = 0.21). The multivariate analysis failed to reveal an independent prognostic value for hif-1alpha expression in the whole patient group. The only significant parameter for all endpoints was the T stage (T3/T4 versus T1/T2: DMFS, relative risk = 3.16, P = 0.01; DFS, relative risk = 2.57, P = 0.03; OS, relative risk = 3.03, P = 0.03). Restricting the univariate and multivariate analyses to T1/T2 tumors, hif-1alpha expression was a significant parameter for DFS and DMFS. CONCLUSIONS: hif-1alpha is expressed in the majority of patients with node-positive breast cancer. It can serve as a prognostic marker for an unfavorable outcome in those with T1/T2 tumors and positive axillary lymph nodes.

OBJECTIVE: To investigate the expression of hypoxia- inducible 1 alpha (HIF-1alpha) in human breast cancer and its relationship with vascular endothelial growth factor (VEGF) protein, proliferating cell nuclear antigen (PCNA ) protein, other tumor biomarkers and clinical pathologic factors. METHODS: The immunohistochemical staining (SP) was used to measure the expression of HIF-1alpha, VEGF and PCNA in human breast fibroadenoma, usual hyperplasia and breast carcinoma. RESULTS: HIF-1alpha was not found expressing in breast fibroadenoma and hyperplastic lesions. In contrast, the positive rate of HIF-1alpha was found in the ductal carcinoma in situ 55% (DCIS, 11/20) and the invasive breast carcinoma 85% (51/60). VEGF positivity in breast carcinoma was 81.3% (65/80). The total positive rate of PCNA in breast carcinoma was 75% (60/80), that in DCIS was 65% (13/20) and that in invasive carcinoma was 78.3% (47/66). Increased levels of HIF-1alpha were statistically significantly associated with increased expression of VEGF (r = 0.762, P <0.01) and PCNA (r = 0.693, P < 0. 01). Conclusion The upregulated expression of HIF-1alpha and VEGF in breast carcinoma has a close relationship with tumor angiogenesis, tumor cell proliferation, lymph node metastasis, ER status and stages of histology. This suggests that HIF-1alpha plays an important role in the carcinogenesis and progression of breast carcinoma; is wished to become a new target for radiotherapy, chemotherapy and biotherapy of tumor, which will offer the new ways to diagnosis and treatment of tumor.

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.

INTRODUCTION: The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs. METHODS: The expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1alpha and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1alpha or pFAK antibodies, respectively. RESULTS: pFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1alpha was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1alpha and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1alpha and VEGF2. CONCLUSIONS: The expression of pFAK, HIF-1alpha, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.

The aim of this article is to identify nuclear co-localization of COX-2 and HIF-1alpha in human-bone metastasis of breast cancer, index of transcriptionally activated cells and functional for gene expression. In particular, we verified whether hypoxia exerted a direct role on metastasis-gene expression or through COX-2 signaling, due to the relevance for clinical implications to individuate molecular targets for diagnosis and therapy. The experiments were performed in vitro with two metastatic clones, 1833 and MDA-231BO, and the parental MDA-MB231 cells, in vivo (1833-xenograft model), and in human-bone metastasis specimens. In 1833 cells in vitro, COX-2 signaling pathway was critical for nuclear HIF-1alpha-protein expression/translocation, mechanisms determining HIF-1 activity and gene expression. The data were corroborated by immunohistochemistry in human-bone metastasis specimens. COX-2 and HIF-1alpha showed wide co-localization in the nucleus, indicative of COX-2-nuclear import in transcriptionally activated metastatic cells and consistent with COX-2-HIF-1alpha functional interaction. A network of microenvironmental signals controlled COX-2 induction and HIF-1 activation downstream. In fact, hypoxia through HGF and TGF-beta1 autoregulatory loops triggered a specific array of transcription factors responsible for COX-2 transactivation. The novelty was that HGF and TGF-beta1 biological signals were produced by hypoxic metastatic cells and, therefore, the microenvironment seemed to be modified by metastatic-cell engraftment in the bone. In agreement, HIF-1alpha expression in bone marrow supportive cells occurred in metastasis-bearing animals. Altogether, the data supported the pre-metastatic-niche theory. Our observations might be useful to design therapies against bone metastasis, by affecting the phenotype changes of metastatic cells occurring at the secondary growth site through COX-2-HIF-1 interaction.

Intratumoral hypoxia has been correlated with distant metastatic potential. Two hypoxia inducible factors (HIFs), HIF-1alpha and HIF-2alpha, are induced by hypoxia, and high expression of these proteins has been correlated to angiogenesis and distant metastasis. Thymosin beta4 (Tbeta4) is frequently highly expressed in cancer, and this overexpression correlates with malignant progression. The objective of this study was to investigate the clinical correlation of HIF-alpha with Tbeta4 and the intracellular functional roles of Tbeta4 on HIF-alpha activation. We examined HIF-1alpha, HIF-2alpha and Tbeta4 expressions in clinical human breast carcinoma (n=70) by immunohistochemistry. We show that high expression of HIF-1alpha and HIF-2alpha strongly correlates with Tbeta4 expression (Pexpression of Tbeta4 correlates significantly with patients with lymph node metastasis (P<0.05) of human breast cancer. Additionally, we demonstrate that hypoxia up-regulates intracellular Tbeta4 protein, which then affects HIF-alpha activity, which is the key in regulating VEGF expression. We confirmed that hypoxia-induced intracellular Tbeta4 and HIF-alpha activities were reduced by interference of Tbeta4 expression using Tbeta4 shRNA lentivirus. Vascular epidermal growth factor (VEGF)-A, a well-recognized lymphangiogenic cytokine, was also down-regulated, but VEGF-C and VEGF-D expressions were not affected. These findings suggest that the overexpression of Tbeta4 is strongly associated with HIF-1alpha and HIF-2alpha expression and is also clinicopathologically involved with lymph node metastatic potential of breast cancer through the modulation of HIF-alphaactivation and induction of VEGF-A. Ultimately, these results highlight Tbeta4 as a potentially therapeutic target in malignant cancers.

Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.

BACKGROUND: Hypoxia up-regulated expression of tissue factor (TF) may facilitate tumor cell metastasis, but transcriptional mechanisms remain undefined. MATERIAL AND METHODS: To verify the role of Egr-1 in hypoxia-induced TF expression in breast cancer cells, quantitative PCR and Western blot analysis were performed. The secretion of VEGF under hypoxia was detected by enzyme-linked immunosorbent assay (ELISA). Egr-1 and HIF-1alpha siRNA were transiently transfected into breast cancer cells to evaluate their specific roles. RESULTS: The increased Egr-1 expression occurring in hypoxic breast cancer cells can up-regulate TF expression and stimulating protein 1(Sp1) was not responsible for the hypoxia-induced expression of TF. HIF-1alpha mediated the hypoxia-induced up-regulation of TF expression through vascular endothelial growth factor (VEGF). The regulatory effects of Egr-1 on TF under hypoxia were independent of HIF-1alpha. Either Egr-1 or HIF-1alpha was responsible for hypoxic induction of tumor cells adhesion. HIF-1alpha, but not Egr-1, had a pivotal role in human breast cancer cells invasion. Both Egr-1 and HIF-1alpha were critical to angiogenesis induced by hypoxic conditions in MDA-MB-231 and HUVEC co-cultures. In nude mice, both Egr-1 and HIF-1alpha small interfering RNA (siRNA) could decrease extravasation of MDA-MB-435 cells in the lung after tail vein injection. CONCLUSIONS: Hypoxia-induced expression of TF in human breast cancer cells depends on Egr-1 and HIF-1alpha, and both of these proteins may play an important role in breast cancer metastasis, either directly or indirectly through the TF pathway.

PURPOSE: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) plays an important role in tumoral adaptation to hypoxic conditions by serving as a transcription factor for several crucial proteins, including vascular endothelial growth factor and carbonic anhydrase IX (CAIX). Here, we evaluated the significance of HIF-1 alpha in renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: Immunohistochemical analysis was done on a tissue microarray constructed from paraffin-embedded primary tumor specimens from 357 patients treated by nephrectomy for RCC. Nuclear expression was evaluated by a single pathologist who was blinded to outcome. The expression levels were associated with pathologic variables and survival. RESULTS: HIF-1 alpha expression was greater in RCC than in benign tissue. Clear cell RCC showed the highest expression levels. In clear cell RCC, HIF-1 alpha was significantly correlated with markers of apoptosis (p21, p53), the mammalian target of rapamycin pathway (pAkt, p27), CXCR3, and proteins of the vascular endothelial growth factor family. HIF-1 alpha was correlated with CAIX and CAXII in localized, but not in metastatic RCC. HIF-1 alpha expression predicted outcome in metastatic patients: patients with high HIF-1 alpha expression (>35%) had significantly worse survival than patients with low expression (< or =35%); median survival, 13.5 versus 24.4 months, respectively (P = 0.005). Multivariate analysis retained HIF-1 alpha and CAIX expression as the strongest independent prognostic factors for patients with metastatic clear cell RCC. CONCLUSIONS: HIF-1 alpha is an important independent prognostic factor for patients with metastatic clear cell RCC. Because HIF-1 alpha and CAIX are independently and differentially regulated in metastatic clear cell RCC, both tumor markers can be complementary in predicting prognosis.

