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Hypoxia inducible factor 1α gene (HIF-1α) splice variants: potential prognostic biomarkers in breast cancer
- Jean-Philippe Dales†1, 2Email author,
- Nathalie Beaufils†3,
- Monique Silvy4,
- Christophe Picard4,
- Vanessa Pauly5,
- Vincent Pradel5,
- Christine Formisano-Tréziny1,
- Pascal Bonnier6,
- Sophie Giusiano2,
- Colette Charpin2 and
- Jean Gabert1, 3
© Dales et al; licensee BioMed Central Ltd. 2010
Received: 26 May 2010
Accepted: 12 July 2010
Published: 12 July 2010
Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known.
Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival.
HIF-1α isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1α TAG ) and HIF-1α 736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1α TAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1α TAG (P = 0.048), and total HIF-1α (P = 0.048) mRNA levels. HIF-1α TAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1α TAG mRNA levels correlated with shortened metastasis free survival (P = 0.01).
Our results show for the first time that mRNA expression of a HIF-1α TAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.
See commentary: http://0-www.biomedcentral.com.brum.beds.ac.uk/1741-7015/8/45
Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed master transcriptional regulator of many genes regulating mammalian oxygen homeostasis [1, 2]. HIF-1 alters transcription of genes mainly involved in energy metabolism, neovascularisation, survival, internal pH and cell migration, and has become recognised as a strong promoter of tumour growth [3, 4]. HIF-1 is a heterodimeric protein composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits . These subunits are members of the basic helix-loop-helix (bHLH) transcription factor superfamily containing a PAS [PER (Period Clock) -ARNT-SIM (Single-minded)] domain . The HIF-1α subunit is specific to HIF-1 and is the unique oxygen-regulated subunit that determines HIF-1 activity [6, 7] whereas HIF-1β is a common subunit for several transcription factors. In human tumours, HIF-1α protein is overexpressed because of intratumoural hypoxia and genetic alterations affecting key oncogenes and tumour suppressor genes [8, 9]. This overexpression correlates with tumour angiogenesis, treatment failure and patient mortality .
Our group and others have previously reported that HIF-1α protein overexpression is a marker of poor prognosis in primary breast cancer patients [10, 11]. HIF-1α is known to be mainly post-transcriptionally regulated by protein ubiquitination and interaction with the Von Hippel-Lindau tumour suppressor protein, and then degraded by the proteasome. Hypoxia stabilises HIF-1α protein by relaxing its ubiquitin-proteasome degradation  and affects subcellular localisation, DNA binding capacity and transcriptional activation function of the HIF-1 complex. HIF-1α protein is composed of 826 amino acids . Its N-terminal region contains the bHLH and the PAS domains, which are essential for DNA binding and dimerisation . Its C-terminal region contains two transactivation domains and a nuclear localisation signal . In the middle section of HIF-1α lies a Pro-Ser-Thr oxygen-dependent degradation domain (ODDD, amino acids 401 to 603), which is responsible for the stability of the HIF-1α protein .
