Nuclear HIF-1alpha Expression Research Articles

Ets‐1 and hypoxia inducible factor‐1α inhibition by angiotensin II type‐1 receptor blockade in hormone‐refractory prostate cancer

Accumulating evidences have suggested that the renin-angiotensin system (RAS) participates in the regulation of tumor angiogenesis. We previously demonstrated that hormone-refractory prostate cancer (HRPC) showed significantly higher angiotensin II (Ang II) type-1 receptor (AT1R) expression, and that the AT1R blocker (ARB) exerted protective effects by inhibiting angiogenesis. However, the downstream transcriptional factors induced by Ang II in prostate cancer cells have not been fully elucidated yet. Three human prostate cancer cell lines: LNCap, C4-2 and C4-2AT6 were used and analyzed. C4-2AT6 cells were established by culture in androgen-ablated conditioned medium for 6 months. C4-2AT6 cells showed significantly higher AT1R expression, accompanied by higher HIF-1alpha and Ets-1 expression in the nucleus. In C4-2AT6 cells, VEGF production was significantly higher than in C4-2 cells and LNCaP cells. These results suggested that HRPC exhibited aggressive angiogenic properties, accompanied by up-regulated HIF-1alpha and Ets-1. Ang II stimulated VEGF production in C4-2 cells and C4-2AT6 cells but not in LNCaP cells. ARB significantly inhibited VEGF production. Western blot analysis demonstrated that AngII induced nuclear expression of HIF-1alpha and Ets-1 in C4-2 and C4-2AT6 cells, but not in LNCaP cells. ARB significantly inhibited HIF-1alpha and Ets-1 induction in C4-2 and C4-2AT6 cells. This study suggests that AT1R blockade may have a significant impact on HRPC through the inhibition of HIF-1alpha and Ets-1 and the resulting suppression of angiogenesis. Our results provide the molecular basis of the clinical benefit of ARB as an angiogenic inhibitor in HRPC.

Association of hypoxia inducible factor-1 alpha and carbonic anhydrase IX overexpression and survival in HER2/neu positive hormone-unresponsive breast cancer patients

e22078 Background: Hypoxia is common in solid tumors with overexpression of hypoxia inducible factor-1 alpha (HIF-1 alpha) and carbonic anhydrase IX (CA IX). In our study, we evaluated HIF-1alpha and CA IX expression in Her-2/neu positive hormone- unresponsive breast cancer patients. Methods: One hundred and eleven patients with operable breast cancer were identified who had paraffin-embedded tissue blocks available. Estrogen and progesterone receptors and Her-2/neu expression were evaluated by immunohistochemistry (IHC). FISH was used for IHC 2+ cases. We assigned two groups with 56 patients who have ER and PR receptor positive, Her-2/neu negative (group 1) and with 55 patients who have ER and PR negative, Her-2/neu positive (group 2). HIF 1 alpha and CA IX expressions were investigated and results were compared for both groups. Additionally, we investigated the association between HIF 1 alpha and CA IX expressions with stage, grade, lymph node metastasis, tumor dimension and menopause status. Results: Median age of the patients was 52 years (range, 34–77) in group 1 and 47 years (range, 27–83) in group 2. Nuclear HIF-1alpha expression was detected in 26 (46.4%) and 46 patients (83.6%) in group 1 and 2, respectively (p<0.0001). Membranous CA IX expression was detected in 25 (46.4%) and 37 (67.3%) patients in group 1 and 2, respectively (p<0.013). In group 1, median disease free survival (DFS) has not been reached. In group 2, median DFS was 46 months (95%CI: 42.98–49.02) (p= 0.0308). In group 1, median overall survival (OS) was 108 months (95%CI: 90.65–125.35) whereas in group 2, median OS was 75 months (95 % CI: 64.94- 85.06) (p= 0.0339). Conclusions: HIF-1 alpha and CA IX overexpressions were more frequently observed in ER and PR negative, Her-2/neu positive breast cancer. This was associated with shorter disease-free and overall survival. No significant financial relationships to disclose.

