The Clinicopathologic and Prognostic Value of Hypoxia-Inducible Factor-2Α in Cancer Patients

Published OnlineFirst December 27, 2018; DOI: 10.1158/1055-9965.EPI-18-0881

Review Cancer Epidemiology, Biomarkers The Clinicopathologic and Prognostic Value of & Prevention Hypoxia-Inducible Factor-2a in Cancer Patients: A Systematic Review and Meta-Analysis Deqing Luo1, Hui Liu1, Dasheng Lin1, Kejian Lian1, and Hongyue Ren2

Hypoxia-inducible factor-2a (HIF2a) plays an important analysis. The results indicated that the increased expression role in the development of tumors. However, the clinico- of HIF2a could predict unfavorable overall survival of pathologic and prognostic significance of HIF2a in cancer cancer patients on both univariate analysis (HR, 1.64; patients remains controversial. Therefore, we performed a 95% CI, 1.41–1.92, P < 0.001) and multivariate analysis meta-analysis to investigate the relationship between the (HR, 2.21; 95% CI, 1.70–2.87, P < 0.001). Moreover, HIF2a HIF2a status and clinical outcome in human cancer. Studies overexpression was associated closely with tumor differ- were screened online using electronic databases. The pooled entiation, tumor–node–metastasis stage, and lymph metas- risk ratios or hazard ratios (HR) with their 95% confidence tasis. In addition, there was no obvious evidence for intervals (CI) were calculated from available publications. significant publication bias in this meta-analysis. Our study Subgroup analysis, sensitivity analysis, heterogeneity, and indicated that HIF2a might be an indicator of poor prog- publication bias were also conducted. A total of 854 nosis and clinicopathologic featuresoftumorsandcould studies with 4,345 patients were obtained in this meta- serve as a novel biomarker in human cancer.

different functions, such as angiogenesis, proliferation, and Introduction tumor stem cells. In addition, HIF2a exhibits distinct roles in hypoxic activation of target genes compared with HIF1a.In Oxygen is essential for energy metabolism, which drives cel- clinical specimens, HIF2a involved in the development of lular biological activity (1). Region of hypoxia serves as a common many human cancers, such as pancreatic cancer (11), colorectal microenvironment in the development and progression of many cancer (12), and ovarian cancer (13). Moreover, HIF2a was solid tumors (2). Hypoxia is associated with a series of molecular directly or indirectly related to the regulation of progression of biology of tumors, such as proliferation, invasion, migration, and tumors (14, 15). It was reported that HIF2a expression was chemoradiotherapy resistant (3, 4). The mediator of hypoxia in associated with poor prognosis in patients with various cancers, tumor is hypoxia-inducible factors (HIF), which are heterodi- including lung cancer (16), gastric carcinoma (17), pancreatic b meric complex composed of a constitutive subunit (HIF1 ) and a cancer (18), and breast cancer (19). However, some studies a a a regulated subunit including HIF1 , HIF2 , or HIF3 (5). Among indicated that there was no significance between HIF2a expres- a a them, so far HIF1 and HIF2 have received more attention (6), sion and the prognosis of tumors (20, 21). Hence, the results and increasing evidence has demonstrated the structure, function, of different studies are controversial, and the prognostic value a and regulation of HIF2 in human cancer (7, 8). of HIF2a expression in cancer remains unknown. a HIF2 shares approximately 48% of amino-acid sequence In this study, a meta-analysis from eligible studies was per- a a a homology with HIF1 (9). As opposed to HIF1 protein, HIF2 formed to investigate the relationship between HIF2a expression < is active under mild or physiologic hypoxia ( 5% O2; ref. 10). and the overall survival (OS) on both univariate and multivariate a During the process of tumor development, HIF2 shows analyses in cancer patients. Furthermore, we made subgroup analysis to assess the roles of HIF2a in clinicopathologic factors of cancer. 1Department of Orthopaedic Surgery, The Affiliated Southeast Hospital of Xia- men University, Orthopaedic Center of People's Liberation Army, Zhangzhou, Materials and Methods Fujian Province, China. 2Department of Pathology, The Affiliated Southeast Hospital of Xiamen University, Orthopaedic Center of People's Liberation Army, This analysis was conducted according to the Preferred Report- Zhangzhou, Fujian Province, China. ing Items for Systematic Reviews and Meta-analyses (PRISMA) Note: Supplementary data for this article are available at Cancer Epidemiology, guidelines (22). Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). Corresponding Author: Hongyue Ren, The Affiliated Southeast Hospital of Search strategy Xiamen University, Orthopaedic Center of People's Liberation Army, Zhangzhou This meta-analysis was limited to assess the prognostic impli- 363000, Fujian Province, China. Phone/Fax: 86-596-2975570; E-mail: cation of HIF2a expression in human cancer patients. A compre- [email protected] hensive literature search of the following databases was per- doi: 10.1158/1055-9965.EPI-18-0881 formed prior to May 27, 2018: PubMed, Web of Science, Cochrane 2018 American Association for Cancer Research. Library databases, Chinese National Knowledge Infrastructure,

