Expression of hypoxia-inducible factor-1alpha is associated with tumor vascularization in human colorectal carcinoma

HIF-1 is reported to transactivate expression of VEGF, which is an important angiogenic factor. To determine whether HIF-1alpha plays a role in angiogenesis through its regulation of VEGF, we examined expression of HIF-1alpha and its relation to clinicopathologic features, VEGF expression and prognosis of patients with colorectal carcinoma. Expression of HIF-1alpha and VEGF was examined in 4 colorectal carcinoma cell lines (COLO320DM, COLO201, DLD-1, WiDr) and 149 colorectal carcinoma tissues (10 fresh specimens, 139 archival, paraffin-embedded specimens). HIF-1alpha protein levels were increased by hypoxia in 3 of 4 colorectal carcinoma cell lines (COLO201, DLD-1, WiDr), and VEGF mRNA levels were also increased by hypoxia in the same cell lines. In 8 of 10 patients with colorectal cancer, expression of HIF-1alpha and VEGF was increased in tumor tissues compared to corresponding normal mucosa. Of 139 archival specimens of colorectal carcinoma, 81 (58.3%) expressed HIF-1alpha protein at a high level. HIF-1alpha expression was correlated with tumor invasion, tumor stage, lymphatic invasion, venous invasion and liver metastasis. Moreover, HIF-1alpha expression was correlated significantly with VEGF expression and microvessel density. Although there was a tendency for poorer prognosis in patients with high HIF-1alpha-expressing tumors, this correlation was not statistically significant. These findings suggest that HIF-1alpha may play a role in angiogenesis and tumor progression via regulation of VEGF in human colorectal carcinoma.     

PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells

Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer.     

Clinicopathologic significance of hypoxia-inducible factor 1alpha overexpression in gastric carcinomas

BACKGROUND: Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a key role in responses to hypoxia and expression of HIF-1alpha downstream genes leads to both an adapted metabolism and increased oxygen supply. We investigated the clinical significance of HIF-1alpha expression in gastric carcinoma. METHODS: We examined HIF-1alpha, vascular endothelial growth factor (VEGF), and insulin-like growth factor-2 (IGF-2) expression patterns immunohistochemically in 126 specimens of gastric carcinoma. CD34 antigen levels were also examined by immunohistochemistry to determine microvessel density (MVD) within tumors and correlations between HIF-1alpha expression, clinicopathological features, and survival were examined. RESULTS: HIF-1alpha expression correlated with tumor size (P<0.005), depth of invasion (P=0.018), VEGF expression (P=0.03), and intra-tumor MVD (P<0.005). IGF-2 expression was more prevalent in HIF-1alpha positive than in HIF-1alpha negative tumors and the 5-year survival rate was 58.4% for HIF-1alpha positive patients and 81.5% for HIF-1alpha negative patients (P=0.009). HIF-1alpha expression is an independent prognostic factor in gastric carcinoma (P=0.032). CONCLUSIONS: Overexpression of HIF-1alpha in gastric carcinomas may upregulate its downstream gene products leading to VEGF-mediated angiogenesis, and resulting in a poor prognosis for patients.     

Sulforaphane inhibited expression of hypoxia-inducible factor-1alpha in human tongue squamous cancer cells and prostate cancer cells

