HIF1α isoforms in benign and malignant prostate tissue and their correlation to neuroendocrine differentiation

Neuroendocrine (NE) cells in benign and malignant prostate tissue are characterized by expression of the NE marker chromogranin A and their specific morphology, including cytoplasmic extensions and dendritic processes. In addition, they lack androgen receptor and are therefore androgen-independent [1, 2]. The majority of prostatic adenocarcinomas contain tumor cells with NE features which are present focally throughout the tumors. Several studies have shown that an increased number of NE cells in prostate cancer tissue and increased levels of circulating chromogranin A, an NE marker usually expressed in NE cells, are associated with a poor prognosis, tumor progression and androgen-independent disease [1, 2]. It has been hypothesized that NE cells might affect the growth and proliferation of prostate tumors in an androgen receptor-independent way, probably through the secretion of NE peptides and autocrine/paracrine signaling pathways [1, 3‐6]. NE tumor cells in prostate cancer may also have a role in tumor angiogenesis. Increased expression of vascular endothelial growth factor (VEGF) in malignant NE cells and a higher density of microvessels around nests of such cells compared to areas of prostate cancer without these cells have been reported [7]. In a previous study, we demonstrated the presence of cytoplasmic hypoxia inducible factor 1α (HIF1α) in a population of NE cells in benign and malignant prostate tissues [8]. HIFs are a family of transcription factors that are essential for angiogenesis and the adaptation of cells to hypoxia in both physiological and pathological processes [9, 10]. The possible mechanisms behind HIF1α stabilization and the function of cytoplasmic HIF1α in NE cells of benign and malignant prostate tissues are not known. However, the complicated regulation of HIFs has been extensively studied in recent years. HIF1, which is composed of two subunits, HIF1α and β, binds to the HRE of promoter regions and activates many genes involved in angiogenesis, glycolysis and anaerobic metabolism. In normoxia, the HIF1α subunit is hydroxylated on specific proline residues (402 and 564) within the oxygen dependent domain (ODD) by specific oxygen-sensitive prolyl hydroxylases and, then ubiquitinated through the von Hippel-Lindau (VHL) dependent pathway and degraded instantly in the proteasome [9, 10]. However, in hypoxic conditions, HIF1α is stabilized by impaired function of these prolyl hydroxylases. After its interaction with a series of small proteins, such as P300 and CBP, the HIF1α and β complex translocates to the nucleus using its nuclear localizing signal (NLS) domains at C-and N-terminus and activates the cell's adaptive response to hypoxia [9, 10]

There have been several previous reports on HIF1α isoforms lacking several exons or displaying different exons than the wild type protein. Some of these isoforms encode cytoplasmic HIF1α protein or proteins with altered transcriptional activity compared to the wild type protein. Regarding the N-terminal domain, two HIF1α isoforms with different first exons have been identified: HIF1α1.2, a protein with a different first and second exon which is 59 amino acids shorter than wild type HIF1α and encodes a cytoplasmic protein specifically expressed in the human testis [11]; HIF1α1.3, which is present in activated T-lymphocytes and encodes a functional protein with weaker transcriptional activity that the wild type protein [12]. At the C-terminal domain, isoforms lacking either exon 12 [13] or exons 11 and 12 [14] have been reported. Both isoforms have been shown to be stable cytoplasmic proteins and inhibit the function of full-length HIF1α. Another shorter HIF1α isoform lacking exon 14 has also been reported [15]. This isoform was shown to be 3-fold less active than full-length HIF1α.

The aim of this study was to elucidate the mechanism behind the cytoplasmic stabilization of HIF1α in NE cells of benign and malignant prostate tissues. We have investigated whether the cytoplasmic HIF1α corresponds to any specific known isoform. Based on the sequences published by the National Center for Biotechnology Information (NCBI), we designed primers to amplify known HIF1α isoforms and determined their presence in benign prostate hyperplasia and prostate cancer with and without NE differentiation.