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Figure 1. Flowchart of the study selection process.

and Wanfang databases. The articles were identified using the meta-analysis; (iii) no sufficient data to estimate the HR and 95% search strategy: (cancer OR sarcoma OR tumor OR neoplasm) confidence intervals (95% CI). AND (HIF2a OR HIF2a OR HIF2 OR HIF2 OR hypoxia-inducible factor 2a OR hypoxia-inducible factor-2a) AND (prognosis OR Data collection survival OR outcome). The language of enrolled studies was The following information was independently extracted by two restricted to English and Chinese. Citation lists of included studies investigators: first author's name, publication year, region, histo- were screened manually to ensure sensitivity of the search strategy. logic type, expression type of HIF2a, patient cases, number of HIF2a positive, percentage of HIF2a positive, analysis type, and Criteria for inclusion and exclusion follow-up time. The Newcastle–Ottawa scale (NOS) score was used All studies included in this meta-analysis were accorded with for assessing the quality, which ranged from 0 to 9. Studies with the following inclusion criteria: (i) case–control studies focus on scores 5 were regarded as high-quality studies; otherwise, studies the relationship of HIF2a expression with the prognosis and were considered to have a low quality. In addition, clinicopatho- clinicopathologic characteristics of human cancer patients; (ii) logic characteristics included gender (male vs. female), tumor studies were reported HIF2a expression status, with availability of differentiation (poor vs. well/moderate), tumor–node–metastasis OS data on univariate or multivariate analysis, reported either as (TNM) stage (III/VI vs. I/II), and lymph metastasis (yes vs. no). hazard ratios (HR) or as Kaplan–Meier (KM) curves; (iii) studies were published in English or Chinese with the full text. In Statistical analysis addition, the major exclusion criteria were as follows: (i) over- All analyses were performed using STATA 12 software lapped articles or studies with overlapping data; (ii) reviews or (STATA Corp.). The effect of HIF2a expression on OS of cancer

