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The role of hypoxia inducible factor-1alpha in gynecological cancer

Accepted 5 May 2010. published online 07 June 2010.
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Abstract

Understanding the mechanisms of carcinogenesis and progression of gynecological tumors is important as these insights might lead to improved diagnostic tools for the pathologist, improved prediction of prognosis, response to therapy, and eventually better biology-based disease management, thereby improving prognosis and quality of life for the individual patient. Hypoxia is an important event in carcinogenesis because it renders a more aggressive phenotype with increased invasiveness and proliferation, formation of metastases and poorer survival. Although selecting patients with hypoxic tumors may therefore be clinically important, there is no consensus as to the method best suited for routine assessment of hypoxia. One of the potential tumor hypoxia markers is hypoxia inducible factor 1 (HIF-1). HIF-1 is the key cellular survival protein under hypoxia, and is associated with tumor progression and metastasis in various solid tumors. In this review, we show that in gynecological cancers, HIF-1A is emerging as an important factor in carcinogenesis, and that overexpression of HIF-1A and its target genes CA9 and SLC2A1 seems associated with shorter progression free- and overall survival. Since hypoxia and HIF-1A expression are associated with treatment failure, targeting HIF-1A could be an attractive therapeutic strategy with the potential for disrupting multiple pathways crucial for tumor growth. Currently, HIF-1A inhibitors are being studied in clinical trials in recurrent ovarian- and cervical cancer, and trials in other gynecological cancers are expected.

Keywords: Hypoxia, Hypoxia inducible factor-1alpha, Endometrial cancer, Ovarian cancer, Cervical cancer, Prognosis, Therapy

Article Outline

• Abstract

• 1. ntroduction

• 2. Tumor biology

• 2.1. Hypoxia markers

• 3. Endometrial cancer

• 3.1. HIF-1A and carcinogenesis

• 3.2. HIF-1A and prognosis

• 4. Cervical cancer

• 4.1. HIF-1A and carcinogenesis

• 4.2. HIF-1A and prognosis

• 5. Ovarian cancer

• 5.1. HIF-1A and carcinogenesis

• 5.2. Impact of HIF-1A on response to chemotherapy

• 5.3. HIF-1A and prognosis

• 6. Vaginal cancer and vulvar cancer

• 7. Polymorphisms of the HIF-1A gene in gynecological cancer

• 8. HIF-1A and hypoxia as a target for cancer therapy

• 9. Conclusion

• Conflict of interest

•

• References

• Biography

• Copyright

1. Introduction

The American Cancer Society estimates that 80.720 women will have been diagnosed with, and 28.120 women will have died of, cancer of the female genital tract in 2009 in the USA. Gynecological malignancies account for 11% of all new cancers in women [1]. Understanding the mechanisms of carcinogenesis and progression of gynecological tumors is important as these insights might lead to improved diagnostic tools for the pathologist, improved prediction of prognosis, response to therapy, and eventually better biology-based disease management, thus in the end improving prognosis and quality of life for the individual patient. The presence of hypoxic regions within tumors has been shown to adversely affect the outcome of chemo- and radiotherapy and is associated with a worse prognosis. Although selecting patients with hypoxic tumors may therefore be clinically important, there is no consensus as to the method best suited for routine assessment of hypoxia. One of the potential tumor hypoxia markers is hypoxia inducible factor 1 (HIF-1). HIF-1 is the key cellular survival protein during hypoxia and is associated with tumor progression and metastasis in various solid tumors [2], [3]. HIF-1A expression could therefore be used to identify patients who are at risk of developing recurrent disease and who may benefit from adjuvant therapy [4], [5], [6], [7]. Moreover, HIF-1A has been proposed as a predictor of response to therapy as HIF-1A expression is associated with resistance to radiotherapy and certain forms of chemotherapy [8]. Lastly, HIF-1A itself might be a therapeutic target in view of its role in carcinogenesis and progression [8]. However, results on HIF-1A in gynecological cancers are not always consistent. This review will therefore put the role of HIF-1A and its target genes carbonic anhydrase 9 (CA9) and solute carrier family 2 (SLC2A1; also known as Glut-1) in the carcinogenesis of gynecological cancers into perspective. It will also describe the current knowledge on HIF-1A as a biomarker of aggressive behavior and as a novel therapeutic target in gynecological cancer.

2. Tumor biology

Solid tumors outgrow their own vasculature beyond the size of several cubic millimeters, resulting in hypoxia. Hypoxia is an important event in carcinogenesis because it renders a more aggressive phenotype with increased invasiveness and proliferation, formation of metastases and poorer survival [9], [10]. Furthermore, it has been shown that hypoxia induces resistance to radiotherapy and chemotherapy in tumors [11], [12], [13], [14]. There is no gold standard for measuring hypoxia, although the Eppendorf pO2 histograph has been regarded as the reference standard for assessing tumor oxygen levels [15]. However, this method has found limited clinical application because O2 microelectrode measurements are invasive and applicable only to tumors accessible to needle electrodes. There is, therefore, great interest in alternative methods of establishing tumor hypoxia. Recent studies have focused on molecular markers of hypoxia such as hypoxia inducible factor 1 (HIF-1), on measuring the level of binding of hypoxia-specific probes as Pimonidazole binding and on developing non-invasive imaging techniques.