BACKGROUND: Hypoxia-inducible factor 1 alpha (HIF-1alpha) is involved in processes promoting carcinogenesis of many tumors. However, its role in the development of colorectal cancer is unknown. To investigate the significance of HIF-1alpha during colorectal carcinogenesis and progression we examined its expression in precursor lesions constituting the conventional and serrated pathways, as well as in non-metastatic and metastatic adenocarcinomas. METHODS: Immunohistochemistry and Western blot is used to analyse HIF-1alpha expression in normal colonic mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well as in non-metastatic and metastatic colorectal adenocarcinomas. Eight colorectal carcinoma cell lines are tested for their HIF-1alpha inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry. RESULTS: In normal mucosa, HPP and TA-LGD HIF-1alpha was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1alpha were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1alpha overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1alpha expression was strongly accumulated in perinecrotic regions. In these cases HIF-1alpha activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1alpha was also seen in periinflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1alpha expression and nuclear translocation. CONCLUSION: We conclude that HIF-1alpha expression occurs in early stages of colorectal carcinogenesis and achieves a maximum in the invasive stage independent of the metastatic status. Perinecrotic activation of HIF-1alpha in invasive tumors underlines a dual role of HIF-1alpha by regulating both pro-survival and pro-death processes. HIF-1alpha up-regulation in response to LPS-mediated stimulation and periinflammatory expression in invasive carcinomas suggest its involvement in inflammatory events. These patterns of HIF-1alpha inducibility could contribute indirectly to the acquisition of a metastatic phenotype.

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is the key regulator of cellular responses to hypoxia and plays a central role in tumour growth. Recently, two single nucleotide polymorphisms (SNPs) in the HIF-1 alpha gene, C1772T and G1790A, were shown to cause significantly higher transcriptional activity than did the wild-type. This study aimed to investigate the effect of these SNPs on the prognosis of colorectal cancer (CRC). PATIENTS AND METHODS: DNA from 336 CRC patients was genotyped. Genotypes of each polymorphism were tested for association with disease-free survival (DFS) using univariate and multivariate Cox-regression analysis. RESULTS: Genotype frequencies were: CC 75.6%, CT 18.8% and TT 1.8% for HIF1A1 C1772T and GG 93.2%, GA 2.7% and AA 0% for G1790A. A statistically significant association between DFS and clinicopathological features was observed. However, no association was found between HIF1A1 C1772T (p=0.44; risk ratio of recurrence, RR=1.19, 95% confidence interval, CI=0.77 to 1.83) and G1790A (p=0.89; RR=0.92, 95% CI=0.29 to 2.90) polymorphisms and DFS in univariate and multivariate Cox-regression analysis. CONCLUSION: These results suggest that HIF1A1 C1772T and G1790A polymorphisms are not involved in the progression or metastasis of CRC.

Deep vein thrombosis associated with advanced cancer is known as Trousseau's syndrome. We hypothesized that thrombin, an activator of protease-activated receptor (PAR)-1 and PAR-4 contributes to tumor metastasis. In this study, we demonstrated that thrombin and the PAR-1 activating peptide (AP) SFLLRN, but not the PAR-4 AP GYPGKF, induced HIF-1alpha activities, protein expression, and cell motility in colorectal cancer cells, and these actions were significantly inhibited by the PAR-1 antagonist SCH79797. Moreover, thrombin-induced HIF-1alpha activity and cell motility were blocked by inhibiting important mediators of signaling transduction, including the ERK, PI3K, and mTOR pathways. These results showed that thrombin induced HIF-1alpha protein expression through PAR-1 and HIF-1alpha translational de novo protein synthesis. Twist can regulate epithelial-mesenchymal transition (EMT) and increase tumor metastasis. However, we observed that thrombin-induced HIF-1alpha increased Twist mRNA and its protein level was mediated by the modulation of PAR-1 activation and the HIF-1alpha translational pathway. In addition, Twist could increase N-cadherin but not E-cadherin to promote tumor metastasis. Overexpression of dominant-negative HIF-1alpha reversed thrombin-mediated Twist and Twist-induced N-cadherin expression. Moreover, siTwist inhibited Twist-induced N-cadherin and Thrombin-induced cell motility. In conclusion, our study showed that thrombin-induced HIF-1alpha upregulated Twist at the transcriptional level to enhance cell motility. These findings show that thrombin upregulates Twist via HIF-1alpha to make tumor cells malignant and also establish a link between the coagulation disorder and cancer metastasis.

We investigated the clinicopathologic significance of HIF-1, CXCR4, and VEGF expression using immumohistochemistry in human colon cancer. HIF-1, CXCR4, and VEGF high expression levels were correlated positively with TNM stage, lymph node involvement, and distant metastasis Furthermore, we found that combined high expression of any two of the three molecules (P = .028 for HIF-1/CXCR4, P = .007 for HIF-1/VEGF, and P = .004 for CXCR4/VEGF) had stronger correlation with lymph node metastasis than did each alone. However, a relationship with distant metastasis is seen only with the combinations CXCR4/VEGF (P = .069 for HIF-1/CXCR4, P = .062 for HIF-1/VEGF, and P = .035 for CXCR4/VEGF) as compared with those of single molecule high expression alone. Combined expression of all three molecules strongly correlates with lymph node metastasis and distant metastasis. The mRNA expression of HIF-1, CXCR4, and VEGF were quantified by real-time PCR in different colon cancer tissue samples, the experiment results shown that fresh colon tissue samples significantly overexpressed CXCR4 and VEGF mRNA compared with negative control. Therefore, the disease-free survival of all patients after curative resection can be considered in association with all three markers expression.

The purpose of this study is to investigate the clinical and histopathologic significance of hypoxia-inducible-factor 1alpha (HIF-1alpha) expression in oesophageal squamous cell carcinoma. One hundred and thirty surgically resected specimens of OSCC were immunohistochemically assessed for HIF-1alpha expression with monoclonal antibody. High HIF-1alpha immunostaining was detected in 40 specimens. The percentage of high HIF-1alpha expression cases increased with tumour stage according to pTNM system. High HIF-1alpha expression correlated with pTNM stage, depth of tumour invasion, lymph node metastasis, distant metastasis, lymphatic invasion and positive surgical margin. The overall survival rate was worse in patients with high HIF-1alpha pattern than in patients with low-expression pattern. Univariate analyses identified high HIF-1alpha positivity, depth of tumour invasion, lymph node metastasis, distant metastasis, lymphatic invasion, and a positive surgical margin as risk factors. Multivariate analyses indicated that depth of tumour invasion, lymph node metastasis and positive surgical margin, but not HIF-1alpha, were independent prognostic factors. Survival in patients with a high HIF-1alpha expression was significantly worse than in those with low expression in patient treated with adjuvant therapy.

OBJECTIVE: To investigate the expression of HPA and HIF-1alpha in human esophageal squamous cell carcinoma and their relationship with cancer development, invasion and metastasis. METHODS: The expression of HPA mRNA and HIF-1alpha protein in 23 mucosa specimens of incisal margin, 26 para-tumorous dysplastic tissues and 70 esophageal squamous cell carcinoma specimens were detected by in situ hybridization assay and immunohistochemical staining, respectively. RESULTS: The positive expression of HPA mRNA and HIF-1alpha protein were significantly increased as the epithelial cells progressed into carcinoma (P < 0.05). The expression of HPA mRNA and HIF-1alpha protein in the esophageal squamous cell carcinoma were significantly correlated with the invasion depth, lymph node metastasis and clinical staging (P < 0.05), while it was not correlated with the differentiation of tumors (P > 0.05). There was a close correlation between the expression of HPA mRNA and HIF-1alpha protein in the carcinoma tissues (P < 0.01). CONCLUSION: The high expression of HPA mRNA and HIF-1alpha protein is correlated with carcinogenesis, progression, invasion and metastasis of esophageal squamous cell carcinoma. There may be a positive cooperation between two of them in the carcinogenesis and development of esophageal carcinoma. The determination of HPA mRNA and HIF-1 alpha will be useful in predicting tumor biological behavior.