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Table 1. Main characteristics of the studies included in the meta-analysis Case HIF2aþ HIF2aþ Follow-up NOS Study Year Region Histologic type Expression (n) (n) (%) Analysis (months) Score Quality Bangoura G 2007 Asian HCC Protein 315 219 69.5 UA/MA 85 6 High Biswas S 2012 Non-Asian RCC Protein 112 46 41.1 UA/MA 180 6 High Chen CB 2011 Asian Chondrosarcoma Protein 34 18 52.9 UA/MA 98 8 High Cleven A 2007 Non-Asian CRC Protein 133 43 32.3 UA/MA 150 7 High Gao ZJ 2017 Asian Lung cancer Protein 140 64 45.7 UA 100 5 High Griffiths EA 2008 Non-Asian Gastric and esophageal Protein 172 106 61.6 UA 118 7 High cancers Holmquist-Mengelbier L 2006 Non-Asian Neuroblastoma Protein 72 23 31.9 UA 90 4 Low Jubb AM 2009 Non-Asian Colon cancer Protein 159 85 53.5 UA/MA 140 7 High Koukourakis MI 2006 Non-Asian HNSCC Protein 198 116 58.6 UA/MA 90 4 Low Leek RD 2002 Non-Asian Breast cancer Protein 139 45 32.4 MA 126 7 High Li W 2016 Asian Osteosarcoma mRNA 82 42 51.2 MA 136 8 High Li WZ 2017 Asian PDAC Protein 283 148 52.3 UA/MA 62 6 High Lim E 2017 Asian OSCC Protein 56 23 41.1 UA 60 4 Low Liu YZ 2009 Asian RCC Protein 60 38 63.3 UA/MA 72 4 Low Luan YJ 2013 Asian Lung cancer Protein 45 11 24.4 UA/MA 69 7 High Pan TT 2015 Asian HCC Protein 90 69 76.7 UA/MA 66 8 High Qu HX 2016 Asian Neuroblastoma Protein 30 15 50.0 UA 50 4 Low ROH JL 2008 Asian SDC Protein 21 7 33.3 UA 120 4 Low Sandlund J 2009 Non-Asian RCC mRNA 168 NR NR MA 247 3 Low Sathornsumetee S 2008 Non-Asian Malignant astrocytoma Protein 45 NR NR MA 100 4 Low Sivridis E 2002 Non-Asian Endometrial carcinoma Protein 81 14 17.3 UA 182 5 High Sun HX 2010 Asian HCC Protein 254 122 48.0 UA/MA 60 6 High Szendroi A 2016 Non-Asian RCC mRNA 69 36 52.2 UA 120 4 Low Tong WW 2015 Asian Gastric carcinoma Protein 127 69 54.3 UA 60 5 High Wang XC 2011 Asian HCC Protein 104 72 69.2 UA/MA 62 4 Low Wang HX 2014 Asian Breast cancer Protein 102 62 60.8 UA/MA 50 5 High Wang Q 2014 Asian Epithelial ovarian cancer Protein 40 32 80.0 UA 30 3 Low Wei LF 2011 Asian Lung cancer Protein 51 37 72.5 UA 80 4 Low Winter SC 2006 Non-Asian HNSCC Protein 139 21 15.1 UA/MA 100 6 High Xie M 2015 Asian RCC Protein 86 NR NR UA/MA 20 3 Low Yang SL 2014 Asian HCC Protein 126 17 13.5 UA 60 4 Low Yang J 2017 Asian Pancreatic cancer Protein 70 47 67.1 UA/MA 25 6 High Yao QY 2015 Asian HCC Protein 63 47 74.6 MA 62 4 Low Yoshimura H 2004 Asian CRC Protein 87 26 29.9 UA 120 5 High Yuan K 2009 Asian Lung cancer Protein 140 64 45.7 UA/MA 50 5 High Yun F 2016 Asian Lung cancer Protein 112 53 47.3 UA/MA 65 4 Low Zang L 2016 Asian DLBCL Protein 90 58 64.4 UA/MA 50 6 High Zhang Q 2017 Asian Pancreatic cancer Protein 90 69 76.7 UA/MA 90 6 High Zhou JC 2014 Asian RCC Protein 63 43 68.3 UA 150 4 Low Zhu GQ 2010 Asian OSCC Protein 97 59 60.8 UA 60 5 High Abbreviations: CRC, colorectal cancer; DLBCL, diffuse large B-cell lymphoma; HCC, hepatocellular cancer; HIF2aþ: hypoxia-inducible factor-2a–positive expression; HNSCC, head and neck squamous cell cancer; MA, multivariate analysis; NOS, Newcastle–Ottawa scale; NR, not reported; OSCC, oral squamous cell carcinoma; PDAC, pancreatic ductal adenocarcinoma; RCC, renal cell cancer; SDC, salivary duct carcinoma; UA, univariate analysis. was calculated as HR with 95% CIs. If the study only showed Results KM graphs, KM curves were read by Engauge Digitizer version Search results and study characteristics 4.1 (http://digitizer.sourceforge.net/). Risk ratios (RR) and A total of 854 articles related with HIF2a and cancer prognosis corresponding 95% CIs were combined to evaluate the rela- were identified from online database searches. According to the tionship between HIF2a expression and clinicopathologic literature selection criteria (Fig. 1), 40 studies were included in this characteristics, including gender, tumor differentiation, TNM meta-analysis (16–21, 23–56), including 4,345 cancer cases and stage, and lymph metastasis. A heterogeneity test of pooled HRs 2,066 HIF2a-positive cases. The main characteristics and clinico- was conducted using c2-based Cochran Q test and I2 index. An pathologic factors of the included studies were summarized I2 value > 50% indicated significant heterogeneity among in Table 1 and Supplementary Table S1, respectively. All studies studies; then a random-effects model was used. Otherwise, a included in the meta-analysis were retrospective studies pub- fixed-effects model was applied to pooled data (I2 < 50%). lished between 2002 and 2017. Of these studies, 28 were Asian To detect heterogeneous studies, a sensitivity analysis was and 12 were non-Asian. The type of cancers included breast performed to evaluate the influence of individual studies on cancer, chondrosarcoma, colorectal cancer, diffuse large B-cell the stability of pooled results. Publication bias was assessed by lymphoma (DLBCL), endometrial carcinoma, gastric and esoph- visually assessing a Begg funnel plot and by quantitatively ageal cancers, hepatocellular cancer (HCC), head and neck squa- performing Begg test and Egger test. All statistical tests were mous cell cancer (HNSCC), lung cancer, malignant astrocytoma, two sided, and statistical significance was defined as P value less neuroblastoma, osteosarcoma, oral squamous cell carcinoma than 0.05. (OSCC), pancreatic cancer, renal cell cancer (RCC), and salivary