As HIF-1 regulates cellular oxygen homeostasis, it plays a key role in hypoxic conditions that occur during embryogenesis, cardiovascular disease, and tumor development [4], [16]. HIF-1 is a transcription factor composed of the subunits HIF-1A and HIF-1β, which are basic helix-loop-helix DNA-binding proteins. The activity of HIF-1 is predominantly regulated at the post-translational level by regulating HIF-1A protein stability. At normal oxygen tension, HIF-1A is hydroxylated in the oxygen-dependent degradation domain (ODDD) by prolyl hydroxylases (PHD). Hydroxylated HIF-1A is recognized by the Von Hippel–Lindau (VHL) protein, ubiquitinated and destined for degradation by proteasome. This process is inhibited during hypoxia [17]. Under hypoxia, stabilized HIF-1A subunits heterodimerize with β-subunits to form the active HIF-1 complex that activates gene transcription by binding to the consensus HIF responsive element (HRE); 5′-RCGTG-3′ in promoters and enhancers of target genes [18]. Among these are glucose transporters, glycolytic enzymes, and genes involved in gluconeogenesis, high-energy phosphate metabolism, growth factors, erythropoiesis, haem metabolism, iron transport, vasomotor regulation and nitric oxide synthesis [18], [19], [20], [21]. Protein products of the HIF-1 target genes help the cell to survive the hypoxic stress by increasing oxygen delivery (angiogenesis) and by switching to anaerobic glycolysis [18], [19], [20], [21]. One of the most inducible and most uniformly HIF-1A induced genes under hypoxia is CA9. CA9 is a membrane-associated carbonic anhydrase that plays a role in pH regulation [22]. SLC2A1 is known as a transmembrane glucose transporter that operates as a passive carrier transporting glucose down a concentration gradient and is activated by HIF-1 in hypoxic conditions [23]. Increased angiogenesis is an effect of HIF-1A through upregulation of vascular endothelial growth factor (VEGF). VEGF acts through its tyrosine kinase receptors to modulate motility and proliferation of endothelial cells and vascular permeability [24]. Although HIF-1A usually induces prosurvival (CA9, SLC2A1 and VEGF) genes, a role of HIF-1A in regulation of apoptosis has also been described. HIF-1A promotes cell death through an increase in p53 or other proapoptotic proteins like BNIP3 [25]. As a result of this dual function of HIF-1α, a “stop-and-go” strategy as a dynamic balance to maintain overall cell growth and survival has been proposed [26]. Hypoxia-induced HIF-1A also affects the expression of genes involved in metastasis formation. Hepatocyte growth factor (HGF) for example is a cytokine which stimulates proliferation and invasion through its receptor, the proto-oncogene c-MET [27]. Invasive cell growth is promoted by HIF-1A induced c-Met transcription and sensitizes cells to HGF stimulation [28], [29], [30]. Taken together, the adaptive response to hypoxia in primary tumors resembles in many ways the so-called metastatic phenotype which explains the poor prognosis of hypoxic cancers [31]. Recently, it has also been reported that hypoxia alters the expression levels of several mircoRNAs (MirRs) [32], [33], [34]. MiR-210 is suggested to be regulated via HIF-1A, thereby extending the HIF transcriptional repertoire. MiR-210 overexpression has been found in multiple cancer types and has been associated with adverse clinical outcome in breast cancer patients [35], [36]. The exception is represented by ovarian carcinomas, that tend to have gene copy deletions of the miR-210 locus and therefore exhibit decreased expression of miR-210 [37].

In normoxic conditions, HIF-1A expression can also be induced by other mechanisms, including infection with oncogenic viruses; loss-of-function mutations in tumor suppressor genes such as Von Hippel–Lindau (VHL); or signaling by receptor tyrosine kinases, prostaglandin E2 receptor or nitric oxide [38]. Furthermore, genetic alterations in the EGFR [39], RAS, and PI-3K/Akt [40], [41], [42], [43], [44] as well as loss of p53 function [45] have been shown to lead to increased non-hypoxic HIF-1 activity. Other possible mechanisms contributing to normoxic HIF-1 expression like oncogenic mutation or amplification of HIF-1A gene have rarely been reported in solid cancers [46], [47]. A polymorphism in HIF-1A (P582S) has been found associated with increased HIF activity and poor prognosis in prostate cancer, but its significance with cancer risk is still incompletely understood [48], [49]. HIF-1A polymorphisms in gynecological tumors will be discussed later.