This study aims to find good markers for predicting the prognosis of patients with esophageal squamous cell carcinoma (ESCC). Vasculogenic mimicry (VM) and the expression of hypoxia inducible factor-lalpha (HIF-lalpha)/E-cad protein in ESCC were investigated by immunostaining. The association between VM, HIF-lalpha/E-cad and clinicopathologic characteristics and 5-year-survival rate of patients with ESCC was analyzed. A total of 160 ESCC specimens were involved in this study and 28 specimens of normal esophageal mucosa served as controls. VM channels were identified in 78 (48.75%) of the 160 ESCC specimens and none of the normal esophageal mucosa was found to have VM. The rates of high-expression of HIF-lalpha and E-cad in ESCC were 43.75% and 38.75%, while the rates in control were 17.86% and 71.43%, respectively (P<0.05 for all). VM and the expression levels of HIF-lalpha and E-cad were significantly related to lymph node metastasis, serosa infiltration, PTNM staging and 5-year-survival rates of patients with ESCC (P<0.05 for all). VM was positively correlated with HIF-lalpha but negatively with E-cad, and HIF-lalpha was negatively correlated with E-cad (P<0.001 for all). The 5-year-survival rate of patients with ESCC was 6.41% (5/78) in VM group and 65% (52/82) in non-VM group, 7.14% (5/70) in high HIF-lalpha expression group and 57.78% (52/90) in low HIF-lalpha expression group. Oppositely, the 5-year-survival rate in high E-cad expression group was 80.65% (50/62) and that in low E-cad expression group was 7.37% (7/98) (P<0.05 for all). Cox multifactor regression analysis indicated that lymph node metastasis, PTNM stage, VM and expression levels of HIF-lalpha and E-cad were independent risk factors of patients with ESCC (P<0.05 for all). Combined detection of VM, HIF-lalpha and E-cad plays an important role in predicting the invasion, metastasis and prognosis of patients with ESCC.

AIM: To investigate the implication of the hypoxia inducible factor HIF-1alpha mRNA in gastric carcinoma and its relation to the expression of vascular endothelial growth factor (VEGF) protein, tumor angiogenesis invasion/metastasis and the patients survival. METHODS: In situ hybridization was used to examine expression of HIF-1alpha mRNA, and immunohistochemical staining was used to examine expression of VEGF protein and CD34 in 118 specimens from patients with gastric carcinoma. RESULTS: The positive rates of HIF-1alpha mRNA and VEGF protein were 49.15% and 55.92%, respectively. Positive expressions of HIF-1alpha and VEGF in stage T3-T4 tumors and those with vessel invasion, lymph node metastasis and distant metastasis were dramatically stronger than stage T1-T2 cases and those without vessel invasion, lymph node metastasis and distant metastasis. The mean microvascular density (MVD) in stage T3-T4 tumors and those with vessel invasion, lymph node metastasis and distant metastasis was significantly higher than stage T1-T2 tumors and those without vessel invasion, lymph node metastasis and distant metastasis. The mean MVD in tumors with positive HIF-1alpha and VEGF expression was significantly higher than that in tumors with negative HIF-1alpha and VEGF expression. The expression of HIF-1alpha was positively correlated with VEGF protein. There were positive correlations between MVD and expression of HIF-1alpha and VEGF. The mean survival time and the 5-year survival rate in cases with positive expression HIF-1alpha and VEGF and MVD value >or= 41.5/0.72 mm2 were significantly lower than those with negative expression of HIF-1alpha and VEGF and MVD value < 41.5/0.72 mm2. CONCLUSION: Overexpression of HIF-1alpha is found in gastric carcinoma. HIF-1alpha may induce the angiogenesis in gastric carcinoma by upregulating the transcription of VEGF gene, and take part in tumor invasion and metastasis. They can be used as prognostic markers of gastric cancer in clinical practice.

Hypoxia-inducible factor-1 (HIF-1) is a major determinant of invasion and metastasis in several tumor types. We previously reported that HIF-1alpha contributed to multidrug resistance in gastric cancer. However, the role of HIF-2alpha on the progression of gastric cancer is seldom reported. In this study, we first examined the possible role of HIF-1alpha and HIF-2alpha in the process of invasiveness and metastasis of gastric cancer, using immunohistochemistry of 80 gastric cancer tissues, western blot and real-time PCR of 8 fresh gastric cancer tissues. The results showed that HIF-1alpha and HIF-2alpha were significantly correlated with the clinical stage and were highly expressed in metastatic gastric cancers compared to nonmetastatic ones. Western blot analysis revealed that hypoxia (1% O(2), 8 h) induced HIF-1alpha and HIF-2alpha expression in different gastric cancer cell lines, including SGC7901, AGS, MGC803 and MKN45. Adhesion and invasion assays found that hypoxia caused an increase in adhesive and invasive abilities of gastric cancer cells. Small interfering (si) RNA against HIF-1alpha and HIF-2alpha in SGC7901 cells significantly inhibited hypoxia-induced adhesive and invasive abilities. Finally, the JNK inhibitor SP 600125 abolished hypoxia-induced HIF-1alpha and HIF-2alpha expression, and inhibited the adhesive and invasive abilities of gastric cancer cells exposed to hypoxia in a dose-dependent manner. Taken together, the present work suggested that HIF-1alpha and HIF-2alpha were involved in metastasis and invasion of gastric cancer cells under hypoxia, with the involvement of JNK signal pathway.

BACKGROUND: The aim of this study was to investigate the relationship between the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), Ras-related C3 botulinum toxin substrate 1 (Rac1), and vascular endothelial growth factor (VEGF), as well as their correlation with angiogenesis and prognosis in gastric carcinoma. MATERIAL AND METHODS: The expression of Rac1, HIF-1alpha, VEGF, and CD34 (described in terms of microvessel density, MVD) was determined by immunohistochemical staining of tissues from 60 radically resected gastric cancer specimens. RESULTS: The proportion of specimens expressing Rac1, HIF-1alpha, and VEGF was 37/60 (61.7%), 35/60 (58.3%), and 40/60 (66.7%), respectively. The levels of Rac1, HIF-1alpha, and VEGF expression were significantly correlated with Lauren's classification, lymph node metastasis, and pathologic staging (p < 0.05). There were positive correlations between MVD and the expression of Rac1, HIF-1alpha, and VEGF. The mean survival time and 5-year survival rate in cases with positive Rac1, HIF-1alpha, and VEGF expression and MVD >/= 26.3 were significantly shorter than those with negative Rac1, HIF-1alpha, and VEGF expression and MVD < 26.3. CONCLUSION: Rac1, HIF-1alpha, and VEGF play an important role in tumor invasion and metastasis, especially in tumor angiogenesis. Thus, testing the expression of Rac1, HIF-1alpha, and VEGF may be a useful index for treatment and prognosis.

The molecular mechanisms underlying the peritoneal dissemination of gastric cancer remain unclear. Using in vivo metastatic models, this study attempted to clarify the role of hypoxia inducible factor (HIF)-1alpha in the development of peritoneal dissemination of gastric cancer. HIF-1alpha knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9. Using KD and control (SC) cells, the presence of peritoneal dissemination was assessed in orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. A series of in vitro analyses were also conducted. Finally, tumor angiogenesis was immunohistochemically analyzed. In the o.i. model, peritoneal dissemination was more frequently observed in the SC mice (93%) compared to the KD mice (13%) (P<0.001). In the i.p. model, peritoneal dissemination occurred at a high rate in both types of mice; however, a greater number of nodules was observed in the KD mice (P=0.017). The in vitro assays showed that HIF-1alpha exerts unfavorable effects on anoikis resistance and adhesion to extracellular matrix. Angiogenesis and vascular invasion were more aggressive in the SC gastric tumors. Vascular invasion was present in the intratumoral regions of the disseminated nodules in the SC o.i., but not the i.p., mice. HIF-1alpha was found to be crucial for the development of peritoneal dissemination in o.i. model, which mimics natural metastasis. In contrast, HIF-1alpha played an inhibitory role in suppressing peritoneal dissemination in the i.p. model. These results indicate that peritoneal dissemination in o.i. mice may not act through a seeding mechanism. An immunohistochemical analysis demonstrated HIF-1alpha-activated angiogenesis and vascular invasion in stomach tumors. Furthermore, the results showed that the disseminated nodules observed in SC o.i. mice were formed via extravasation of cancer cells. We provide a possible mechanism in which peritoneal dissemination of gastric cancer develops via a vascular network whereby HIF-1alpha activates tumor angiogenesis.

OBJECTIVE: To investigate the protein and mRNA expression of HIF-1alpha and COX-2 in laryngeal squamous carcinoma, and their correlation with clinicopathologic parameters. METHOD: The protein and mRNA expression of HIF-1alpha and COX-2 in fresh laryngeal squamous carcinoma tissues and adjacent normal tissues from 76 patients were examined by immunohistochemistry and reverse transcription polymerase chain reaction. RESULT: 1) The protein positive expression rate of HIF-1alpha and COX-2 in tumor samples were significantly higher than that in normal tissues (P < 0.05); The protein positive expression rate of HIF-1alpha were higher in moderately and poorly differentiated tumors than in well differentiated tumors (P < 0.05); While the protein positive expression rate of COX-2 were not associated with T stage (P > 0.05); 2) The mRNA positive expression rate of HIF-1alpha and COX-2 were 67.11% and 75.00% respectively, their expression were significantly higher in tumors than that in normal tissues (P < 0.05). The mRNA expression of COX-2 were not detected in normal tissues. The mRNA positive expression rate of HIF-1alpha were associated with T stage, but were not associated with differentiated clinicopathologic stage. 3) The protein positive expression rate of HIF-1alpha were associated with that of COX-2. CONCLUSION: HIF-1alpha and COX-2 could up regulate each other to promote the development and metastasis of laryngeal squamous carcinoma.