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Table 2. Analysis for HIF2a and clinicopathologic features of patients with cancer Number of Number of Number of Pooled data Test for heterogeneity Clinicopathologic features studies (n) cases (n) HIF2aþ (n) RR 95% CI P value x2 P value I2 (%) Gender (male vs. female) 17 2,103 1,168 0.94 0.87–1.02 0.116 15.67 0.476 0 Differentiation (poor vs. well/moderate) 11 1,319 732 1.42 1.23–1.65 <0.001 22.77 0.012 56.1 TNM stage (III/VI vs. I/II) 10 1,241 693 1.50 1.35–1.66 <0.001 10.41 0.319 13.5 Lymph metastasis (yes vs. no) 9 961 525 1.38 1.24–1.54 <0.001 8.35 0.400 4.2 Abbreviations: HIF2aþ, hypoxia-inducible factor-2a–positive expression; TNM stage, tumor–node–metastasis stage (designed by the American Joint Committee on Cancer).

duct carcinoma (SDC). Among these studies, 38 studies detected Association between HIF2a and clinicopathologic features the protein expression level of HIF2a, and 2 studies reported the To further investigate the effect of HIF2a on the prognosis of mRNA expression of HIF2a. Overall, sample sizes ranged from 21 cancer, we analyzed clinicopathologic features including gender, to 315 cases, and the follow-up of these studies ranged from 20 to differentiation, TNM stage, and lymph metastasis. All the detailed 247 months. Patients' OS on univariate and multivariate analyses data are summarized in Table 2 and Supplementary Fig. S1A– were reported in 35 studies and 25 studies, respectively. Moreover, S1D. The results indicated that high expression of HIF2a was not 23 studies were evaluated as high quality and 17 as low quality. related with gender (RR, 0.94; 95% CI, 0.87–1.02, P ¼ 0.116;

Figure 2. Forest plot of meta-analysis of OS on univariate analysis in association with HIF2a expression.