Prognostic significance of Hypoxia-Inducible Factor 1 alpha(HIF-1alpha) expression in serous ovarian cancer: an immunohistochemical study

BackgroundThe hypoxia-inducible factor (HIF) has emerged as an attractive target for cancer therapy. The few publications addressing the prognostic significance of Hypoxia-Inducible Factor 1α (HIF-1α) cellular expression in ovarian cancer produced contradictory findings which are not permissible to widely acceptable conclusions and clinical applications. Our study was designed to investigate this by including a comparatively large number of cases and by using a combination of antibodies to analyze immunohistochemically the expression of HIF-1α.MethodsOne hundred (n = 100) neoplastic and 20 benign (controls) pathological samples from paraffin-embedded tissue were included. They were classified after surgery as stage I (n = 23) and stage III G3 (n = 55). Also 22 borderline serous adenocarcinoma patients and 20 benign controls were stained. The mean follow up was 3 years. Only patients with the diagnosis of serous carcinoma of stage III, G3 who received 6 cycles of postoperative TC (175–180 mg/m2 paclitaxel and carboplatin after calculating the area under the concentration curve) with complete medical records (n = 55) were selected for survival analysis. The survival analysis of the samples compared two groups after the patients were dichotomized by HIF-1α final score to positive and negative.ResultsThe frequency of the nuclear expression of HIF-1α in benign tumours was significantly lower (median: no expression) than in borderline and ovarian cancer tumours combined (p < 0.001). HIF-1α expression in serous ovarian carcinoma was not stage dependent. The overall survival of patients with tumours that stained strongly for HIF-1α was significantly shorter than that of patients with tumours that stained weakly or were negative for HIF-1α (p = 0.01). Kaplan-Meier survival curves confirmed that HIF-1α "positive" had decreased overall survival compared to HIF-1α "negative" patients (p = 0.003) and this was an independent adverse prognostic factor (multivariable analysis p = 0.006). HIF-1α "positive" patients displayed a shorter median progress free interval (PFI) (not statistically significant p > 0.05). Interestingly the overall PFI of the subgroup of patients that have undergone suboptimal cytoreduction at primary surgery (n = 21) with tumours that stained strongly for HIF-1α was significantly worse than that of patients with tumours that stained weakly or were negative for HIF-1α (p = 0.03).ConclusionOur report confirms the prognostic value of HIF-1α when restricted to poorly differentiated serous ovarian carcinoma. In addition it shows that this association is elusive, since it is not only methodology-related but it can be antibody-depended. There is adequate evidence to speculate that targeting HIF-1α could improve the long-term prognosis of these patients In order to increase the overall sensitivity of the immunoassay, maintaining acceptable levels of specificity, a panel of antibodies should be used.

Hypoxia-inducible factor 1 alpha expression increases during colorectal carcinogenesis and tumor progression

BackgroundHypoxia-inducible factor 1 alpha (HIF-1α) 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-1α 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.MethodsImmunohistochemistry and Western blot is used to analyse HIF-1α 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-1α inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry.ResultsIn normal mucosa, HPP and TA-LGD HIF-1α was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1α were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1α overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1α expression was strongly accumulated in perinecrotic regions. In these cases HIF-1α activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1α was also seen in periiflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1α expression and nuclear translocation.ConclusionWe conclude that HIF-1α 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-1α in invasive tumors underlines a dual role of HIF-1α by regulating both pro-survival and pro-death processes. HIF-1α up-regulation in response to LPS-mediated stimulation and periinflammatory expression in invasive carcinomas suggest its involvement in inflammatory events. These patterns of HIF-1α inducibility could contribute indirectly to the acquisition of a metastatic phenotype.

Overexpression of hypoxia‐inducible factor‐1α predicts an unfavorable outcome in urothelial carcinoma of the upper urinary tract

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in cell hypoxia adaptation. Overexpression of HIF-1alpha subunit has been reported to be involved in the carcinogenesis, progression and metastasis of many human cancers. We evaluated the clinical significance of HIF-1alpha expression in urothelial carcinoma (UC) of the upper urinary tract. Ninety-eight cases (mean age = 63.5 +/- 11.7, range = 23-84 years) of renal pelvic or ureter UC were included in the present study. Those who had distant metastasis at diagnosis, other cancer, urolithiasis, incomplete clinical information or had received radiotherapy or chemotherapy before surgery were excluded. Nuclear HIF-1alpha expression were evaluated by immunohistochemistry staining on a paraffin-embedded section of the tumor and non-malignant upper urinary tract specimens and scored by two qualified pathologists. Clinical data were collected retrospectively. Positive HIF-1alpha expression was found in 65 (66.3%) of the cancer specimens. No HIF-1alpha expression was found in normal urothelial specimens. Tumor HIF-1alpha expression score was significantly correlated with tumor T stage (P < 0.001), N stage (P < 0.001) and grade (P = 0.004). Tumor necrosis was associated with high tumor T stage (P < 0.001), N stage (P = 0.002) and grade (P < 0.001). Higher HIF-1alpha score (negative vs 3-5 vs 6-7) was a significant predictor for cancer-specific survival (Cox regression hazard ratio = 2.23, P = 0.004), and tumor recurrence (Cox regression hazard ratio = 1.58, P = 0.036). Our findings indicate that HIF-1alpha immunostaining may have an important role in predicting prognosis in upper urinary tract urothelial carcinoma.