Although HIF-1α protein regulation has been well studied, HIF-1α messenger regulation has only recently begun to be documented . HIF-1α is encoded by the HIF1A locus, which is located on human chromosome 14q21-q24 and encodes for 15 exons, resulting in a principal transcript of about 8 kb . Different functional domains of the HIF-1α protein are encoded by separate exons. In addition to the originally described wild type of HIF-1α (HIF-1αWT) , eight alternative splice variants of HIF-1α (HIF-1α827, HIF-1α736, HIF-1α557, HIF-1α516, HIF-1α785, HIF-1α417, HIF-1αTE and an isoform with alternative exon 1 (I.2 isoform)) with different activity have been reported in human cell lines [17–23]. All HIF-1α splice variants have been shown to be translated, to dimerise with HIF-1β and to be active in human cells. Only one isoform (HIF-1α 736 ) has been documented in human breast cancer . The aim of this study was to analyse HIF-1α splice variant expression in human breast cancer and to evaluate its clinical relevance in terms of prognosis. In this study we quantified the mRNA levels of 3 isoforms (HIF-1α 736 , HIF-1α 557 and HIF-1α 516 ) in breast specimens of 53 primary cancers and 29 normal tissues or benign lesions by real-time quantitative reverse transcription PCR (RT-qPCR) assays. HIF-1α 827 is similar to wild type except for an additional three base pairs TAG insertion at the exon 1 to 2 junction involving a difference of two amino acids upstream of the bHLH domain . HIF-1α 736 is also characterised by an additional three base pairs TAG insertion at the exon 1 to 2 junction, and the lack of exon 14, which produces a frame shift and introduces a stop codon in the coding sequence after the Ile735 . HIF-1α 557 loses exon 12 and HIF-1α 516 lacks exons 11 and 12 [18, 19]. HIF-1α 557 has been shown to be induced by zinc ions. HIF-1α 557 and HIF-1α 516 have been shown to function as dominant negative isoforms of HIF-1α in vitro. HIF-1α 785 is deprived of exon 11 and was described as being induced in vitro by phorbol-12-myristate-13-acetate . HIF-1α 417 isoform is deprived of exon 10 . The HIF-1α TE variant with alternative exon 1 (I.1 isoform) was previously found to be expressed in the testis . For these reasons, both these isoforms were thought to be out of the scope of this study on breast tissues samples. We also developed tools to quantify expression of HIF-1α splice transcripts characterised by insertion of a three base pairs TAG at the exon 1 to 2 junction (which we designated HIF-1α TAG ) involving a difference of two amino acids upstream of the bHLH domain (HIF-1α 827 and HIF-1α 736 isoforms) . The last known isoform was not quantified because it was described in humans after the end of our study . Expression levels of these isoforms were also compared to standard prognostic clinicopathological parameters, microvessel density of tumours and clinical outcome.
Patients and breast tissue samples
A series of 29 normal tissues or benign lesions (normal/benign) of breast tissue and 53 primary invasive breast carcinomas tissue specimens were selected. All frozen tissues were available in the tumour library of the Department of Pathology (Hôpital Nord, Assitance Publique-Hôpitaux de Marseille, France). Each patient gave informed consent and the study was approved by our institutional review board. Within 15 min of excision fresh tissue samples were frozen in liquid nitrogen by the pathologist (freezing delay shorter than 15 min). All tissue specimens were then stored at -80°C and were never thawed (freezers under temperature control 24 h a day). Specimens of normal breast tissues were obtained from adjacent tumour-free tissue taken from 12 breast cancer patients. Benign lesions were obtained from women undergoing surgery for fibroadenoma (n = 13) or adenosis (n = 4). Normal/benign tissues were obtained from 29 patients aged from 12 to 79 years (mean 44.7 years, SD 16.9). Malignant tumour specimens represented 53 early invasive breast adenocarcinomas diagnosed between 9 September 1989 and 1 May 1996. Tumour tissue specimens were obtained from patients aged from 30 to 84 years (mean 54 years, SD 11.4). Surgery was in all cases the first treatment. The patients underwent axillary node excision combined with wide local excision with margin clearance or mastectomy according to current European recommendations in the department of Oncologic Gynaecology in Hôpital de la Conception, Marseille. For this first step of treatment, patient management was handled by the same group of surgeons (PB). Likewise, radiotherapy, chemotherapy and hormone therapy were applied according to criteria currently used at that time. Each of the respective areas was identified microscopically and dissected immediately upon receipt of the breast specimen by the pathologist. The tumour content of each specimen was verified by histological examination of frozen section by haematoxylin staining. Histological examination of surgical specimens was also performed on paraffin embedded sections stained with haematoxylin, eosin and saffronin. Tumour grading, initially assessed by using the grading methods of Bloom and Richardson , was re-evaluated according to Elston and Ellis . Duration of follow-up ranged from 9 to 162 months (mean 80 months, SD 43). For statistical purposes, we selected approximately equal numbers of long (n = 31) and short metastasis-free survival patients (n = 22).
Clinical and pathological parameters
Lymph node status, tumour size, peritumoural vascular invasion, histological type and histological grade of tumours were obtained from pathological reports. The oestrogen receptor (OR) and progesterone receptor (PgR) status was determined by immunohistochemistry on paraffin-embedded sections as previously described [27, 28].