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random-effects model: c2 ¼ 15.67, I2 ¼ 0%, P ¼ 0.476). However, there was significant association of high HIF2a expression the overexpression of HIF2a was significantly associated with with lower OS on multivariate analysis (HR, 2.21; 95% CI, tumor differentiation (RR, 1.42; 95% CI, 1.23–1.65, P < 0.001; 1.70–2.87, P < 0.001; heterogeneity: random-effects model: random-effects model: c2 ¼ 22.77, I2 ¼ 56.1%, P ¼ 0.012), TNM c2 ¼ 90.39, I2 ¼ 73.4%, P < 0.001). stage (RR, 1.50; 95% CI, 1.35–1.66, P < 0.001; random-effects model: c2 ¼ 10.41, I2 ¼ 13.5%, P ¼ 0.319), and lymph metastasis Test of heterogeneity (RR, 1.38; 95% CI, 1.24–1.54, P < 0.001; random-effects model: There was some evidence for heterogeneity in OS data with c2 ¼ 8.35, I2 ¼ 4.2%, P ¼ 0.400). both univariate analysis and multivariate analysis; therefore, we created Galbraith plots to identify potential sources of Correlation between HIF2a and OS in patients with cancer heterogeneity. As shown in Supplementary Fig. S2A, the studies Thirty-five studies demonstrated the association between by Biswas, Sun, Szendroi, and Xie should be the main con- HIF2a expression and OS with univariate analysis in human tributors to heterogeneity in OS data with univariate analysis. cancer patients. As shown in Fig. 2, relevant results indicated When these four studies were omitted, the statistical signifi- that HIF2a overexpression was markedly correlated with a cance of the combined HRs was not substantially altered, but I2 poor OS survival with univariate analysis among cancer decreased from 58.3% to 0% (Supplementary Fig. S3A). Cor- patients (HR, 1.64; 95% CI, 1.41–1.92, P < 0.001; heteroge- respondingly, the main sources of heterogeneity in OS data neity: random-effects model: c2 ¼ 81.51, I2 ¼ 58.3%, P < with multivariate analysis were studies by Sun, Wang, Xie, and 0.001). In addition, we further analyzed the relationship Yao (Supplementary Fig. S2B). After omission of the above between expression of HIF2a and OS with multivariate anal- four studies, I2 decreased from 73.4% to 37.4% (Supplemen- ysis among 25 studies. As shown in Fig. 3, results indicated that tary Fig. S3B).

Figure 3. Forest plot of meta-analysis of OS on multivariate analysis in association with HIF2a expression.