2.1. Hypoxia markers

As cervical cancer is one of the tumors rather easily assessable for invasive measurement of oxygenation status, many researchers have focused on cervical cancer in their search for markers of hypoxia. Direct measurement of hypoxia via oxygen electrodes has been performed in several cancers showing a wide range of oxygen levels within tumors [50]. Not all tumors showed hypoxia, and almost all tumors harbored normoxic tumor parts. Among molecules involved in the so-called hypoxic response of tumor cells, HIF-1A, CA9 and SLC2A1 can be detected immunohistochemically. Immunohistochemical staining with hypoxia markers is advantageous because the reagents are generally available and can retrospectively be applied for research purposes on paraffin-embedded tissue. Conflicting data concerning the correlation between HIF-1A expression and O2 oxygen measurements in cervical cancer have been found. Two studies reported a positive correlation between HIF-1A expression and tumor oxygenation, as determined with the Eppendorf pO2 histograph [51], [52]. However, these associations were weak. No correlation between HIF-1A protein expression and tumor oxygenation was found by others [53], [54], [55]. A correlation between Eppendorf electrode measurements and Pimonidazole binding, CA9 and SLC2A1 expression has been described in cervical carcinoma [56], [57], [58], [59]. However, these correlations were again weak and the results were disputed by others [54], [60]. The different surrogate hypoxia markers did however show significant marker colocalization confirming that hypoxia is likely to be the major factor in their expression. Firstly, possible causes for Eppendorf and hypoxia marker mismatch patterns include differences in marker sensitivity with respect to degree and duration of hypoxia, tumor heterogeneity as well as effects of factors other than hypoxia on expression of endogenous markers. Secondly, the relationship between marker expression and oxygen electrode measurements is a complex one, as these methods do not sample the same tumor microenvironment or provide directly comparable measures of hypoxia. Oxygen electrode measurements are further likely to be dominated by the level of acute (perfusion limited) rather than chronic (diffusion limited) hypoxia. In contrast, HIF-1A and its target genes, probably better reflect chronic hypoxia.

Immunohistochemical analyses of paraffin-embedded tissue sections have shown HIF-1A to be highly expressed in many tumor types [4], [61]. Because of the biological function of HIF-1A, only nuclear staining should be taken into account when assessing HIF-1A expression. The presence of cytoplasmic HIF-1A is probably unrelated to HIF-1 activity; stable HIF-1A is thought to rapidly translocate to the nucleus were it heterodimerizes with HIF-1β subunits to form the active HIF-1 complex.

3. Endometrial cancer

3.1. HIF-1A and carcinogenesis

A unique feature of the endometrium is the cycle-specific change in vascularization. It has been postulated that menstruation results from vasoconstriction of spiral arteriole with resultant hypoxia leading to necrosis [62]. This focal hypoxia in perimenstrual endometrium could result in locally increased HIF-1A. HIF-1A was expressed with increasing intensity from premenstrual (secretory) through to menstrual phase endometrium. No HIF-1A was detected in proliferative phase endometrium [63]. In another study, HIF-1A was undetectable in the majority of samples [64]. In the HIF-1A positive cases, expression was only seen in small foci within the tissue, suggesting that if hypoxia does occur at this time, it is not widespread [64]. The precise functional role of HIF-1 regulation in initiating menstruation or in the subsequent regeneration and repair of the endometrium is still unknown.

In postmenopausal women, HIF-1A was increasingly expressed from inactive endometrium through hyperplasia to endometrioid carcinoma, paralleled by activation of its downstream genes (CA9, SLC2A1 and VEGF) and increased angiogenesis. Perinecrotic, chronic hypoxia associated HIF-1A expression was absent in inactive endometrium, rare in endometrial hyperplasia and frequent in endometrioid carcinoma. These results point to the importance of hypoxia and the subsequent stabilization of HIF-1A in endometrioid endometrial carcinogenesis [16], [61], [65], [66]. The mechanism of tumorigenesis of type I (endometrioid endometrial) carcinoma differs from that of type II carcinoma. Type I endometrial cancers often develop in a background of atypical complex hyperplasia, are estrogen dependent and characterized by mutations in PTEN. Type II tumors generally arise from atrophic endometrium and often contain p53 mutations. More expression of HIF-1A was observed in type II than in type I endometrial carcinoma [67], [68]. p53 accumulation has been associated with HIF-1A overexpression in different human tumors [69]. However, p53 expression was not associated with HIF-1A expression type II endometrial carcinomas [67], [68] and could thus be not the reason for this difference. Moreover, loss of PTEN tumor suppressor gene is thought to be a cause of non-hypoxia-mediated HIF-1A expression [70], [71], [72]. We showed that, although over 60% of the type I tumors showed extensive loss of PTEN by immunohistochemistry, this was not correlated with HIF-1A expression (unpublished data).

Correlation of HIF-1A with FIGO stage, tumor grade, or myometrial invasion is still under discussion [61], [67], [73], [129].