Hypoxia-inducible factor 1α is closely linked to an aggressive phenotype in breast cancer

The aim of this study is to clarify characteristics of invasive breast cancer with expression of Hypoxia-induced factor 1alpha (HIF-1alpha) which is induced by hypoxia and signal transduction of growth factors. We examined, by immunohistochemical analysis, the expression of HIF-1alpha in normal breast tissue, benign disorders and breast cancer. In invasive breast cancer, we investigated the correlation between expression of HIF-1alpha and clinicopathological and biological factors. We also studied the prognostic value of HIF-1alpha in breast cancer. HIF-1alpha was mainly detected in tumor cell nuclei. In the 171 cases of invasive breast cancer examined, nuclear HIF-1alpha expression was detected in 63 (36.8%) cases. Immunoreactive nuclear HIF-1alpha was correlated with tumor size (p = 0.0013), lymph node metastasis (p = 0.0005), tumor stage (p = 0.0031) and histological grade (p = 0.0074). Elevated HIF-1alpha levels was also associated with hormone receptor negativity (p = 0.0025), HER2 overexpression (p = 0.0053), high Ki67 labeling index (p = 0.0002), increased levels of VEGF (p < 0.0001), COX-2 overexpression (p = 0.0029) and increased nuclear p53 (p = 0.0048). In terms of the possible use of HIF-1alpha as a prognostic indicator, patients who had increased HIF-1alpha levels in their tumor showed shorter disease-free survival (DFS) (p < 0.0001) and overall survival (OS) (p = 0.0002) than those lacking HIF-1alpha in univariate analysis. In multivariate analysis of DFS and OS, HIF-1alpha was identified an independent prognostic factor. These findings suggest that HIF-1alpha was closely linked to an aggressive phenotype in breast cancer. It may be useful to study the expression of HIF-1alpha using immunohistochemical analysis for better understanding of the tumor characteristics of breast cancer.

Expression of hypoxia-inducible factor 1 α, hypoxia-inducible factor 2 α, and von Hippel–Lindau protein in epithelial ovarian neoplasms and allelic loss of von Hippel-Lindau gene: nuclear expression of hypoxia-inducible factor 1 α is an independent prognostic factor in ovarian carcinoma

The hypoxia-inducible factor (HIF) is a transcriptional factor with important roles in tumor biology. To clarify the possible involvement of the HIF-alpha subunit and von Hippel-Lindau (VHL) protein in the development and progression of ovarian carcinoma, we analyzed the immunohistochemical expressions of HIF-1alpha, HIF-2alpha, and VHL in 107 cases of epithelial ovarian tumors. In addition, we examined loss of heterozygosity (LOH) at VHL gene loci. The frequency of the cytoplasmic expression of HIF-2alpha in carcinomas was higher than that in benign and borderline tumors (P < .0001). Furthermore, the nuclear expression of HIF-1alpha and the cytoplasmic expression of HIF-2alpha were significantly higher in tumors of FIGO (International Federation of Gynecology and Obstetrics) stages III and IV than in those of stages I and II. On the other hand, the cytoplasmic expression of HIF-1alpha did not show differences among histological malignancies. There was a positive correlation between nuclear HIF-1alpha expression and vascular endothelial growth factor (rho = 0.320, P < .001). Although LOH at the VHL gene locus was frequent in ovarian carcinomas (24%), there is no significant correlation between LOH and loss of VHL expression. In 22 clear cell carcinomas, VHL expression showed a significantly negative correlation with the nuclear expression of HIF-1alpha (rho = -0.529, P = .0153). The log-rank test showed that nuclear positive immunostaining for HIF-1alpha (P = .002) and cytoplasmic positive immunostaining for HIF-2alpha (P = .0112) in tumor cells are associated with poor prognosis of patients with ovarian carcinoma. Multivariate analysis also showed that the nuclear expression of HIF-1alpha is an independent prognostic factor. These results show that the HIF-alpha subunit represents an important biomarker in the evaluation of the prognosis of patients with ovarian carcinoma.

