aHIF but not HIF-1alpha transcript is a poor prognostic marker in human breast cancer.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is part of a transcriptional factor that regulates genes involved in metabolic and vascular adaptation of tumours to oxygen restriction. A splicing variant lacking exon 14 (sHIF-1alpha) encodes a truncated protein that competes with the normal HIF-1alpha protein, decreasing its activity. A natural antisense transcript (aHIF) complementary to the 3'-untranslated region of HIF-1alpha mRNA was described recently. With a semiquantitative multiplex reverse transcriptase-PCR (RT-PCR) assay, we assessed transcript concentrations of HIF-1alpha, sHIF-1alpha and aHIF in 110 patients with invasive breast carcinoma. We found a strong positive association between HIF-1alpha and sHIF-1alpha, sHIF-1alpha and aHIF, and an inverse correlation between HIF-1alpha /sHIF-1alpha and aHIF. aHIF transcript expression was associated with poor disease-free survival in univariate (P = 0.0038) and multivariate (P = 0.0016) analyses in this series of high-risk primary breast carcinomas. In our series of breast cancer patients, aHIF, and not HIF-1alpha transcript, is a marker of poor prognosis.

Cayre A ，Rossignol F ，Clottes E ，Penault-Llorca F ... -  《-》

Natural antisense transcripts of HIF-1alpha are conserved in rodents.

A natural antisense transcript (aHIF), which sequence is strictly complementary to the 3' untranslated region (3'UTR) of HIF-1alpha mRNA, has been identified in human and shown to be overexpressed in renal carcinomas. We searched for aHIF in different rodent tissues. Two candidate expressed sequence tag (EST) were identified in silico and their PCR products (1.1 and 1.0 kb) were cloned and sequenced in mouse and rat, respectively. These transcripts were rigorously complementary to the 3'UTR of rodent HIF-1alpha mRNA and were broadly expressed in all mouse and rat tissues we tested. The conservation of aHIF in rodents underlined its potential importance in cell regulations. Therefore the responses of aHIF and HIF-1alpha transcripts were investigated in various types of hypoxic conditions. In freshly isolated rat renal tubules, aHIF RNA level was increased by acute hypoxia and low in normal supply of oxygen. In a rat strain raised in chronic hypobaric altitude hypoxia, aHIF transcript was greatly induced in the oxidative-type soleus and heart muscles of 3 month-old animals. By contrast, in the glycolytic-type extensor digitorum longus muscle aHIF transcript amount was lowered by hypoxia whereas HIF-1alpha transcript was highly expressed. In brain, where oxidative glycolysis takes place, HIF-1alpha mRNA and its antisense transcript levels were high and not significantly changed by altitude. Tumour cell lines cultured for 6 h in conditions mimicking hypoxia expressed lower amounts of HIF-1alpha mRNA. In two rat cell lines, aHIF transcript levels were greatly augmented after a 6-h incubation in these conditions, whereas in a mouse cell line, aHIF level was significantly reduced.

Rossignol F ，de Laplanche E ，Mounier R ，Bonnefont J ，Cayre A ，Godinot C ，Simonnet H ，Clottes E ... -  《GENE》

Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in tumor growth and metastasis by regulating energy metabolism and inducing angiogenesis to survive cellular hypoxia. Increased levels of HIF-1alpha, the O(2)-regulated subunit of HIF-1, were noted during breast carcinogenesis. In this study, the prognostic value of HIF-1alpha expression and its correlation with various clinicopathologic variables in patients with invasive breast carcinoma were investigated. Expression levels of HIF-1alpha, HER-2/neu, estrogen receptor, and progesterone receptor were analyzed in 150 patients with early-stage breast carcinoma by immunohistochemistry. HER-2/neu gene amplification was investigated with automated fluorescent in situ hybridization. The mitotic activity index, histologic grade, and tumor type were assessed in hematoxylin and eosinstained specimens. Clinical data included disease-free survival, overall survival, lymph node status, and tumor size. The data were analyzed with two-sided univariate and multivariate tests, with P values < 0.05 considered significant. High levels of HIF-1alpha had an association of borderline significance with decreased overall survival (P = 0.059) and disease-free survival (P = 0.110) that was ascribed completely to the subgroup of women with lymph node negative tumors (n = 81 patients; P = 0.008 and P = 0.004, respectively). HER-2/neu immunoreactivity (P < 0.001) and gene amplification (P < 0.001), vascular endothelial growth factor expression (P = 0.016), and Ki-67 expression (P < 0.001) were correlated strongly with HIF-1alpha positivity, although none of those factors had an independent effect on survival. Increased levels of HIF-1alpha were associated independently with shortened survival in patients with lymph node negative breast carcinoma. Therefore, the use of immunohistochemical assessment of HIF-1alpha as a new predictor of poor outcome may improve clinical decision-making regarding adjuvant treatment of patients with lymph node negative breast carcinoma.