Tumour microvascular density
Immunodetection studies were performed on frozen sections 5 ?m thick and automated immunoperoxidase procedures as previously reported .
RNA extraction and reverse transcription
Homogenising Trizol procedures have been performed in accordance with current protocols. Frozen tissue (25 to 50 mg) was homogenised in 0.5 ml Trizol reagent (Life Technologies, Carlsbad, California) with lysing matrix D in a FastPrep machine (Q-BIOgene, MP Biomedicals, Illkirch, France) for six cycles of 30 s at a setting of 6 m/s as recommended by the manufacturer. Chloroform (100 μl) was added to each sample, which were mixed vigorously for 15 s and incubated for 3 min at room temperature. Phases were separated by centrifugation (12,000 g for 15 min at 4°C) and the aqueous phase was recovered. Isopropanol (250 μl) was added to each of the samples, which were then mixed by inversion and incubated for 10 min at room temperature. Total RNA was pelleted by centrifugation (12,000 g for 10 min at 4°C), washed with 1 ml of ice-cold 75% ethanol, air dried and resuspended in 15 μl Rnase-free distilled water. Then, 1 μg of RNA was reverse transcribed with 200 U MMLV Reverse Transcriptase following the Europe Against Cancer (EAC) protocol .
Generation of standard plasmids
pcDNA3 plasmids containing HIF-1α 827 and HIF-1α 736 sequences with the three base pairs TAG insertion were kindly provided by J Pouyssegur (Institut de Signalisation, Biologie du Développement et Cancer, Centre Lacassagne, Nice, France). For other splice variants, cDNA from human embryonic kidney (HEK)293 cells was amplified using the following primers, respectively localised in exons 1 and 2: forward, 5'-CCGGCGGCGCGAACGACAAG-3', reverse, 5'-TGCGAACTCACATTATGTGG-3', or primers localised in exons 10 and 14, respectively: forward, 5'-TGACCCTGCACTCAATCAAG-3', reverse, 5'-AGTAGCTGCATGTACGTCTG-3'. Conditions for PCR amplification were: 10 min denaturation at 95°C followed by 35 cycles of 30 s at 95°C, 1 min at 55°C, 1 min at 72°C, and the last elongation at 72°C for 10 min. If necessary, DNA bands of interest were isolated and purified from 2% agarose gel and cloned into the pCRII TOPO vector (TOPO-TA cloning kit, Invitrogen, Life Technologies, Carlsbad, California) according to the manufacturers. Plasmids were purified (Nucleospin plasmid, Macherey Nagel, Hoerdt, France) and the cloned inserts were sequenced. Serial dilutions were prepared in Escherichia coli rRNA (Roche Diagnostics, Meylan, France) to generate RT-qPCR standard curves (PCT international application no. 02/00937 of 15 March 2002, in the name of Université de la Méditerranée) according to the EAC strategy .
Optimisation of RT-qPCR
Details of oligonucleotide sequences of primers and probes used to quantify hypoxia inducible factor 1α (HIF-1α) mRNA splice variants by real-time quantitative reverse transcription PCR assays
Sequence (5' to 3')
Primer/probe concentrations (nM)
5 to 6 junction
13 to 15 junction
11 to 13 junction
10 to 13 junction
Amplifications were performed with 5 μl of cDNA of each sample using RT-qPCR optimised conditions as previously described. mRNA from HEK293 cells was used as positive control throughout the study. Standard (106 copies of plasmid) specifics of another splice variant were amplified as negative control throughout the study and NTC was performed. Standard curves were generated by serial dilutions of HIF-1α splice variant plasmids ranging from 1 to 106 copies and used for calculation of copy numbers (CN) for each transcript. Transcripts of the gene coding for the TATA box-binding protein (TPB) were also quantified as endogenous gene controls, and each sample was normalised on the basis of its TBP content as previously described . All experiments were carried out with duplicates for each data point. For each experiment sample, the mRNA copy number of HIF-1α gene (CN HIF-1α ) and endogenous reference TBP gene (CN TBP ) were quantified from standard curves. Final HIF-1α mRNA concentrations were expressed in normalised copy numbers (NCN HIF-1α ), as follows: NCN HIF-1α = CN HIF-1α /CN TBP .