OBJECTIVE: To investigate the expressions and clinical significance of Hypoxia-Inducible Factor-1alpha (HIF-1alpha) and angiopoietin-2 (Ang-2) proteins in laryngeal squamous cell carcinoma. METHOD: Immunohistochemical technique was used to detect the expressions of HIF-1alpha and Ang-2 protein in 52 laryngeal squamous cell carcinoma and 10 normal tissues. RESULT: The positive rates of HIF-1alpha and Ang-2 proteins were significantly higher in laryngeal squamous cell carcinoma than those in normal tissues (P < 0.05). The expressions of HIF-1alpha protein was related with TNM stage and lymph node metastasis (P < 0.05), but had no marked correlations with the sex, age and histological differentiation (P > 0.05). The level of Ang-2 expression was also correlated with TNM stage, lymph node metastasis and histological differentiation of laryngeal squamous cell carcinoma. But not related to the age and sex. There was positive correlations between both expressions of HIF-1alpha and Ang-2 proteins (r = 0. 333, P < 0.01). CONCLUSION: High expressions of HIF-1alpha and Ang-2 proteins in laryngeal squamous cell carcinoma may play an important role in tumor growth, invasion and metastasis.

OBJECTIVE: Analyze the relationship between the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and epidermal growth factor receptor (EGFR) and CD105 micro vessel density (MVD) and their value in evaluating biologic behavior and prognosis in laryngeal cancer. METHODS: Ninety-one cases of laryngeal cancer were analyzed about their clinical and pathology data. In tumor tissue the expression of HIF-1alpha and EGFR was detected by immunohistochemistry and MVD was marked by CD105. RESULTS: The expression of HIF-1alpha was correlated with size, TNM stage, T stage, lymph node metastasis and histological grade (all P<0.05). The expression of EGFR was correlated with TNM stage, lymph node metastasis, histological grade and relapse (all P<0.05). MVD was correlated with type, TNM stage, T stage, lymph node metastasis, metastasis and histological grade (all P<0.05). The expression of HIF-1alpha and EGFR was correlated with MVD (F value was 7.644 and 5.197 respectively, P value was 0.001 and 0.025 respectively). The correlation between the expression of HIF-1alpha and EGFR was significant statistically (r= 0.238, P=0.007). The survival rate of patients of 3 years and 5 years were 56.1% and 44.2% respectively. Survival analysis by Log Rank showed that prognosis of laryngeal cancer patients was correlated with type, TNM stage and the expression of both HIF-1alpha and EGFR. While Cox multiple factors analysis demonstrated that TNM stage and expression of EGFR were independent prognostic factor of laryngeal cancer (P value was 0.049 and 0.041 respectively, RR was 1.300 and 2.417 respectively). CONCLUSIONS: HIF-1alpha and EGFR are key molecular event during development and progression of laryngeal cancer, which act in regulating tumor angiogenesis as well, and show intimate relationship with biological behavior and prognosis of laryngeal cancer.

OBJECTIVE: To detect the expression of hypoxia inducible factor 1 alpha (HIF-1alpha), glucose transporter protein-1 (GLUT-1) and vascular endothelial growth factor (VEGF) in human laryngeal carcinoma tissue, and to study the relationship between hypoxia and HIF-1alpha, GLUT-1, VEGF in human laryngeal carcinoma Hep-2 cells and to explore the effect of HIF-1alpha, GLUT-1 and VEGF as endogenous hypoxic markers on laryngeal carcinoma. METHODS: The expression levels of HIF-1alpha, GLUT-1 and VEGF were detected in 35 cases of laryngeal carcinoma by SP immunohistochemical methods and in Hep-2 cells by SP immunocytochemical methods. The relationship between HIF-1alpha and GLUT-1, VEGF protein expression was analyzed. RESULTS: Of the 35 cases, 16 cases expressed HIF-1alpha, 16 cases expressed GLUT-1, 19 cases expressed VEGF. The expression of HIF-1alpha and VEGF were closely correlated with pathologic grading and lymphnode metastasis. GLUT-1 was correlated with lymphnode metastasis. The expression levels of HIF-1alpha, GLUT-1 and VEGF in Hep-2 cells under hypoxic condition were higher than those under normoxic condition. CONCLUSION: HIF-1alpha may promote the expression of GLUT-1 and VEGF in laryngeal carcinoma, furthermore promote tumor angiogenesis, invasion, and metastasis of the laryngeal carcinoma.

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.

The purpose of this study was to evaluate the relationship between hypoxia-inducible factor-1alpha (HIF-1alpha) protein expression in hepatocellular carcinoma (HCC), and responses of abdominal metastatic lymph nodes (LNs) from HCC patients treated with external beam radiotherapy (EBRT). HIF-1alpha immunohistochemical staining was performed on tissue microarrays (TMAs) of primary HCC specimens from 69 HCC patients with abdominal LN metastases. All patients received abdominal metastatic LN EBRT at the Department of Radiation Oncology at Zhongshan Hospital. A receiver-operating characteristic (ROC)-based approach and logistical regression analysis were used to determine the predictive value of HIF-1alpha expression in primary tumors with HCC metastatic LN EBRT response. Kaplan-Meier curves and log-rank tests were used to analyze patient survival. Cox proportional hazards regression model was used to analyze independent prognostic factors. HIF-1alpha expression was correlated with blood hemoglobin (Hb: r = -0.280, P = 0.020), response of abdominal metastatic LNs to EBRT (r = 0.286, P = 0.017), locoregional recurrence (r = 0.278, P = 0.021), and cancer-specific deaths (r = 0.298, P = 0.013). HIF-1alpha expression was predictive of EBRT response of metastatic LNs [area under the curve (AUC): 0.646; 95% confidence interval (CI): 0.499-0.793; P = 0.047], locoregional recurrence (AUC: 0.657; 95% CI: 0.509-0.805; P = 0.049) and cancer-specific deaths (AUC: 0.671; 95% CI: 0.531-0.812; P = 0.035). Patients with tumors exhibiting high HIF-1alpha expression had significantly poorer overall survival (OS) than those with low tumor expression of HIF-1alpha (P = 0.016). Multivariate analysis showed that Hb (P = 0.035), vascular invasion (P = 0.026), Child-Pugh score (P < 0.001), intrahepatic tumor control (P < 0.001), and HIF-1alpha (P = 0.020) were independent prognosis factors for OS of HCC patients after receiving abdominal metastatic LN EBRT. HIF-1alpha expression in primary HCCs was associated with EBRT response of abdominal metastatic LNs and poor prognosis.

Angiopoietin-like protein 4 (ANGPTL4) plays complex and often contradictory roles in vascular biology and tumor metastasis, but little is known about its function in hepatocellular carcinoma (HCC) metastasis. In the present study, we showed that hypoxia-inducible factor 1alpha (HIF-1alpha) directly up-regulates ANGPTL4, and its stableness positively correlates with ANGPTL4 expression in HCC tissue. Overexpression of ANGPTL4 significantly increased HCC cell transendothelial migration in vitro and intrahepatic and distal pulmonary metastasis in vivo, whereas silencing ANGPTL4 expression or treatment with a neutralizing antibody specific for ANGPTL4 protein resulted in a reduced transendothelial migration. We also found that serum ANGPTL4 is higher in HCC patients, compared to healthy control, and correlates with intrahepatic metastasis and histological grade. Further, secreted ANGPTL4 promotes transendothelial migration and metastasis of HCC cells in vitro and in vivo through the up-regulation of vascular cell adhesion molecule-1 (VCAM-1) of human umbilical vein endothelial cells and the activation of the VCAM-1/integrin beta1 axis. CONCLUSION: ANGPTL4 is a target gene of HIF-1alpha and acts as an important regulator in the metastasis of HCC. Serum ANGPTL4 correlates with tumor progression and metastasis and might be used to indicate prognosis in HCC patients.

AIM: To study the expression of HIF-1alpha in serum of primary hepatocellular carcinoma (PHC) and effects on tumor invasion and metastasis. METHODS: The serum HIF-1alpha level from 60 cases of PHC patients and 60 cases of normal healthy individuals were detected by ELISA. RESULTS: The serum HIF-1alpha level of the PHC group was (161.14+/-80.79) pg/mL, the control group was (22.67+/-8.47) pg/mL, compared the different was significant (P<0.05). The serum HIF-1alpha level of distant metastasis of tumor group was (225.15+/-108.16) pg/mL, the no distant metastasis of tumor group was (101.26+/-55.18) pg/mL, compared the different was significant (P<0.05). The serum HIF-1alpha level of portal vein tumor thrombus group was (255.45+/-122.23) pg/mL. The no portal vein tumor thrombus group was (139.97+/-71.49) pg/mL, compared the different was significant (P<0.05). The serum HIF-1alpha level of tumor capsule group was (99.97+/-55.75) pg/mL. The no tumor capsule group was (187.36+/-91.52) pg/mL, compared the different was significant (P<0.05). The serum HIF-1alpha level was no significantly different among the different age, gender, TNM stage and tumor diameter. CONCLUSION: The serum HIF-1alpha has high expression in PHC. The serum HIF-1alpha expression has close relation with the whether distant metastasis of tumor, portal vein tumor thrombus and tumor capsule. The serum HIF-1alpha level can predict the invasion and metastasis of PHC.