Previous studies show that a number of natural compounds from our diet have anticancer effects. Sulforaphane is the most characterized isothiocyanates (ITCs), which are identified in cruciferous vegetables. Sulforaphane is viewed as a conceptually promising agent in cancer prevention. Because of its ability to induce cancer cell apoptosis, it inhibits progression of benign tumors to malignant tumors and interrupts metastasis. However, the effect of sulforaphane on tongue cancer cell proliferation has not yet been reported, and the mechanisms that sulforaphane inhibits cancer development are still unclear. Hypoxia-inducible factor 1 (HIF-1) expression is associated with tumorigenesis and angiogenesis. It regulates the expression of many genes including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, and lactate dehydrogenase A. In our study, we investigated the effects of sulforaphane on expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was overexpressed in many human malignant tumors, human tongue squamous cell carcinoma and prostate cancer DU145 cells. Sulforaphane inhibited hypoxia induced expression of HIF-1alpha via inhibiting synthesis of HIF-1alpha. Sulforaphane was also found to inhibit hypoxia induced HIF-1alpha expression through activating JNK and ERK signaling pathways, but not AKT pathway. Inhibition of HIF-1alpha by sulforaphane resulted in decreasing expression of VEGF. Taken together, these results suggest that sulforaphane is an effective chemopreventive compound against tongue cancers and prostate cell angiogenesis in vitro, and that the HIF-1alpha target provides a new sight into the mechanisms of sulforaphane's inhibition against tumor cell proliferation.     

Hypoxia inducible factor-1alpha is a prognostic marker in premenopausal patients with intermediate to highly differentiated breast cancer but not a predictive marker for tamoxifen response

Hypoxia is common in many solid tumours, including breast cancer. Hypoxia triggers the expression of hypoxia inducible factor-1alpha (HIF-1alpha), and HIF-1alpha has been associated with an impaired prognosis in breast cancer and down-regulation of the oestrogen receptor (ER), potentially affecting the treatment efficiency of antioestrogens. The role of HIF-1alpha regarding prognostic and treatment predictive information in breast cancer has not been established and we therefore analyzed HIF-1alpha using immunohistochemistry in a cohort of 377 premenopausal stage II breast cancers arranged in a tissue microarray. The patients were included in a randomized trial with either 2 years of tamoxifen or no adjuvant treatment. The tamoxifen treatment effect could be studied in subgroups of breast cancer and pure prognostic information could be scrutinized for untreated control patients. HIF-1alpha was scored as positive in 24% of the tumours and correlated positively to tumour size, Nottingham histological grade (NHG), Ki-67, Her2 and cyclin E expression and negatively to lymph node status, cyclin D1, ER and PR (progesterone receptor) expression. Surprisingly, there was no difference in tamoxifen response for patients with high or low HIF-1alpha expressing tumours. In lymph node-positive patients as well as NHG 1/2 tumours, high HIF-1alpha protein expression was significantly associated with an impaired recurrence-free survival (p=0.014, 0.018). When analyzing the subgroup of NHG 1/2 tumours, a high HIF-1alpha expression was the only independent significant prognostic marker in multivariate analysis, including standard prognostic markers, suggesting that HIF-1alpha might be a useful prognostic marker in this subgroup of breast cancer, with a rather good but diverse prognosis.     

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

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

Hypoxia-inducible factor-1 alpha in non small cell lung cancer: relation to growth factor, protease and apoptosis pathways

Hypoxia-inducible factor (HIF)-1 alpha is the regulatory subunit of HIF-1 that is stabilized under hypoxic conditions. Under different circumstances, HIF-1 alpha may promote both tumorigenesis and apoptosis. There is conflicting data on the importance of HIF-1 alpha as a prognostic factor. This study evaluated HIF-1 alpha expression in 172 consecutive patients with stage I-IIIA non small cell lung cancer (NSCLC) using standard immunohistochemical techniques. The extent of HIF-1 alpha nuclear immunostaining was determined using light microscopy and the results were analyzed using the median (5%) as a low cut-point and 60% as a high positive cut-point. Using the low cut-point, positive associations were found with epidermal growth factor receptor (EGFR; p = 0.01), matrix metalloproteinase (MMP)-9 (p = 0.003), membranous (p < 0.001) and perinuclear (p = 0.004) carbonic anhydrase (CA) IX, p53 (p = 0.008), T-stage (p = 0.042), tumor necrosis (TN; p < 0.001) and squamous histology (p < 0.001). No significant association was found with Bcl-2 or either N- or overall TMN stage or prognosis. When the high positive cut-point was used, HIF-1 alpha was associated with a poor prognosis (p = 0.034). In conclusion, the associations with EGFR, MMP-9, p53 and CA IX suggest that these factors may either regulate or be regulated by HIF-1 alpha. The association with TN and squamous-type histology, which is relatively more necrotic than other NSCLC types, reflects the role of hypoxia in the regulation of HIF-1 alpha. The prognostic data may reflect a change in the behavior of HIF-1 alpha in increasingly hypoxic environments.     