3.2. HIF-1A and prognosis

As shown in Table 1, contradictory results have been described as to the prognostic value of HIF-1A overexpression in endometrial carcinoma. Although HIF-1A was significantly higher expressed in recurrent endometrial carcinoma when compared with their primary tumors, it was not an independent predictor for recurrent endometrial carcinoma [67], [73]. In stage 1 endometrial cancers, HIF-1A was associated with a worse prognosis [16]. However, others did not find prognostic impact of HIF-1A expression [65]. Immunohistochemical studies are difficult to compare because of a variation in definition of HIF-1A positivity. In some studies [16], [67], both nuclear and cytoplasmic HIF staining was scored. The significance of cytoplasmic HIF-1A, however, is still not elucidated; as said, stable HIF-1A is thought to rapidly translocate to the nucleus were it heterodimerizes with HIF-1β subunits to form the active HIF-1 complex. This is supported by the findings of Miyazawa et al. [74], who showed that the cytoplasmic expression of HIF-1A was only moderately correlated with DNA-binding HIF-1 level in the nucleus. The intensity of HIF-1A expression may not correlate with the activity and thus should, to our opinion, rather be ignored. Acs et al. [65] used a semiquantitative scoring method taking also the scoring intensity into account. However, intensity of HIF-1A staining may not directly be correlated to HIF-1 activity.

Table 1.

HIF-1A and prognosis or risk of recurrence in cancers of the female genital tract.


Author	Marker	N	% Positive cells	HIF-1α scoring method	Prognostic value for survival	Prognostic value for recurrence
Endometrial cancer
Pijnenborg [73]	HIF-1A	40	73%	N/A	N/A	No
Pansare [67]	HIF-1A	149	75%	Nuclear and cytoplasmic	N/A	No
Acs [65]	HIF-1A	107	74%	Nuclear	No	No
Sivridis [16]	HIF-1A	81	49%	Nuclear and cytoplasmic	Yes	N/A
Ozbudak [66]	Glut-1	100	87%	Membrane	No	No

Ovarian cancer
Shimogai [94]	HIF-1A	66	N/A	RT-PCR, mRNA expression	Yes	No
Osada [89]	HIF-1A	107	33%	Nuclear	Yes	N/A
Nakai [99]	HIF-1A	52	69%	Western blot on FFT, NIH analysis to quantify	Yesa	N/A
Nakayama [95]	HIF-1A	60	72%	RT-PCR, mRNA expression	No	No
Birner [93]	HIF-1A	102	69%	Semiquantitatively; percentage of positive tumor cells and staining intensity	Nob	No
Cantuaria [100]	Glut-1	104	86%	Membrane	N/A	Yes

Cervical cancer
Dellas [55]	HIF-1A	44	73%	Nuclear; semiquantitatively; percentage of positive tumor cells and staining intensity	Yes	No
Ishikawa [87]	HIF-1A	38	45%	Nuclear and cytoplasmic	Yes	Yes
Mayer [53]	HIF-1A	34	100%	Nuclear; computer assisted image analysis technique.	No	No
Hutchison [52]	HIF-1A	99	96%	Nuclear; semiquantitatively; percentage of positive tumor cells and staining intensity	No	No
Bachtiary [79]	HIF-1A	67	72%	Nuclear; semiquantitatively; percentage of positive tumor cells and staining intensity	Yes	Yes
Burri [86]	HIF-1A	91	94%	Nuclear; semiquantitatively; percentage of positive tumor cells and staining intensity	Yes	No
Birner [75]	HIF-1A	91	81%	Nuclear; semiquantitatively; percentage of positive tumor cells and staining intensity	Yes	Yes
Kirkpatrick [88]	CAIX	118	N/A	Staining intensity and the extent of all tumor cells	Yes	N/A
Loncaster [58]	CAIX	130	71%	Membrane	Yes	No
Hedley [56]	CAIX	110	70%	Immunofluorescence	N/A	No
Airley [59]	SLC2A1	93	N/A	Cytoplasmic and membrane	N/A	Noc

N/A=not examined/reported.

HIF-1A overexpression was only a significant prognostic indicator in stage III/IV patients who underwent suboptimal resection at primary surgery and were indicated for postoperative chemotherapy.

HIF-1A overexpression alone had no impact on the prognosis, but HIF-1A overexpression and non-functional p53 indicated dismal prognosis.

SLC2A1 is of prognostic significance for metastatic-free survival.

Moreover, these studies did not consider the different expression patterns throughout the tumors (diffuse versus perinecrotic) that have been shown in to be prognostically crucial [5], [129]. Seeber [129] show that nuclear perinecrotic HIF-1A expression was significantly associated with a shorter disease-free survival in endometrioid endometrial carcinoma. This significance of expression pattern could be explained by the fact that perinecrotic HIF-1A expression is thought to be hypoxia driven, whereas diffuse HIF-1A expression may rather be due to non-hypoxic stimuli [5]. We showed that perinecrotic HIF-1A expression is more often accompanied by activation of its downstream factors SLC2A1 and CA9, indicating it to be more active than diffuse HIF-1A [5], [129]. Fig. 1 shows an example of nuclear HIF-1A in a diffuse and perinecrotic expression pattern.

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Fig. 1. Immunohistochemical staining of HIF-1A in endometrioid endometrial carcinoma. Typical patterns are shown: (A) perinecrotic HIF-1A expression (10× magnification) and (B) diffuse HIF-1A expression (10× magnification). Asteriks indicates necrosis.