Cigarette smoking is not only a documented risk for lung carcinogenesis but also promotes lung cancer development. Nicotine, a major component of cigarette smoke but not a carcinogen by itself, has been found to induce proliferation, invasion and metastasis of non-small cell lung cancer (NSCLC). Here we reported that proinvasive effect of nicotine is analogous to that of hypoxia and involves stabilization and activation of hypoxia-inducible factor (HIF)-1alpha, a key factor in determining the presence of HIF-1 and expression of its downstream metastasis-associated genes. Furthermore, nicotine-induced upregulation of HIF-1alpha was dependent on mitochondria-derived reactive oxygen species (ROS). Ecotopic expression of mitochondrial targeted catalase effectively prevented nicotine-induced accumulation of HIF-1alpha protein. In addition, we demonstrated that the effect of nicotine in upregulation of HIF-1alpha was mediated by Dihydro-beta-erythroidine (DhbetaE)-sensitive nicotine acetylcholine receptors (nAChRs) and required synergistic cooperation of Akt and mitogen-activated protein kinase (MAPK) pathways. These results suggest that exposure to nicotine could mimic effects of hypoxia to stimulate HIF-1alpha accumulation and activity that might underlie the high metastatic potential of lung cancer.

We evaluated the role of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and hypoxia-inducible factor 1-a (HIF1-a) in melanoma angiogenesis and investigated their expression in dysplastic nevi, as potential melanoma precursors. In addition, we examined a possible correlation of VEGF expression with PlGF and HIF1-a. These factors were detected immunohistochemically in 95 melanomas of all types and stages and in 28 dysplastic nevi. We used 10 intradermal melanocytic nevi as controls. HIF1-a was expressed in 93 out of 95 (97.89%) melanomas and in none of the dysplastic or control nevi. HIF1-a expression was more intense in melanocytes around necrotic areas but did not correlate with melanoma type, the Clark staging or the Breslow thickness. A strong positive association was detected between HIF1-a and VEGF expression in all cases. VEGF was detected in 82 out of 95 (86.31%) melanomas and in 21 out of 28 (75%) dysplastic nevi, whereas it was expressed weakly in neoplastic cells of the controls. Its expression was more intense in melanomas, especially in nodular melanomas of elevated stage and thickness. PIGF was detected in 46 out of 95 (48.42%) melanomas and in none of the nevi. expression did not correlate with melanoma staging nor thickness; however, it was more intense in superficial spreading melanomas, where a weak positive association between VEGF and PlGF was also detected. There was no association between HIF1-a and PlGF in any melanoma type. Hypoxia, through the expression of HIF1-a, plays a key role in melanoma progression; it activates VEGF secretion, which induces angiogenesis and metastasis. The role of PlGF seems to be limited.

cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Pharmacologic or genetic blockades of the HIF-1alpha pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten-deficient, Braf-mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1alpha or HIF2alpha abrogates metastasis without affecting primary tumor formation. HIF1alpha and HIF2alpha drive melanoma invasion and invadopodia formation through PDGFRalpha and focal adhesion kinase-mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1alpha and HIF2alpha activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling.

metastasis-associated protein 1 (MTA1) is highly upregulated in cancer cells with metastatic potential; however, the molecular mechanism by which MTA1 increases the metastatic potential of cancer cells is unknown. We characterized the functional consequences of MTA1 overexpression in cancer cells with an emphasis on its potential role as a deacetylator of hypoxia-inducible factor-1alpha (HIF-1alpha). MTA1 increased the expression of HIF-1alpha protein, but did not increase the expression of its mRNA. Glutathione S-transferase pull-down and coimmunoprecipitation assays demonstrated direct interaction of MTA1 with HIF-1alpha both in vitro and in vivo. Immunoprecipitation and acetylation assays also showed that MTA1 has deacetylation activity on HIF-1alpha in vivo. Moreover, MTA1 increased the transcriptional activity of HIF-1alpha and enhanced the expression of vascular endothelial growth factor, a target molecule of HIF-1alpha. Conditioned medium collected from MTA1 transfectants also increased angiogenesis in vitro and in vivo, probably through enhanced HIF-1alpha stabilization. These results indicate that MTA1 enhances angiogenesis by stabilization of the HIF-1alpha protein, which is closely related to the increased metastatic potential of cancer cells with high MTA1 expression.

Stabilization of the hypoxia-inducible factor-1alpha (HIF-1alpha) transcription complex, caused by intratumoural hypoxia, promotes tumour progression and metastasis, leading to treatment failure and mortality in different types of human cancers. The transcription factor TWIST is a master regulator of gastrulation and mesoderm-specification and was implicated recently as an essential mediator of cancer metastasis. Notably, HIF-1alpha- and TWIST-null mice show similarities in their phenotypes. Here, we have shown that hypoxia or overexpression of HIF-1alpha promotes epithelial-mesenchymal transition (EMT) and metastastic phenotypes. We also found that HIF-1 regulates the expression of TWIST by binding directly to the hypoxia-response element (HRE) in the TWIST proximal promoter. However, siRNA-mediated repression of TWIST in HIF-1alpha-overexpressing or hypoxic cells reversed EMT and metastastic phenotypes. Co-expression of HIF-1alpha, TWIST and Snail in primary tumours of patients with head and neck cancers correlated with metastasis and the worst prognosis. These results provide evidence of a key signalling pathway involving HIF-1alpha and TWIST that promotes metastasis in response to intratumoural hypoxia.

The hypoxic tumor environment has been shown to be critical to cancer metastasis through the promotion of angiogenesis, induction of epithelial-mesenchymal transition (EMT), and acquisition of invasive potential. However, the impact of hypoxia on the expression profile of the proteolytic enzymes involved in invasiveness is relatively unknown. Membrane-type 4 matrix metalloproteinase (MT4-MMP) is a glycosyl-phosphatidyl inositol-anchored protease that has been shown to be overexpressed in human cancers. However, detailed mechanisms regarding the regulation and function of MT4-MMP expression in tumor cells remain unknown. Here, we demonstrate that hypoxia or overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) induced MT4-MMP expression in human cancer cells. Activation of SLUG, a transcriptional factor regulating the EMT process of human cancers, by HIF-1alpha was critical for the induction of MT4-MMP under hypoxia. SLUG regulated the transcription of MT4-MMP through direct binding to the E-box located in its proximal promoter. Short-interference RNA-mediated knockdown of MT4-MMP attenuated in vitro invasiveness and in vivo pulmonary colonization of tumor cells without affecting cell migratory ability. MT4-MMP promoted invasiveness and pulmonary colonization through modulation of the expression profile of MMPs and angiogenic factors. Finally, coexpression of HIF-1alpha and MT4-MMP in human head and neck cancer was predictive of a worse clinical outcome. These findings establish a novel signaling pathway for hypoxia-mediated metastasis and elucidate the underlying regulatory mechanism and functional significance of MT4-MMP in cancer metastasis.

BACKGROUND: Vascular endothelial growth factor A (VEGFA), a critical mediator of tumor angiogenesis, is a well-characterized target of hypoxia-inducible factor 1 (HIF-1). Murine arrest-defective protein 1A (mARD1A(225)) acetylates HIF-1alpha, triggering its degradation, and thus may play a role in decreased expression of VEGFA. METHODS: We generated Apc(Min/+)/mARD1A(225) transgenic mice and quantified growth of intestinal polyps. Human gastric MKN74 and murine melanoma B16F10 cells overexpressing mARD1A(225) were injected into mice, and tumor growth and metastasis were measured. VEGFA expression and microvessel density in tumors were assessed using immunohistochemistry. To evaluate the role of mARD1A(225) acetylation of Lys532 in HIF-1alpha, we injected B16F10-mARD1A(225) cell lines stably expressing mutant HIF-1alpha/K532R into mice and measured metastasis. All statistical tests were two-sided, and P values less than .05 were considered statistically significant. RESULTS: Apc(Min/+)/mARD1A(225) transgenic mice (n = 25) had statistically significantly fewer intestinal polyps than Apc(Min/+) mice (n = 21) (number of intestinal polyps per mouse: Apc(Min/+) mice vs Apc(Min/+)/mARD1A(225) transgenic mice, mean = 83.4 vs 38.0 polyps, difference = 45.4 polyps, 95% confidence interval [CI] = 41.8 to 48.6; P < .001). The growth and metastases of transplanted tumors were also statistically significantly reduced in mice injected with mARD1A(225)-overexpressing cells than in mice injected with control cells (P < .01). Moreover, overexpression of mARD1A(225) decreased VEGFA expression and microvessel density in tumor xenografts (P < .04) and Apc(Min/+) intestinal polyps (P = .001). mutation of lysine 532 of HIF-1alpha in B16F10-mARD1A(225) cells prevented HIF-1alpha degradation and inhibited the antimetastatic effect of mARD1A(225) (P < .001). CONCLUSION: mARD1A(225) may be a novel upstream target that blocks VEGFA expression and tumor-related angiogenesis.