Bos R ，van der Groep P ，Greijer AE ，Shvarts A ，Meijer S ，Pinedo HM ，Semenza GL ，van Diest PJ ，van der Wall E ... -  《CANCER》

Comparative expression analysis of hypoxia-inducible factor-alpha and its natural occurring antisense in breast cancer tissues and adjacent noncancerous tissues.

Hypoxia-inducible factors (HIFs) have been shown to be upregulated in tumor tissues and linked with tumor progression and metastasis in breast cancer. Among regulatory mechanisms for HIF expression is a natural occurring antisense named aHIF, which has been shown to be overexpressed in breast cancer and influence the level of the HIF-1α transcript. In the present study, we analyzed the expression of HIF-1α and aHIF in breast cancer tissues versus adjacent noncancer tissues (ANCTs) in relation with the clinical and biological behavior of the tumors. aHIF has been shown to be expressed in 67.4% of invasive ductal carcinoma samples, while none of ANCTs showed its expression. HIF-1α has been expressed in all of tumors and 90% of ANCTs. Comparison of HIF-1α expression level between tumor and ANCT tissues showed a total upregulation in tumor samples. No statistically significant association has been found between the level of HIF-1α expression in tumor samples and clinicopathologic and demographic characteristics such as age, tumor size, estrogen receptor status, progesterone receptor status, HER2/neu expression level, lymph node status, histological grade, and stage except for a weak correlation between HIF-1α expression and Ki-67 status. Besides, we could not detect any significant correlation between relative expression of HIF-1α and aHIF in tumor samples. Collectively, these data suggest that aHIF overexpression can be used as a potential biomarker in breast cancer. However, further studies are needed for the evaluation of its mechanism of action in regulation of HIF-1α expression in different pathological conditions. HIF-1α overexpression results in the upregulation of several genes that participated in cancer-associated pathways such as proliferation, angiogenesis, and glucose metabolism. We showed that HIF-1α is upregulated in breast tumor samples compared with adjacent noncancerous tissues. Its expression has been associated with Ki-67 status. Its natural occurring antisense is only expressed in tumor tissues. Thus, it can be used as a potential biomarker in breast cancer.

Tasharrofi B ，Soudyab M ，Nikpayam E ，Iranpour M ，Mirfakhraie R ，Sarrafzadeh S ，Geranpayeh L ，Azargashb E ，Sayad A ，Ghafouri-Fard S ... -  《-》

Natural antisense transcripts of hypoxia-inducible factor 1alpha are detected in different normal and tumour human tissues.

Hypoxia-inducible factor 1 is a heterodimeric transcription factor, made up of two subunits called HIF-1alpha and aryl receptor nuclear translocator, that regulates the expression of genes associated with adaptation to reduced oxygen pressure. The HIF-1alpha messenger RNA (mRNA) and protein are both up-regulated in common human cancers where this transcription factor is known to be involved in tumour progression. In renal carcinoma, a natural antisense of HIF-1alpha transcript (aHIF) that is complementary to the 3'untranslated region of HIF-1alpha mRNA has been described. Here, we provide a grown work for further characterisation of this natural antisense and we show that: (1) aHIF is widely expressed in normal foetal and adult human tissues as in tumour tissues. Foetal aHIF expression level is higher than adult one and high enough to affect the HIF-1alpha mRNA/aHIF transcripts ratio; (2) aHIF could expose AU rich elements present in the 3' untranslated region of HIF-1alpha mRNA and thus possibly increase the degradation speed of HIF-1alpha mRNA; and (3) aHIF promoter possesses several putative hypoxia response elements which could explain the overexpression of aHIF under hypoxic conditions, creating a negative loop of regulation of HIF-1alpha expression.

Rossignol F ，Vaché C ，Clottes E 《GENE》