Differences among groups of breast tissue specimens (normal/benign tissues vs carcinomas) in terms of their HIF-1α mRNA levels were assessed using Kruskal-Wallis and Mann-Whitney tests with Bonferroni correction. Since levels of expression in breast cancer specimens showed non-Gaussian distribution, non-parametric tests were used for the analysis of correlation with clinicopathological parameters. Metastasis-free survival (MFS) time, defined as the time from surgery until diagnosis of metastasis, was used as a follow-up endpoint. Regression models (univariate and multivariate) were used to compare survival and identify predictors of survival. All preoperative predictors (clinicopathological and biological parameters) were included in the analysis. The predictors with P value < 0.20 in the univariate model were tested in the multivariate regression model. Then backward conditional method was used for variable selection by the Cox multivariate regression model. Finally, variables with adjusted P values < 0.05 were kept into the final model. All statistical analyses were performed with SPSS V.13.0.1 software (SPSS, Chicago, IL, USA).
Distribution of clinical and histopathological characteristics of breast cancer patients
No. of patients
No. of patients with metastasis
Positive lymph node:
1 to 3
4 to 10
Pathological tumour size, mm:
11 to 20
21 to 50
Peritumoural vascular invasion:
Optimisation and validation of RT-PCR assays
Primers and probes sets for HIF-1α variants were tested using RT-qPCR on the HEK293 RNA and specific standard plasmids. Primer concentration optimisation assays were carried out to determine the optimal concentrations for each primer and probe set. To test the specificity and crossreaction of the primers used for RT-qPCR, experiments were performed using genomic DNA, standard plasmids specific of another splice variant, and nNTC. No amplification was observed with genomic DNA of PBMCs, specific standard plasmids of other splice variants and NTC. Serial dilutions of HIF-1α variants specific standard plasmid were used to generate a standard curve to assess the intra-assay and interassay reproducibility and sensitivity. For each variant, excellent reproducibility and linearity of standard curves were found. All runs exhibited amplification slopes between -3.31 and -3.55, which correspond to efficiencies higher than 92% and coefficients of correlation between 0.993 and 1. The assays sensitivity was found at 10 copies for all variants. We did not succeed in quantifying the HIF-1α 417 isoform.
Differential expression of HIF-1αsplice variants in normal/benign and malignant breast tissues
HIF-1αsplice variants expression correlates with prognostic clinicopathological parameters of breast cancer
Relationship between the hypoxia inducible factor 1α (HIF-1α) splice variant's expression levels (normalised copy numbers) and clinicopathological factors or microvessel density in tumour tissue specimens from 53 breast cancer patients
HIF-1α TAG ,
HIF-1α 736 ,
Lymph node status:
Pathological tumour size:
< 20 mm
≥ 20 mm
Peritumoural vascular invasion:
Tumour microvessel density (low vs high)
Expression of HIF-1α TAG splice variant correlates with patient survival
Univariate and multivariate (backward conditional selection) Cox regression analysis of metastasis-free survival (MFS) in breast cancer patients considering clinicopathological characteristics, tumour microvessel density and hypoxia inducible factor 1α (HIF-1α) TAG mRNA levels
Univariate analysis of MFS,
Exp B (95% CI)
Multivariate analysis of MFS,
Exp B (95% CI)
1.32 (0.52 to 3.37)
Positive lymph node:
1 to 3
8.00 (2.56 to 24.81)
5.56 (1.75 to 17.63)
4 to 9
7.50 (1.70 to 30.00)
6.68 (1.48 to 30.14)
1.20 (0.14 to 10.00)
0.48 (0.05 to 4.25)
Pathological tumour size, mm:
10 to 20
1.10 (0.10 to 8.70)
21 to 49
2.40 (0.30 to 18.90)
2.70 (0.20 to 42.90)
1.70 (0.70 to 4.40)
Peritumoural vascular invasion:
3.40 (0.80 to 14.80)
0.33 (0.13 to 0.85)
0.20 (0.07 to 0.60)
0.23 (0.07 to 0.72)
Tumour microvessel density
1.02 (0.07 to 0.60)
HIF-1α TAG mRNA levels
1.03 (1.00 to 1.05)
Several isoforms of HIF-1α resulting from alternative splicing have been described in various human tumour cell lines or tissues. Only one isoform (HIF-1α 736 ) has been documented in human breast cancer . Moreover, only a few studies have been performed to link HIF-1α mRNA expression and tumour-specific parameters [32, 33]. Specific antibodies allowing HIF-1α splice variant detection at the protein level are not currently available. We used a real-time quantitative reverse transcription PCR assay to measure mRNA expression levels of 4 alternatively spliced transcripts of HIF-1α in breast specimens of 53 primary cancers and 29 normal tissues or benign lesions.