Celastrol, a natural biologically active compound isolated from Tripterygium wilfordii Hook F root extracts, has been shown to possess antitumor properties and therefore, is an interesting candidate for the development of novel chemotherapeutic cancer agents. In this study, we have demonstrated that Celastrol is a potent inhibitor of hypoxia-induced angiogenic and metastatic activity as shown by a decrease in the proliferation of both endothelial and cancer cells, blocking of migration as well as of tube formation of endothelial cells, and by inhibition of cancer cell invasion under hypoxic conditions. Moreover, Celastrol decreased hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels under both normoxia and hypoxia and inhibited hypoxia-induced accumulation of nuclear HIF-1alpha protein. Meanwhile, inhibition of nuclear HIF-1alpha protein levels were accompanied by a reduction in the transcriptional activity of HIF-1alpha target genes, including VEGF. In addition, the inhibitory effect of Celastrol on HIF-1alpha protein was partly due to its suppression of HSP90 activity. We conclude that Celastrol regulates HIF-1alpha at multiple levels that may together or individually contribute to its antitumor activity against hypoxia-induced angiogenesis and metastasis.

The hypoxia-inducible transcription factor-1alpha (HIF-1alpha) is a major regulator of angiogenesis, carcinogenesis, and various processes by which cells adapt to hypoxic conditions. Therefore, the identification of critical players regulating HIF-1alpha is not only important for the understanding of angiogenesis and different cancer phenotypes, but also for unraveling new therapeutic options. We report a novel mechanism by which HIF-1alpha is degraded after glycogen synthase kinase-3 (GSK-3)-induced phosphorylation and recruitment of the ubiquitin ligase and tumor suppressor F-box and WD protein Fbw7. Further, experiments with GSK-3beta and Fbw7-deficient cells revealed that GSK-3beta and Fbw7-dependent HIF-1alpha degradation can be antagonized by ubiquitin-specific protease 28 (USP28). In agreement with this, Fbw7 and USP28 reciprocally regulated cell migration and angiogenesis in an HIF-1alpha-dependent manner. Therefore, we have identified a new pathway that could be targeted at the level of GSK-3, Fbw7, or USP28 to influence HIF-1alpha-dependent processes like angiogenesis and metastasis.

Intratumoral hypoxia and expression of hypoxia-inducible factor-1alpha (HIF-1alpha) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1alpha enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1alpha or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1alpha-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1alpha controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. SIGNIFICANCE: Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

BACKGROUND: HIF-1alpha is a major regulator in tumor progression and metastasis which responds to hypoxia. Many studies have demonstrated that hypoxia-inducible factor1-alpha (HIF-1alpha) polymorphisms are significantly associated with cancer metastasis, but the results are inconsistent. We conducted a comprehensive meta-analysis to estimate the associations between HIF-1alpha C1772 T polymorphism and cancer metastasis. METHODS: Comprehensive searches were conducted on PubMed and EMBASE database. Fifteen studies were included in the meta-analysis. We used the OR and 95%

In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5'-UTR is sufficient for significant translational up-regulation. On the other hand, the 3'-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3'-UTR-dependent down-regulatory machinery predominates over the 5'-UTR-dependent up-regulation of HIF1A. However, 5'-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5'-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A.

OBJECTIVE: To examine the expression of HIF-1alpha and CD34 in nasopharyngeal carcinoma and nasopharyngitis tissues, evaluate the role of HIF-1alpha and microvessel density (MVD) in nasopharyngeal carcinoma, probe the relationship between HIF-1alpha and MVD. METHOD: The expression of HIF-1alpha was detected in 58 cases with nasopharyngeal carcinoma and 20 samples of nasopharyngitis tissues by immunohistochemical staining SABC methods and SP methods. MVD in tumor was counted by immunostaining with CD34 and analyzed with clinicopathologic parameters. RESULT: The positive rate of HIF-1alpha was 79.31% and 10.00% respectively in nasopharyngeal carcinoma and nasopharyngitis tissues, there was significant difference between them (P<0.01). MVD was 39.74+/-7.41 and 18.43+/-3.24 respectively in nasopharyngeal carcinoma and nasopharyngitis tissues, there was significant difference between them (P<0.01). The expression of HIF-1alpha and MVD were significantly higher in nasopharyngeal carcinoma and in lymph node metastasis group than in no-metastasis group. In different clinical stage, the expression of HIF-1alpha (P<0.05) and MVD (P<0.01) was different. The expression of HIF-1alpha was positively correlated with MVD (P<0.01). CONCLUSION: HIF-1alpha may be involved in carcinogenesis, invasion and metastasis of NPC. Visualization plays a key role in invasion and metastasis of NPC. The expression of HIF-1alpha was positively correlated with tumor visualization in NPC. HIF-1alpha is likely to be a potential target of anticancer therapy.

BACKGROUND AND OBJECTIVE: Vasculogenic mimicry (VM), found in many high invasive ability tumors, is associated with tumor invasion and metastasis. Many genes exhibit abnormal levels of expression in these tumors. This study aims to find good markers for predicting the invasion and metastasis of non-small cell lung cancer (NSCLC). METHODS: VM and expression of CD82/KAI1 and HIF-1alpha were examined via immunohistochemistry and histochemistry of 160 NSCLC and 20 normal lung tissue specimens. RESULTS: In NSCLC, positive rates of 37.5%, 48.8%, and 36.9% were obtained for CD82/KAI1, HIF-1alpha, and VM, respectively. In normal lung tissue, positive rates of 95.0%, 0, and 0 were obtained for CD82/KAI1, HIF-1alpha, and VM, respectively. A significant difference was found between the NSCLC and normal lung groups (P<0.01). VM and the expression levels of CD82/KAI1 and HIF-1alpha were significantly related to tumor differentiation, lymph node metastasis, clinical staging, and postoperative survival time (P<0.01 for all). A negative correlation was found between the expression levels of CD82/KAI1 and HIF-1alpha; a similar relationship was observed between CD82/KAI1 and VM. A positive relationship between the expression of HIF-1alpha and VM was revealed; there was a significant relationship between microvessel density (MVD) and the expression of CD82/KAI1 or HIF-1alpha or VM. VM and overexpression of HIF-1alpha were related to poor prognosis: the survival rates were significantly lower in positive patients than in negative patients (both P<0.01). The survival rates of the CD82/KAI1-positive and CD82/KAI1-negative groups were significantly different (P<0.01). The five-year survival rate was significantly different between the group with MVD>/=22 and the group with MVD<22 (P<0.01). pTNM stage, positive expressions of CD82/KAI1 and HIF-1alpha, and VM were independent prognostic factors of NSCLC (P<0.01). CONCLUSIONS: VM and the expressions of CD82/KAI1 and HIF-1alpha in NSCLC are related to differentiation, lymph node metastasis, clinical staging, and prognosis. The combined detection of CD82/KAI1, HIF-1alpha, and VM has an important role in predicting the progression and prognosis of NSCLC.

BACKGROUND: The number of blood vessels correlates with metastasis in solid tumors, including oral squamous cell carcinoma (OSCC). Hypoxia inducible factor-1alpha (HIF-1alpha) could play a role in tumor lymphangiogenesis by regulating the lymphatic expression of vascular endothelial growth factor-C (VEGF-C). HIF-1alpha protein expression, VEGF-C protein expression, lymphatic vessel density (LVD) and blood vessel density (BVD) in OSCC were investigated. MATERIALS AND METHODS: HIF-1alpha and VEGF-C protein expression were investigated by means of immunohistochemistry in samples from 65 cases of OSCC. The density of the lymphatic microvessels and blood microvessels immunohistochemically stained by LYVE-1 and CD34 antibody, respectively, was calculated. The association between the HIF-1alpha expression and the clinicopathological parameters was evaluated. RESULTS: HIF-1alpha overexpression occurred in 43 out of the 65 tumor samples (66.2%), while VEGF-C overexpression was observed in 34 out of the 65 tumor samples (52.3%). Higher LVD was found in both high HIF-1alpha and high VEGF-C expression cases. HIF-1alpha overexpression was significantly correlated with VEGF-C overexpression (p = .018, Chi-square test), higher LVD (p < 0.001, Mann-Whitney U-test), and regional lymph nodal involvement (p = 0.004, Chi-square test) as well as UICC TMN classification (p = 0.043, Chi-square test), respectively. In addition, higher BVD existed in the high HIF-1alpha and VEGF-C expression groups (p < 0.001, Mann-Whitney U-test). CONCLUSION: HIF-1alpha might play a crucial role in regional lymph node metastasis as a regulator of lymphangiogenesis and angiogenesis in OSCC with a possible novel pathway involving VEGF-C. Therefore, HIF-1alpha might be a particularly promising target for controlling regional lymph node metastases by a combination of antiangiogenic and anti-lymphangiogenic effects in OSCC.

Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominant-negative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3'-UTR of HIF-1alpha and HIF-2alpha. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a-mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR-199a, and HIF, with implications in cancer metastasis.

OBJECTIVE: Hypoxia-inducible factor 1alpha (HIF-1alpha) has been universally detected in many types of cells and plays a key role in modulation of tumor-related genes. The purpose of this study was to explore the relationship between HIF-1alpha messenger RNA (mRNA) expression and biologic characteristics in pancreatic cancer, such as tumor angiogenesis, tumor cell proliferation, differentiation, apoptosis, and metastasis. METHODS: Reverse transcriptase-polymerase chain reaction was used to detect HIF-1alpha mRNA expression. The expressions of survivin, proliferating cell nuclear antigen (PCNA), and CD34 proteins were measured immunohistochemically. The correlation between the expression levels of HIF-1alpha mRNA and survivin, PCNA, and CD34 were analyzed. RESULTS: There was very significant difference in the expression of HIF-1alpha between the pancreatic cancer tissue and adjacent normal tissue (P < 0.01), but no significant difference was found among tumor histopathologic grades (P > 0.05). There was significant difference in the expression of HIF-1alpha mRNA between Japanese Pancreatic Society stages I to II and stages III to IV (P < 0.05). The expression of HIF-1alpha mRNA was closely correlated with the expression of survivin, PCNA, and CD34 proteins. CONCLUSION: The expression level of HIF-1alpha mRNA is surmised to have a significant correlation with tumor angiogenesis, cell proliferation, apoptosis, and metastasis. Inhibition of HIF-1alpha may be an important and approachable therapeutic target for pancreatic cancer.

PURPOSE: Pancreatic cancer is characterized by intratumoral hypoxia, early and aggressive local invasion, and metastatic potential. Hypoxia-inducible factor-1 (HIF-1) is the major transcriptional activator of hypoxia-responsive genes and intratumoral hypoxia is associated with increased risk of metastasis. However, the behavior of the cells having HIF-1 activity during the malignant progression in pancreatic cancer has not been tested. EXPERIMENTAL DESIGN: We orthotopically transplanted pancreatic cancer cells stably transfected with a HIF-1-dependent luciferase reporter gene and monitored HIF-1 activity in vivo in control and POP33-treated mice. POP33 is a novel prodrug, which has potential to increase caspase-3 activity and induce apoptosis in HIF-1-active/hypoxic cells. RESULTS: In vivo optical imaging showed that HIF-1 activity proceeded along with local invasion, the peritoneal dissemination, and the liver metastasis. HIF-1-active hypoxic cells were selectively eradicated by POP33. Moreover, selective killing of HIF-1-active hypoxic cells significantly suppressed malignant progression, resulting in a significant improvement in survival rate. CONCLUSIONS: These results show that HIF-1-active cells constitute a large proportion of invading and metastatic cells and suggest that eradication of these cells may improve the outcome in advanced pancreatic cancer, a condition for which no effective therapy currently exists.

OBJECTIVE: To explore the correlation between the expressions of hypoxia inducible factor-1alpha (HIF-1alpha) and insulin in pancreatic cancer. METHODS: HIF-1alpha and insulin expression was detected by immunohistochemistry in the center and the edge of pancreatic adenocarcinoma specimens of 65 cases. Western blot was used to detect HIF-1alpha expression and insulin level in the center and the edge of pancreatic adenocarcinoma specimens of 28 cases. The relationship between HIF-1alpha expression and insulin level in the pancreatic cancer was analyzed. RESULTS: The results of immunohistochemistry and Western blot showed that HIF-1alpha protein expression was high in both the center and the edge of pancreatic cancers (P > 0.05), and insulin level was significantly higher at the edge of specimen than that in the center (P < 0.05). HIF-1alpha protein and insulin levels were positively correlated at the edge of cancer tissue (r = 0.374, P < 0.05), but no significant correlation between them in the center of cancerous tissue (r = -0.145, P > 0.05). CONCLUSION: Insulin may promote the local invasion and metastasis of pancreatic cancer by up-regulating HIF-1alpha.

BACKGROUND: Hypoxia plays a crucial role in the development of solid tumors. Hypoxia-inducible factor-1alpha (HIF-1alpha) is essential for this process, and has been suggested to be a target for cancer therapy. New therapeutic approaches for pancreatic cancer are needed owing to the extremely poor prognosis, in large part as a consequence of high rates of metastasis. The mechanism remains to be explored. AIM: To illustrate the role of hypoxia-inducible factor-1alpha in pancreatic cancer metastasis and the value of the molecule as a target for pancreatic cancer therapy. MATERIALS AND METHODS: To address this shortcoming, we used both in vitro and in vivo approaches to evaluate the overall effects of HIF-1alpha on pancreatic cancer. We used a plasmid encoding small interfering RNAs (SiRNAs) to silence HIF-1alpha expression in the Panc-1 pancreatic cancer cell line, and used a series of assays to detect changes in gene expression at the protein and mRNA levels, cell proliferation, cell apoptosis, and the abilities of cells to migrate under both hypoxia and normoxia conditions. RESULTS: Both in vitro and in vivo analysis suggested that hypoxia significantly promotes cell proliferation and migration, resulting in metastasis. Pancreatic cancer cells in which HIF-1alpha expression was inhibited were less invasive, with reduced resistance to hypoxia, impaired migration, and reduced capacity to cause metastasis. CONCLUSIONS: HIF-1alpha may be a dominant factor driving the metastatic progression of pancreatic cancer and can be a potent therapeutic target for the disease.

BACKGROUND: Intratumoral hypoxia and epithelial-mesenchymal transition are involved in tumor invasion and metastasis. AIMS: This study investigated the molecular mechanisms that relay the hypoxia signal into the epithelial-mesenchymal transition and metastasis. METHODS: Morphology analysis and tumor cell migration and invasion assays were performed to detect phenotypic changes of pancreatic cancer cells under normoxic and hypoxic conditions after lentiviral HIF-1alpha shRNA transfection. Quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry were used to detect gene expression in pancreatic cancer cell lines and tissues or normal pancreatic tissues. Luciferase, gel shift, and ChIP assays were used to assess gene regulation. RESULTS: Under hypoxic conditions, these tumor cells underwent typical morphological and molecular changes to epithelial-mesenchymal transition. Moreover, Snail expression was induced by hypoxic conditions and was regulated by HIF-1alpha expression at the transcriptional level through HIF-1alpha-binding to the second site of hypoxia-responsive elements of the Snail gene promoter. In addition, Snail expression was associated with HIF-1alpha expression in pancreatic cancer tissues, and expression of both was associated with tumor metastasis and poor patient survival. CONCLUSIONS: Our study provides key evidence that HIF-1alpha and Snail are responsible for hypoxia-induced metastasis phenotypes in pancreatic cancer and that HIF-1alpha and Snail expression can be used as biomarkers to predict tumor metastasis and patient survival.

OBJECTIVES: Several clinical studies have indicated that the rates of invasive growth and metastatic disease in cancer depend on the degree of hypoxia, which is mediated by hypoxia-inducible factor 1 alpha (HIF-1alpha). To determine its potential role as a marker for prostate cancer (CaP) diagnosis, HIF-1alpha mRNA levels were measured in blood samples of patients diagnosed with different stages of prostatic disease. METHODS: HIF-1alpha mRNA levels were measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and correlated with accurate clinicopathological data. Quantitative data were compared with serum prostate-specific antigen (PSA) measurements to determine variations in the accuracy of the CaP diagnosis. RESULTS: HIF-1alpha mRNA levels were significantly upregulated in patients with localized CaP (LocCaP; n=63; p<0.0001), compared with patients with no evidence of malignancy (NEOM) and benign prostatic hyperplasia (BPH) (n=35 for both patient groups combined). Receiver operator characteristic (ROC) curve analysis demonstrated that HIF-1alpha specificity for the NEOM/BPH diagnosis was 88.6%. Sensitivity for LocCaP was 74.6% with an overall diagnostic efficiency of 79.6%. Specificity of the NEOM diagnosis at PSA levels of 4.0 ng ml(-1) was 28.6% and sensitivity of the LocCap diagnosis was 65.1%, demonstrating a reduced overall diagnostic efficiency, compared with that given by HIF-1alpha measurements, of 52.0%. Levels of HIF-1alpha in patients with metastatic CaP (MetCaP; n=27)) were similar to those in the NEOM/BPH group. CONCLUSIONS: HIF-1alpha is upregulated early in CaP development with subsequent downregulation at later metastatic stages. This study demonstrates increased accuracy of early-stage disease diagnosis using HIF-1alpha qRT-PCR compared with serum PSA measurements. HIF-1alpha may therefore be a useful adjunct, together with other diagnostic markers used in relative qRT-PCR and current diagnostic techniques (including serum PSA and PSA velocity) to minimize unnecessary biopsies indicated by elevated serum PSA levels alone.

The receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our laboratory indicate that Axl and its ligand growth arrest-specific 6 (GAS6) may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial-mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were downregulated. Moreover, CoCl(2), a hypoxia mimicking agent, prevented GAS6-mediated downregulation of Axl in these cell lines. Immunochemical staining of human prostate cancer tissue microarrays showed that Axl, GAS6, and hypoxia-inducible factor-1alpha (Hif-1alpha; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.

BACKGROUND: Castrate-resistant prostate cancer (CRPC) is a lethal condition in patients receiving androgen deprivation therapy for prostate cancer (PC). Despite numerous studies showing the expression of HIF1alpha protein under normoxia in PC cell lines, the role of this normoxic HIF1alpha expression in chemo-resistance and migration has not been investigated previously. As no method is currently available to determine which tumors will progress to CRPC, the role of HIF1alpha in PC and its potential for predicting the development of CRPC was also investigated. METHODS: The effect of HIF1alpha protein knockdown on chemo-resistance and migration of PC3 cells was assessed by cell counting and Transwell assays, respectively. Translation efficiency of HIF1alpha mRNA was determined in PC cells using a HIF1alpha 5'UTR-luciferase construct. Clinical outcomes were correlated following the staining of 100 prostate tumors for HIF1alpha expression. RESULTS: The CRPC-like cell lines (PC3 and DU145) expressed more HIF1alpha protein than an androgen sensitive cell line (LNCaP). Migration rate and chemo-resistance were higher in the PC3 cells and both were decreased when HIF1alpha expression was reduced. Increased translation of HIF1alpha mRNA may be responsible for HIF1alpha overexpression in PC3 cells. Patients whose tumors expressed HIF1alpha had significantly decreased metastasis-free survival and the patients who were on androgen-deprivation therapy had decreased CRPC-free survival on Kaplan-Meier analysis. On multivariate analysis HIF1alpha was an independent risk factor for progression to metastatic PC (Hazard ratio (HR) 9.8, p = 0.017) and development of CRPC (HR 10.0, p = 0.021) in patients on androgen-deprivation therapy. Notably the tumors which did not express HIF1alpha did not metastasize or develop CRPC. CONCLUSIONS: HIF1alpha is likely to contribute to metastasis and chemo-resistance of CRPC and targeted reduction of HIF1alpha may increase the responsiveness of CRPCs to chemotherapy. expression of HIF1alpha may be a useful screening tool for development of CRPC.

PURPOSE: Inactivation of von Hippel-Lindau (VHL) gene in clear-cell renal cell carcinoma (RCC) leads to increased levels of hypoxia-inducible factors (HIF) and overexpression of HIF target genes, such as VEGF and others. VEGF-targeted agents are standard in advanced clear-cell RCC but biomarkers of activity are lacking. EXPERIMENTAL DESIGN: We analyzed tumor tissue samples from metastatic clear-cell RCC patients who received pazopanib as part of clinical trial VEG102616. We evaluated several components of the VHL/HIF pathway: VHL gene inactivation (mutation and/or methylation), HIF-1alpha and HIF-2alpha immunohistochemistry staining, and HIF-1alpha transcriptional signature. We evaluated the association of these biomarkers with best overall response rate (ORR) and progression-free survival (PFS) to pazopanib, a standard first-line VEGF-targeted agent. RESULTS: The VEG102616 trial enrolled 225 patients, from whom 78 samples were available for tumor DNA extraction. Of these, 70 patients had VHL mutation or methylation. VHL gene status did not correlate with ORR or PFS. Similarly, HIF-1alpha (65 samples) and HIF-2alpha (66 samples) protein levels (high vs. low) did not correlate with ORR or PFS to pazopanib. The HIF-1alpha transcriptional signature (46 samples) was enriched in tumors expressing high HIF-1alpha levels. However, the HIF-1alpha gene expression signature was not associated with clinical outcome to pazopanib. CONCLUSIONS: In patients with advanced clear-cell RCC, several potential biomarkers along the VHL/HIF-1alpha/HIF-2alpha axis were not found to be predictive for pazopanib activity. Additional efforts must continue to identify biomarkers associated with clinical outcome to VEGF-targeted agents in metastatic RCC.

BACKGROUND: HIF-1alpha plays a key role in the development and progression of cancer. Its polymorphic variants C1772T and G1790A have been associated with greater susceptibility to cancer and increased tumor progression. METHODS: We determined the distribution of these polymorphisms among 121 patients with glottic cancer and 154 healthy volunteers by PCR-RFLP. We also analyzed the relationship between the presence of these polymorphisms and various clinicopathologic variables. RESULTS: Advanced tumors (T3-T4) were associated with the TT variant (p = 0.036), which was present in 75 % of T4 tumors (p = 0.008). Among patients with nodal metastasis (N+), 41.7 and 22 % were carrying the TT and GA variants, respectively, compared with 9.4 and 2 % of the patients with no metastasis (N0), (p = 0.006 and p = 0.032). CONCLUSIONS: The presence of the TT and GA variants were associated with lymph node metastasis, while the presence of the TT variant can be associated with larger tumor size.

OBJECTIVE: In order to assess the significance of thyroid-stimulating hormone receptor (TSHR), peroxiredoxin1 (Prx1), thioredoxin1 (Trx1), hypoxia inducible factor-1 (HIF-1)alpha in invasion and metastasis of papillary thyroid carcinoma (PTC). METHODS: The 34 paraffin-embedded specimen of classical PTC, 39 of tall cell variant of PTC and normal thyroid tissues adjacent to cancer operated in Tianjin cancer Hospital from 2003 to 2007 were tested. The expressions of TSHR, Prx1, Trx1, HIF-1alpha in the specimen were assayed by immunohistochemical staining. The correlation between the 4 factors (TSHR, Prx1, Trx1, HIF-1alpha) and clinical features of PTC such as gender, age, tumor size, invasion, lymph node metastasis, histological type and T stage were studied. RESULTS: The rates of immuno-positive staining Prx1, Trx1 and HIF-1alpha in PTC were higher than the normal thyroid tissues adjacent to cancer with significant difference respectively (chi2 were 5.49, 6.16 and 40.48, P<0.05). The positive expression rate of TSHR in PTC was lower than the normal thyroid tissues adjacent to cancer (chi2=15.70, P<0.05), in tall cell variant of PTC was lower than classical PTC (chi2=4.24, P<0.05). The positive expression rates of Prx1, Trx1 and HIF-1alpha in PTC with local invasion, with lymph node metastasis, in tall cell variant and in stage T3-T4 were higher than PTC without local invasion, without metastasis, in classical variant and in stage T1-T2 with significant difference respectively (P<0.05). There were correlation between the expressions of Prx1 and Trx1, Trx1 and HIF-1alpha (r1 were 0.664 and 0.652, P<0.05). CONCLUSIONS: The high expressions of Prx1, Trx1 and HIF-1alpha were related to invasion and lymph node metastasis in PTC. TSH may promote the development of thyroid carcinoma by TSH-Prx1-Trx1-HIF-1 signaling pathway.

Hypoxia inducible factor-1alpha (HIF-1alpha) is upregulated by hypoxia, and involved in tumor growth and metastasis in many malignant tumors including papillary thyroid carcinoma (PTC). Metallothionein (MT) is a group of small molecular weight cysteine-rich proteins with a broad variety of functions. SLUG is a member of SNAIL superfamily of zinc finger transcriptional factors implicated in epithelial-mesenchymal transition (EMT). The purpose of this study was to examine HIF-1alpha, MT and SLUG expression in PTC and assess association of their expression with clinicopathological indicators. HIF-1alpha, MT and SLUG protein expression in 129 PTCs, 61 nodular hyperplasia and 118 normal thyroid tissue specimens were analyzed using immunohistochemistry. The protein expression levels of these three molecules were up-regulated in PTCs. High protein expression of HIF-1alpha, MT and SLUG was significantly correlated with high TNM stage (P=0.003, 0.002, 0.024, respectively) and lymph node metastasis (LNM) (P<0.001 for all three molecules). Furthermore, HIF-1alpha, MT and SLUG protein expression were correlated with one another. Concomitant high expression of any two of these three molecules had stronger correlation with high TNM stage (Pexpression of all these three molecules is significantly associated with high TNM stage and LNM as compared with cases not showing such expression (P<0.001). These results demonstrated that the evaluation of HIF-1alpha, MT and SLUG expression in PTC may be useful in predicting the risk of LNM and high TNM stage.