In short, hypoxia and the subsequent stabilization of HIF-1A seem an important event in endometrioid endometrial carcinogenesis. Especially, a nuclear, perinecrotic HIF-1A staining pattern seems to be associated with a shorter disease-free survival.

4. Cervical cancer

4.1. HIF-1A and carcinogenesis

Multiple studies have suggested an important role for HIF-1A in malignant progression of cervical cancer. Birner et al. [75] observed HIF-1A expression in 80% of CIN-III lesions and early-stage invasive cervical cancers. In contrast, there was no expression of HIF-1A in normal cervical samples. In 20% of cases, non-dysplastic squamous cell epithelium directly adjacent to invasive cancer showed weak expression of HIF-1A in the basal and intermediate cells. These observations suggest HIF-1A may be a facilitator of premalignant progression. The human papillomavirus (HPV) might be an important co-player for the development of tumorigenic properties of HIF-1A in cervical cancer. The HPV E6 oncoproteins commonly inactivate p53 [76]. Also, the overexpression of HPV E6 and the loss of p53 promote HIF-1A protein accumulation in human cervical cancer cells [77]. Therefore, the potential tumor-suppressive functions of HIF-1A may be lost already in initial stages through the influence of HPV infection on HIF-1A expression and p53 protein inactivation. As loss of p53 increases HIF-1A expression and oncoproteins E6 of various HPV types differ in their potential to inactivate p53 [78], Bachtiary et al. [79] hypothesized that the extent of HIF-1A expression differs in specimens depending on the inactivation potential of HPV type found in the tissue. However, no association between HIF-1A expression and the HPV type present was found.

Moreover, a dual function of HIF-1A in early cervical carcinogenesis has been proposed. On the one hand, it stimulates tumor growth, on the other hand, it supports hypoxia-mediated apoptosis via stabilization of p53 [25], [80]. In this situation, cells have a higher susceptibility to die because of hypoxia through p53-induced apoptosis. This is supported by the fact that loss of wild-type p53 is associated with a marked reduction in hypoxia-mediated apoptosis [81].

4.2. HIF-1A and prognosis

Several studies have reported on hypoxia as a predictor of adverse outcomes in cervical cancer patients treated with radiation [82], [83]. Differences in local control were not apparent on multivariate analysis, however. Two smaller studies yielded conflicting data regarding the impact of hypoxia on disease-free survival [84], [85]; in contrast to the studies by Höckel and Fyle [82], [83], these former studies demonstrated hypoxia to be a predictor of local control. The reason of the data conflict is not clear, but different definitions of local control may play a role here.

In cervical cancer, no significant association between HIF-1A expression and FIGO stage, histological grade, tumor size and lymphatic node involvement has been established. While multiple studies showed that patients with strong HIF-1A expression had a significantly shorter overall survival time, disease-free interval and only partial response to radiotherapy [55], [75], [79], [86], [87], others [52], [53] could not confirm this. Different results could be due to the varying patient groups included and different HIF-1A scoring methods applied. In the study by Mayer et al. [53] for example, the patient group was small and only patients treated with curative intent were included in the survival analysis. Dellas et al. [55] included advanced stage patients and again others included all patients. Most [52], [55], [75], [79], [86] used a semiquantitative scoring method, combining the percentage of positive nuclei and the intensity of the staining. It would be interesting to reanalyze the prognostic value of HIF-1A ignoring staining intensity and taking HIF-1α-expression patterns into account. Where we noticed the importance of especially the perinecrotic HIF-1A staining pattern in endometrial cancer, Ishikawa et al. [87] only scored HIF-1A in nonnecrotic areas, hereby possibly ignoring the most hypoxic tumor areas.

CA9 is another potentially useful marker, but again, its prognostic ability is not fully clear. In one study, the level of CA9 expression appeared to be a significant prognostic factor for disease-specific and metastasis-free survival [58]. However, in the same study, CA9 expression was not correlated with local control. In line with this study, elevated CA9 expression was associated with more frequent distant metastases in early-stage cervical cancer [88]. Another study showed no impact for CA9, regardless of the hypoxia threshold or the number of tumor measurements [56]. The impact of SLC2A1 expression on prognosis was evaluated in 121 cases of cervical cancer treated with radiotherapy [59]. For the whole group, there was a significantly improved metastasis-free survival in patients with SLC2A1 negative tumors. No such effect was however seen on disease-free or overall survival.

Overall, these results propose HIF-1A expression to be a prognostic marker in cervical cancer. More research taking HIF-1A expression patterns into account is however needed before drawing any definite conclusions. Of the other hypoxia markers described, the prognostic value is less convincing.