Expression pattern of angiogenic factors and prognosis after hepatic resection in hepatocellular carcinoma: importance of angiopoietin‐2 and hypoxia‐induced factor‐1a

Hepatocellular carcinoma (HCC) is a hypervascular tumor and angiogenesis plays an important role in its progression. Angiogenesis is regulated by a balance between pro and antiangiogenic molecules. The aim of this study was to investigate the expressions of angiogenic factors and elucidate their roles in angiogenesis in HCC. We investigated immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), hypoxia-induced factor-1alpha (HIF-1alpha) and thrombospondin-1 (TSP-1) in 60 specimens of surgically resected HCC. We investigated the relationship between their expressions and clinicopathological factors or prognosis. Ang-2 staining had a significant correlation with the grade of differentiation of HCC cells (P=0.0082). VEGF and Ang-2 expression correlated positively with microvessel density (MVD) (P=0.0061 and 0.0374, respectively). MVD of well-differentiated HCC were significantly lower than those of moderately and poorly differentiated HCC. The disease-free survival time of patients with high Ang-2 and/or HIF-1alpha expression was significantly shorter than that of the low expression group (P=0.0278 and 0.0374, respectively). Our study showed that the expression of VEGF and Ang-2 correlated with MVD. Strong Ang-2 expression and/or high nuclear expression of HIF-1alpha is a significant predictive factor for recurrence after curative resection in HCC patients.

Expression of hypoxia-inducible transcription factors in developing human and rat kidneys

Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.

Expression of hypoxia-inducible factor-1 alpha is associated with an unfavorable prognosis in non-small cell lung cancer

7147 Background: Hypoxia-inducible factor (HIF) -1alpha is a transcription factor that mediates adaptive responses of human cells in tissue hypoxia. HIF-1alpha activates various factors supporting tumor growth and progression. HIF-1alpha expression in patients with non-small cell lung cancer (NSCLC), however, has not been well studied. The aim of this study was to determine the expression of HIF-1alpha protein and its relation to clinicopathologic features and prognosis of patients with NSCLC. Methods: We studied retrospectively expression of HIF-1alpha protein in 139 patients with NSCLC by immunohistochemistry. Nuclear expression of HIF-1alpha was measured by observing 1000 cancer cells. The percentage of positive cells was rated as follows: negative expression, cases with <10% positive cells; weak expression, 10–50% positive cells; moderate expression, 50.1–80% positive cells; and strong expression, cases with 80% positive cells. Expression of HIF-1alpha was compared in relation to clinicopathologic findings. Results: HIF-1alpha expression ranged from 0 to 98.8% with a mean value of 41.8%. Forty-two patients (30.2%) showed negative expression, 37 patients (26.6%) showed weak expression, 32 patients (23%) showed moderate expression, and 28 patients (20.1%) showed strong expression. HIF-1alpha expression showed significant correlation with histology, pT status, or pathological stage (p=0.0012, p=0.0097, p=0.008, respectively). Five-year survival rates of negative expression group, weak expression group, moderate expression group and strong expression were 80.2%, 68.4%, 40%, and 20.5%, respectively (p<0.0001, log-rank test). A multivariate analysis of survival time by the stepwise procedure revealed that pathological stage and HIF-1alpha expression were statistically significant prognostic factors (p<0.0001, p=0.0002, respectively). Conclusions: This study shows that HIF-1alpha may be an independent prognostic factor, which can indicate the subsets of patients with poor prognosis, in patients with NSCLC who underwent surgical resection. No significant financial relationships to disclose.