HIF-1alpha and CA IX staining in invasive breast carcinomas: prognosis and treatment outcome

Hypoxia stabilizes HIF-1alpha (Hypoxia Inducible Factor-1alpha), which then triggers the expression of several genes involved in many aspects of cancer progression, including metabolic adaptation, cell survival and angiogenesis. The aim of our study was to evaluate the impact of HIF-1alpha and CA IX (carbonic anhydrase IX) (one of its target genes) expression on prognosis and treatment outcome of patients with breast cancer. Because of the extreme O2-dependent instability of the protein, we first validated HIF-1alpha staining using xenograft tumours that were subjected to experimental conditions mimicking surgical clamping or sitting at room temperature under normoxic conditions after surgical excision but before fixation. Afterwards, the immunohistochemical staining of HIF-1alpha and CA IX was evaluated in 132 invasive breast carcinomas with a 10-year follow-up, and correlated to classical clinicopathological parameters and response to adjuvant therapy. No significant correlation was found between tumour size or nodal status and the expression of HIF-1alpha or CA IX. Statistically significant association was found between HIF-1alpha or CA IX staining and the grade, hormonal receptors loss and the presence of carcinoma in situ. Overexpression of HIF-1alpha and CA IX correlates with a poor prognosis in breast cancer. We show that HIF-1alpha is an independent prognostic factor for distant metastasis-free survival and disease-free survival in multivariate analysis. Furthermore, overexpression of HIF-1alpha or CA IX correlates with a poor outcome after conventional adjuvant therapy. CA IX is, however, a weaker prognostic and predictive factor than HIF-1alpha, and its association with HIF-1alpha does not modify the survival curve neither response to therapy, compared to HIF-1alpha alone.     

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

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

Transforming growth factor-beta and Ras regulate the VEGF/VEGF-receptor system during tumor angiogenesis.

The formation of new microvasculature by capillary sprouting, or angiogenesis, is a prerequisite for solid tumor growth. The genetic alterations required to activate the angiogenic program in tumor angiogenesis are still only vaguely known, but dominantly acting oncoproteins may have a much greater impact than previously realized. Here we have studied the consequences of oncogenic transformation on tumor angiogenesis in a mouse mammary carcinoma model. We provide evidence that the expression of vascular endothelial growth factor (VEGF) and of the VEGF receptor-2 (Flk-1), a signaling system centrally involved in tumor angiogenesis, occurs efficiently in tumors formed by Ras-transformed mammary epithelial cells and that both TGF-beta1 and hypoxia are potent inducers of VEGF expression in these cells. VEGF induction in the tumor periphery is mainly triggered by TGF-beta1, whereas VEGF expression in perinecrotic areas is regulated by both hypoxia and TGF-beta1. As the Ras-transformed tumor cells convert into migrating, fibroblastoid cells that start to produce TGF-beta during tumor progression, the TGF-beta effect on VEGF expression becomes propagated throughout the tumor tissue. Thus, in progressed tumors, areas of TGF-beta1 activation and hypoxia may overlap and hence cooperate to induce VEGF expression and angiogenesis. Nevertheless, the overexpression of VEGF in non-Ras-transformed mouse mammary epithelial cells was not sufficient to promote vascularization in vivo. Based on these findings, we conclude that amongst the multiple mutations that render a normal cell tumorigenic, oncogenic Ras is a major player that in conjunction with the tumor's micro-environment sets the stage for tumor cell invasion and angiogenesis.