5. Ovarian cancer

5.1. HIF-1A and carcinogenesis

A difference in HIF-1A expression, with malignant tumors expressing more HIF-1A than the benign and borderline controls in most studies, suggests a role of HIF-1A in ovarian carcinogenesis [89], [90], [91], [92]. However, Birner et al. [93] found higher expression of HIF-1A in borderline cases compared to carcinomas. Tumor cells expressing HIF-1A and SLC2A1 tended to overlap, but with significant differences between expression pattern. Therefore, it is suggested that SLC2A1 expression in ovarian tumors is in part controlled by HIF-1A and is also strongly affected by other micro-environmental conditions [92]. Based on different data, it should be emphasized that HIF-1A and SLC2A1 expression differs among the different histotypes of ovarian adenocarinomas. Clear cell carcinoma is characterized by the highest HIF-1A expression [74], [89], [90], although others found the highest expression of HIF-1 and SLC2A1 to be in serous papillary ovarian carcinoma [91], [92], [94]. In the latter studies, this was considered to be attributed to the papillary proliferation tumor structure which is believed to lead to a more hypoxic microenvironment.

In contrast with endometrial- and cervical carcinoma, a consistent significant correlation between tumor stage, grade and HIF-1A expression has been described for ovarian carcinoma. HIF-1A expression was significantly higher in tumors of FIGO stages III and IV than in those of stages I and II [65]. With larger tumor bulk in the high stage tumors and subsequent areas of hypoxia this is as expected. HIF-1A expression was also significantly higher in grade 1 ovarian cancers compared with grade 3 tumors [93]. This, and the already mentioned relatively high expression in borderline tumors, is unexpected as cells with a high proliferative rate consume more oxygen, which may result in a relatively hypoxic status and thus more HIF-1A stabilization. The applied semiquantitative (percentage positive cells and staining intensity) determination of HIF-1A expression might be the reason for this. As described, intensity of HIF-1A staining may not directly be correlated to HIF-1 activity and could therefore confuse the correlations found. Quantitative RT-PCR-analysis of HIF-1A in ovarian carcinoma showed a higher HIF-1A mRNA expression in malignant compared to benign epithelium [90], [95]. Further, expression level in poorly differentiated carcinomas was significantly higher than that in well-differentiated carcinomas [95]. However, no correlation was shown between HIF-1A protein expression and gene expression, confirming that HIF-1A activity is mainly regulated at the post-transcriptional level [90], [96].

5.2. Impact of HIF-1A on response to chemotherapy

For ovarian cancer, a platinum-based chemotherapy combination including paclitaxel or docetaxel is at present the most effective first-line treatment modality [97], [98]. The predictive role of HIF-1A for the effect of postoperative chemotherapy is not yet clear. Where Nakai et al. found a higher response rate to platinum-based combination chemotherapy in patients with HIF-1A expression, Birner et al. found no significant influence of HIF-1A expression [93], [99]. This could be due to different patient groups, HIF-1A detection methods and different treatment regimens. Nakai et al. [99] used Western blot analysis to determine HIF-1A expression, thereby however not considering the presence of non-functional HIF-1A in the cytoplasm and thus possibly overestimating the correlation between HIF-1A and response to therapy. SLC2A1 overexpression was associated with a complete response to chemotherapy, regardless of the tumor histology [100]. An explanation of a better response to chemotherapy in SLC2A1 positive patients could lay in the fact that malignant cells usually express higher levels of glucose transporter proteins to satisfy higher request of energy for a rapid proliferation and chemotherapeutic drugs have their preferential effect on actively dividing cells.

5.3. HIF-1A and prognosis

The prognostic significance of HIF-1A in ovarian cancer is not unequivocal. Birner et al. [93] concluded that HIF-1A protein expression alone has no impact on the prognosis for ovarian cancer, whereas in a subgroup of patients with concurrent overexpression of HIF-1A and p53 protein, a significantly shorter overall survival was observed. A shorter 5-year OS has been described in patients with high HIF-expression [89], [94], [99], whereas no effect on survival was noticed by others [93], [95]. As described previously, the method used by Nakai et al. [99] might overestimate the correlation found. Nakayama et al. [95] and Shimogai et al. [94] correlated HIF-1A mRNA levels with survival data. However, as HIF-1 is regulated post-translational through regulation of HIF-1A protein stability, HIF-1A mRNA is not necessarily related to the degree of HIF-1 activity. Another limiting factor in the survival analysis of ovarian carcinoma is that, despite of the varying HIF-1A expression in the different histotypes, there was no subgroup survival analysis performed for the different histological subtypes.

In stages III–IV patients who experienced a clinical complete response to chemotherapy, patients with weak SLC2A1 staining showed a longer DFS compared with those patients with greater than 50% of tumor cells strong positive [100]. This appears to be conflicting with the assumption made in the same study that patients with a strong SLC2A1 staining have a better response to chemotherapy. The authors suggest that this could be due to the fact that even after complete response to chemotherapy, some cancer cells still will be present. Among these, the cells with high SLC2A1 protein levels could have a survival advantage leading to a progression of disease.

In summary, the described results point to HIF-1A as a prognostic factor for OS. Yet, no correlation between DFS and HIF-1A has been found in ovarian cancer. Low SLC2A1 protein levels could be due to less active HIF-1 and result in a longer DFS.