Expression of hypoxia-inducible factor-1 alpha is associated with an unfavorable prognosis in non-small cell lung cancer

7147 Background: Hypoxia-inducible factor (HIF) -1alpha is a transcription factor that mediates adaptive responses of human cells in tissue hypoxia. HIF-1alpha activates various factors supporting tumor growth and progression. HIF-1alpha expression in patients with non-small cell lung cancer (NSCLC), however, has not been well studied. The aim of this study was to determine the expression of HIF-1alpha protein and its relation to clinicopathologic features and prognosis of patients with NSCLC. Methods: We studied retrospectively expression of HIF-1alpha protein in 139 patients with NSCLC by immunohistochemistry. Nuclear expression of HIF-1alpha was measured by observing 1000 cancer cells. The percentage of positive cells was rated as follows: negative expression, cases with <10% positive cells; weak expression, 10–50% positive cells; moderate expression, 50.1–80% positive cells; and strong expression, cases with 80% positive cells. Expression of HIF-1alpha was compared in relation to clinicopathologic findings. Results: HIF-1alpha expression ranged from 0 to 98.8% with a mean value of 41.8%. Forty-two patients (30.2%) showed negative expression, 37 patients (26.6%) showed weak expression, 32 patients (23%) showed moderate expression, and 28 patients (20.1%) showed strong expression. HIF-1alpha expression showed significant correlation with histology, pT status, or pathological stage (p=0.0012, p=0.0097, p=0.008, respectively). Five-year survival rates of negative expression group, weak expression group, moderate expression group and strong expression were 80.2%, 68.4%, 40%, and 20.5%, respectively (p<0.0001, log-rank test). A multivariate analysis of survival time by the stepwise procedure revealed that pathological stage and HIF-1alpha expression were statistically significant prognostic factors (p<0.0001, p=0.0002, respectively). Conclusions: This study shows that HIF-1alpha may be an independent prognostic factor, which can indicate the subsets of patients with poor prognosis, in patients with NSCLC who underwent surgical resection. No significant financial relationships to disclose.

Immunohistochemical study on hypoxiain spontaneous polycystic liver and kidney disease in rats

Hypoxia-inducible factor (HIF) mediates homeostatic responses to hypoxia and activates transcription of hypoxia-inducible genes including vascular endothelial growth factor (VEGF). The aim of this study was to examine the expressions of VEGF, HIF-1alpha and HIF-3alpha in spontaneously occurring hepatorenal polycystic lesions in two Sprague-Dawley (Crj:CD) rats. Hepatic multiple cysts were derived from the interlobular and large bile ducts, while renal cysts were from the collecting ducts and distal tubuli. These findings were confirmed by a lectin peanut agglutinin (PNA) histochemistry. In the polycystic liver, VEGF immunoreaction was strongly evident in the cytoplasm of hepatocytes, whereas expression of HIF-3alpha, but not HIF-1alpha, was found in a few nuclei of hepatocytes. In the polycystic kidney, VEGF immunoreaction was increased in the cytoplasm of collecting ducts and distal tubuli, whereas nuclear expression of HIF-1alpha and HIF-3alpha was evident in the proximal tubuli and thin loop of Henle, respectively. The results suggest that hypoxia-related molecules may be induced by cystic alterations in a heterogeneous appearance.

The Expression and Distribution of the Hypoxia-Inducible Factors HIF-1α and HIF-2α in Normal Human Tissues, Cancers, and Tumor-Associated Macrophages

The cellular response to hypoxia includes the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in diverse processes such as glycolysis and angiogenesis. Induction of the HIF-regulated genes, as a consequence of the microenvironment or genetic changes, is known to have an important role in the growth of experimental tumors. Hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) are known to dimerize with the aryl hydrocarbon receptor nuclear translocator in mediating this response. Because regulation of the alpha chain protein level is a primary determinant of HIF activity, our aim was to investigate the distribution of HIF-1alpha and HIF-2alpha by immunohistochemistry in normal and pathological tissues using monoclonal antibodies (mAb). We raised a new mAb to detect HIF-1alpha, designated 122, and used our previously validated mAb 190b to HIF-2alpha. In the majority of solid tumors examined, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas, nuclear expression of HIF-1alpha and -2alpha was observed in varying subsets of the tumor cells. HIF-2alpha was also strongly expressed by subsets of tumor-associated macrophages, sometimes in the absence of any tumor cell expression. Less frequently staining was observed in other stromal cells within the tumors and in normal tissue adjacent to tumor margins. In contrast, in normal tissue neither molecule was detectable except within subsets of bone marrow macrophages, where HIF-2alpha was strongly expressed.