Interestingly, this work shows for the first time higher expression levels of two HIF-1α splice variants (HIF-1α TAG and HIF-1α 736 ) in OR-negative carcinomas compared to normal/benign tissues. We also investigated the prognostic value of HIF-1α transcript expression levels in breast cancer and found a significant relationship between either clinicopathological characteristics or patient metastasis-free survival. First, we found that HIF-1α TAG mRNAs levels were substantially higher in high grade and steroid hormone receptor-negative tumours. The second and most striking observation was that HIF-1α TAG mRNA levels were indicative of shorter metastasis-free survival, and that this correlated with lymph node status. Contrary to the Cayre et al. report , we did not find significant correlation between total HIF-1α mRNA expression and lymph node status but we observed significant association with tumour grade. This could be because our series was smaller than series of Cayre et al. or because the technology used in our study was more sensitive. In our series, total HIF-1α mRNA expression negatively correlated with OR status. Similar to Cayre et al. we did not find any correlation between total HIF-1α mRNA expression and outcome. Our results showing that HIF-1α 736 mRNA expression does not correlate with clinicopathological characteristics of tumours also concur with earlier findings of the same group . In this study, effect of adjuvant (none, chemotherapy or hormone) treatment on survival and interactions with expression levels of HIF-1α splice variants were not checked because of the limited number of patients in each subgroup. Further experiments in a larger series are required to answer this question.
Alternative splicing is known to play an important role in gene expression regulation by modulating the functional properties of transcription factors . In this regard, alternative splicing can modify DNA-binding properties of transcription factors , introduce or eliminate activating domains or increase the in vivo stability of a given isoform . Moreover, the abundance of specific isoforms is likely to result from differential expression, RNA stability and selective splicing process leading to an increase of some mRNA species. Recent evidence indicates that in several cancers the ratio of splice variants is dramatically altered and that differential expression of alternatively spliced isoforms in cancer patients can have severe implications for clinical outcome . Remarkably, statistical association has previously been reported between HIF-1β splicing variant expression, oestrogen receptors and breast cancer survival . It should be noted that the primers designed on exons 1 and 2 in our study allowed quantification of the HIF-1α TAG sequence present in both HIF-1α 827 and HIF-1α 736 splice variants . The HIF-1α TAG transcript is characterised by insertion of a three base pairs TAG insertion that may be generated by the use of two potential splice acceptor dinucleotides (AG) of intron 1 at a splice junction site as previously described [39, 1]. This splicing results in the replacement of Lys12 by Asn12 and the addition of Arg13 residue located upstream from the bHLH domain of the protein. This replacement may modify the DNA binding affinity of the protein complex as previously shown for Arg14 and Arg15 residues in aryl hydrocarbon receptor (AHR)/aryl hydrocarbon receptor nuclear translocator (ARNT) heterodimer . Further biochemical and structural studies are required for a better understanding of the functional properties of this variant.
To our knowledge, our results suggest for the first time that at least one HIF-1α splice α variant may be a marker for the advanced clinical and oestrogen-resistant stage of breast cancer. Based on their correlation with survival, HIF-1α TAG mRNA levels may be a potential useful prognostic indicator whose value should be further validated in prospective studies.
This work was supported by grants from Institut National du Cancer (ACI2004, PL2006). We are grateful to Jacques Pouyssegur for his generous gifts of plasmids and discussion. Claudine Andonian from the department of Pathology is also acknowledged for her excellent work with collecting and archiving the breast tumour samples.
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