6. Vaginal cancer and vulvar cancer

Cancers of the vulva and vagina are relatively rare and, therefore, little is known about the pathophysiological role of tumor oxygen status in these entities. Contrary to cervical cancer [101] no significant differences in the oxygenation status as measured by the Eppendorf pO2 histography between primary and recurrent vulvar tumors were found. Hypoxia measured by Eppendorf or visualized by CA9 staining neither correlated with metastatic nodal status [102], [103].

No studies concerning the influence of HIF-1A on the carcinogenesis of vaginal- and vulvar cancer are available. Theoretically, in as far as a significant proportion of these cancers are also HPV related, one would expect results to be similar as in cervical cancer.

7. Polymorphisms of the HIF-1A gene in gynecological cancer

It has been shown that polymorphisms of the HIF-1A oxygen-dependent degradation domain (ODDD) may increase risk for developing prostate [48] and colorectal cancer [104], [105], [106] and are associated with unfavorable tumor features and a worse prognosis in colorectal cancer and esophageal squamous cell carcinoma [107], [108], [109]. The single nucleotide polymorphism (SNP) C1772T (also described as C1744T) in the HIF-1A gene coding region, results in an amino acid change at position 582 changing a Proline to a Serine (i.e. P582>S) in the ODD-domain (http://www.ncbi.nlm.1nih.gov/SNP/snp_ref.cgi?rs=11549465). Carriers of this SNP seemed to have an increased risk of developing cervical and endometrial cancer [110]. However, the proportion of allele carriers with the most common polymorphism in the control group was different from ratios described in other studies. Horrée et al. [49] examined whether the C1744T polymorphism increased the risk for endometrioid endometrial cancer. Although the C1744T polymorphism was associated with higher microvessel density and AKT activation it did not lead to increased cancer risk. Interestingly they found that the P582S genotype variation in the ODDD of the HIF-1A protein may occur as a de novo mutation in endometrial cancer. Although the significance of this remains to be established, others have proposed it may increase transactivation of HIF-1A [48].

8. HIF-1A and hypoxia as a target for cancer therapy

The unsatisfactory results obtained with conventional pharmacological treatment encourage further biological and clinical investigations addressed to a better understanding of specific cell targets and signaling transduction pathways involved in endometrial-, ovarian-, and cervical carcinogenesis and to the identification of novel molecular targeted therapies.

As hypoxia in solid tumors leads to resistance to radiotherapy and chemotherapy [11], [12], [13], [14] and HIF-1A expression is associated with treatment failure and/or patient mortality in tumors like cervical and endometrial cancer [8], targeting HIF-1A could be an attractive treatment strategy, with the potential for disrupting multiple pathways crucial for tumor growth. There are 4 major areas of research in hypoxia-related drug therapy: (1) designing drugs that directly inhibit HIF-1 signaling, (2) influencing other signaling cascades that indirectly alter HIF signaling, (3) exploiting the hypoxic microenvironment to increase specificity and decrease toxicity of known drugs and (4) altering regulation of HIF targets genes that are critical for tumor growth. Table 2 gives a limited overview of the current knowledge on agents inhibiting HIF.

Table 2.

HIF-1α targeted therapy.


Class	Inhibitor	Mechanism
Small molecule inhibitors of HIF-1
Topoisomerase inhibitor	Topotecan (topo-I)	HIF transcriptional repression and downregulation of HIF-1-dependent gene expression [123]
HSP90 inhibitor	Geldanamycin	Destabilization of HIF-1A protein and inhibition of DNA binding of HIF-1 [124]
Other	PX-478	Inhibition of HIF-1A on multiple levels [125]

Inhibitors of signal transduction pathways
mTOR inhibitor	CCI-779	Downregulation of HIF-1A by inhibiting mTor [126]
ErbB2 receptor tyrosine kinase inhibitor	Herceptin	Downregulation of VEGF by reduction of HIF-1A synthesis [127]
EGFR tyrosine kinase inhibitor	Iressa	Decreasing VEGF expression by decreasing HIF-A expression [128]

Inhibition of HIF-1 would, of course, hit multiple targets but because of its bi-functional effects, e.g. proapoptotic genes induced by hypoxia, outcome will be difficult to predict. Thus far, selective HIF-1 inhibitors have not been identified. Antisense therapy against HIF-1A has been shown to reduce HIF-1A expression and transcriptional activity; however, at present it is not clinically applicable. Therefore, the potential of HIF-1A as a target for cancer therapy lies in the small molecule inhibitors of HIF-1. Several small molecular inhibitors of the HIF-1 transcriptional activation pathway have also been identified. Although none of these have been shown to directly and specifically target HIF-1 [111], [112], they do decrease HIF-1A protein levels. Some of these HIF-1-inhibitors are in clinical trials at present. Topotecan, a topoisomerase I inhibitor that has been used as a second-line therapy for ovarian cancer, is one such small molecule inhibitor of HIF-1. Topotecan inhibits hypoxic induction of HIF-1A protein and DNA-binding activity [113], [114]. It is being tested in multiple clinical trials, including different studies for patients with recurrent endometrial-, cervical-, and ovarian cancer [115], [116].