HIF1α, a major transcription factor essential in tumorigenesis, has been shown to be over-expressed in many types of tumors, including prostate cancer [16‐18]. Neo-angiogenesis occurs in an uncoordinated fashion in malignant tumors, leading to hypoxia, inactivation of prolyl hydroxylases and inhibition of HIF1α degradation, which, in turn, activates HIF1α [19]. HIF1α protein has also been shown to be stabilized under normoxic conditions by growth factor stimulation or due to mutations in the VHL protein [20]. The accumulation of HIF1α protein has been reported to be an early event in prostate cancer and high-grade prostatic intraepithelial neoplasia (HGPIN) [21, 22]. The accumulation of wild type HIF1α should lead to its nuclear transport, unless regulatory parts are defective or missing. One possible explanation for the cytoplasmic stabilization of the protein is the presence of certain HIF1α isoforms. Indeed, in this study, we have shown that a broad range of HIF1α isoforms are present in both benign prostate hyperplasia and prostate tumors. We have shown by PCR, immunohistochemistry and in situ hybridization that the previously reported testis-specific isoform, HIF1α 1.2 corresponds to cytoplasmic HIF1α in NE-differentiated benign and malignant prostate cells. HIF1α1.2, which lacks one of the NLS domains responsible for nuclear transport, was shown to accumulate in the cytoplasm and has also been shown in a previous study to have inhibitory effects on full-length HIF in human sperms [11]. The authors of that study speculated that inhibition of full-length HIF1α might be essential for the function of hypoxic sperm. A similar function for cytoplasmic HIF1α in benign and malignant prostate cells might be considered. In addition, co-localization of cytoplasmic HIF1α and HIF1β in this study indicates that this isoform might sequester HIF1β in the cytoplasm. Other reports confirm a similar function for cytoplasmic HIF1α. Stimulation of HEK293 cells with zinc induces a cytoplasmic spliced variant of HIF1α without exon 12 [23]. This 557-amino-acid isoform was shown to sequester HIF1β in the cytoplasm and therefore to inhibit wild type HIF1α. Due to a frame shift mutation, the isoform is shortened and lacks both trans activating domains and the C-terminal nuclear localizing signal domain. Another cytoplasmic spliced variant of human HIF1α (516 amino acids in length) represents a stable, short isoform without exons 11 and 12 [14]. Similarly to the variant lacking only exon 12, this isoform was identified as a dominant negative due to its association with HIF1β in the cytoplasm.

Recent studies have shown an association between hypoxia inducible factor and androgen receptor. The androgen receptor activates HIF1α, leading to upregulation of hypoxia inducible factor gene products [24, 25]. In addition, hypoxia has been shown to increase androgen receptor sensitivity [26] and androgen receptor induced expression of the human PSA gene in a prostate cancer cell line [27]. Malignant NE cells which have been shown to increase in number after hormonal treatment, are usually androgen independent and only occasionally express androgen receptor and PSA [1]. As, we have shown here, the NE differentiated tumor cells display an isoform with a possible inhibitory effect on the wild type HIF1α protein, indicating that the androgen independent malignant NE cells might also be independent of HIF1α for their survival and tumorogenesis.

Malignant NE cells have been correlated with increased microvessel density in prostate tumors and display increased expression of VEGF, an important angiogenic regulatory factor. The functional implication of cytoplasmic HIF1α and its relationship to VEGF remain to be elucidated. HIF1α is a key regulatory transcription factor of VEGF, but VEGF has also been shown to be regulated by other transcriptions factors. For example, RaIA uppregulates VEGF-C in prostate cancer cells during androgen ablation [28] and STAT3 binds to the VEGF promoter and directly regulates VEGF [29]. If cytoplasmic HIF1α in NE cells exerts an inhibitory effect on full-length HIF1α, this might indicate that VEGF is up-regulated in NE cells by other transcription factors.

In summary, our results suggest that cytoplasmic HIF1α in androgen independent NE cells of benign prostate tissue and non NE differentiated prostate tumors, corresponds to HIF1α1.2 isoform. Co-localization of this isoform with immunoreactive cytoplasmic HIF1β indicates that the HIF1α 1.2 isoform, which lacks one of the protein's nuclear translocation signals, may sequester HIF1β in the cytoplasm.

The authors declare that they have no competing interests.