Other small molecule inhibitors of HIF-1 activity currently investigated in clinical trials are PX-478, a inhibitor of HIF-1 transcription factor activity [117], and geldanamycin, a HSP90 (heat shock protein 90) inhibitor [118]. HSP90 is involved in the folding of HIF-1A and Geldanamycin induces degradation of HIF-1A [119]. Both are being evaluated in advanced solid tumors. More trials in gynecological cancers are expected.

Some known anticancer agents have shown to inhibit HIF-1 [120], [121]. Duyndam et al. [122] showed in human ovarian cancer cell lines that the conventional anticancer agents cisplatin and doxorubicin can negatively influence HIF-1 activity with a concomitant reduction of VEGF expression.

An inhibitor that targets a pathway activated by HIF-1 is Rencarex®. This CA9 antibody is currently in phase III clinical trials in renal cell cancers, and may find its way into clinical trials in gynecological cancers in the future.

Lack of specificity increases the difficulty in attributing any anti-tumorigenic effects of these drugs specifically to inhibition of HIF-1A. Cell type, oncogenic mutations and microenvironment will also influence HIF-1 behavior. Further work is needed to identify more selective inhibitors of HIF-1 and to translate these developments into clinical trails.

9. Conclusion

Overall, there is convincing evidence of the clinical importance of tumor hypoxia, but detection of hypoxia is still not widespread in routine clinical practice. Since O2 microelectrode measurements are invasive and applicable only to tumors accessible to needle electrodes, there is a great interest in surrogate markers for tumor hypoxia. On the basis of the available data, the suitability of the described endogenous hypoxia markers for the estimation of the oxygenation status of gynecological tumors seems questionable. However, although none of the described hypoxia markers is absolutely hypoxia-specific, the data summarized above suggest these markers may be able to identify patients who have a poorer prognosis or who will be less sensitive to certain therapies. HIF-1A is emerging as an important factor in the carcinogenesis of gynecological tumors and, despite some different results, overall HIF-1A expression seems associated with a shorter progression free survival. Besides, since hypoxia and thus HIF-1A expression are associated with treatment failure, targeting HIF-1A could be an attractive therapeutic strategy. Currently, HIF-1A inhibitors are being studied in multiple clinical trials in recurrent ovarian- and cervical cancer, and trials in other gynecological cancers are expected.

Conflict of interest

All authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed in the manuscript.

Reviewers

Leen J. Blok, MD, Erasmus University Medical Center, Department of Obstetrics and Gynecology, Rotterdam, Netherlands.

Paul Cornes, MD, Bristol Haematology & Oncology Centre, Department of Oncology, Horfield Road, Bristol, BS2 8ED, United Kingdom.

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Paul J van Diest studied Medicine at the VU University medical center (VUMC) in Amsterdam from 1981 to 1988. After his M.D. graduation, he worked as Ph.D. student in the Department of Pathology of the VUMC. After obtaining his Ph.D. in 1990, he worked for another year as postdoc and was then resident in Pathology from 1991 to 1996 in the same department. In 1994 he became Visiting Professor at the University of Ancona, Italy. After obtaining his Board certification in Pathology in 1996, he became Consultant Pathologist at the Department of Pathology of the VUMC and was Head of the Quantitative Pathology Unit. In 1999 he was appointed Associate Professor and functioned also as Head of Pathology, Stichting Artsen Laboratorium, Utrecht, The Netherlands, from 1999 to 2003. In 2001, he was promoted to full Professor of Oncologic Pathology and 2001 to 2003 was appointed as Director of the Oncology Research Institute of the VUMC. In 2003 he moved to Utrecht to become Head of the Department of Pathology at the University Medical Center Utrecht (UMCU) where he is currently still working. Since 2009, he is the chairman of the Division of Laboratories and Pharmacy. He has served in numerous committees of the VUMC and the UMCU and within the Dutch Society of Pathology, and Netherlands Society for Oncology. He has organized several international conferences. He serves on the editorial board of eight international journals, as was editor of the Journal of Clinical Pathology from 1998 to 2002, and reviews for many other journals. He has been active in the board as secretary and president of the International Society for Diagnostic Quantitative Pathology, the European Society of Analytical Cellular Pathology, and the International Society for Cellular Oncology. He has been a dedicated cancer researcher since the start of his career, obtaining numerous grants a.o. from the NIH and the Dutch Cancer Society. He has published 419 papers in peer reviewed journals, personally supervised 30 Ph.D. theses, and has an H-index of 49.

a Department of Gynecological Oncology, University Medical Center Utrecht, Utrecht, The Netherlands

b Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands

c Department of Internal Medicine, University Medical Center Utrecht, Utrecht, The Netherlands

d Department of Radiation Oncology (MAASTRO), GROW – School for Oncology and Developmental Biology, Maastricht University Medical Center, The Netherlands

Corresponding author at: Department of Pathology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands. Tel.: +31 88 7556565, fax: +31 30 2544990.

PII: S1040-8428(10)00123-X

doi:10.1016/j.critrevonc.2010.05.003