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Table 3. Subgroup analysis for HIF2a and OS on univariate analysis in cancer patients Test for Number of Pooled data heterogeneity Subgroups studies (n) Case (n) HIF2aþ (n) HIF2aþ (%) HR 95% CI P value P value I2 (%) Region Asian 26 2,713 1,442 53.2 1.75 1.44–2.11 <0.001 <0.001 61.9 Non-Asian 9 1,135 490 43.2 1.40 1.08–1.82 0.011 0.081 42.9 Histologic type HCC 5 889 499 56.1 1.86 0.98–3.51 0.058 <0.001 88.5 Lung cancer 5 488 229 46.9 1.89 1.46–2.44 <0.001 0.348 10.2 RCC 5 390 163 41.8 0.91 0.58–1.48 0.703 0.020 65.8 CRC 3 379 154 40.6 1.72 1.21–2.43 0.002 0.536 0 Pancreatic cancer 3 443 264 59.6 1.97 1.42–2.75 <0.001 0.268 24 Gastric and esophageal cancers 2 299 175 58.5 1.65 1.21–2.26 0.002 0.776 0 HNSCC 2 337 137 40.7 1.79 1.26–2.53 0.001 0.927 0 Neuroblastoma 2 102 38 37.3 2.53 1.29–4.93 0.007 0.923 0 OSCC 2 153 82 53.6 1.14 0.73–1.80 0.567 0.638 0 Othera 6 368 191 51.9 1.74 1.27–2.39 0.001 0.761 0 Case (n) <100 19 1,232 625 50.7 1.77 1.35–2.33 <0.001 0.001 57.9 100 16 2,616 1,307 50.0 1.57 1.30–1.90 <0.001 0.001 61.2 Follow-up (months) <100 24 2,672 1,441 53.9 1.80 1.48–2.20 <0.001 <0.001 63.3 100 11 1,176 491 41.8 1.36 1.10–1.67 0.004 0.165 29.5 Quality High 21 2,760 1,420 51.4 1.70 1.40–2.07 <0.001 <0.001 62.7 Low 14 1,088 512 47.1 1.54 1.18–2.02 0.002 0.013 51.8 Abbreviations: CRC, colorectal cancer; HCC, hepatocellular cancer; HIF2aþ, hypoxia-inducible factor-2a–positive expression; HNSCC, head and neck squamous cell cancer; OSCC, oral squamous cell carcinoma; RCC, renal cell cancer. a"Other" included chondrosarcoma, salivary duct carcinoma (SDC), endometrial carcinoma, breast cancer, epithelial ovarian cancer, and diffuse large B-cell lymphoma (DLBCL).

Subgroup analyses 0.022). The significant associations also exist in other subgroups, To further explore the sources of high heterogeneity, we con- including the Asian (HR, 2.46; 95% CI, 1.68–3.62, P < 0.001), ducted subgroup analysis for OS data with univariate analysis non-Asian (HR, 1.72; 95% CI, 1.40–2.12, P < 0.001), protein (Table 3; Supplementary Fig. S4A–S4E) according to region, expression of HIF2a (HR, 2.23; 95% CI, 1.69–2.93, P < 0.001), histologic type, number of cases, months of follow-up, and smaller cases (n < 100; HR, 2.63; 95% CI, 1.75–3.96, P < 0.001), quality of the included study. Based on histologic type, HIF2a larger cases (n 100; HR, 1.91; 95% CI, 1.42–2.56, P < 0.001), overexpression resulted in poor OS in lung cancer (HR, 1.89; 95% shorter follow-up time (n < 100; HR, 2.71; 95% CI, 1.74–4.24, P < CI, 1.46–2.44, P < 0.001), colorectal cancer (HR, 1.72; 95% CI, 0.001), longer follow-up time (n 100; HR ¼ 1.90; 95% CI, 1.44– 1.21–2.43, P ¼ 0.002), pancreatic cancer (HR, 1.97; 95% CI, 1.42– 2.51, P < 0.001), high quality (HR, 2.28; 95% CI, 1.71–3.05, P < 2.75, P < 0.001), gastric and esophageal cancers (HR, 1.65; 95% 0.001), and low quality (HR, 1.94; 95% CI, 1.14–3.31, P ¼ 0.015). CI, 1.21–2.26, P ¼ 0.002), HNSCC (HR, 1.79; 95% CI, 1.26–2.53, P ¼ 0.001), and neuroblastoma (HR, 2.53; 95% CI, 1.29–4.93, Sensitivity analyses and publication bias P ¼ 0.007). Clinical associations between HIF2a and OS with Sensitivity analysis was conducted through the sequential univariate analysis were found in the Asian (HR, 1.75; 95% CI, omission of individual studies. Furthermore, no single study 1.44–2.11, P < 0.001), non-Asian (HR, 1.40; 95% CI, 1.08–1.82, could essentially change the results in both univariate analysis P ¼ 0.011), smaller cases (n < 100; HR, 1.77; 95% CI, 1.35–2.33, (Supplementary Fig. S6A) and multivariate analysis (Supple- P < 0.001), larger cases (n 100; HR, 1.57; 95% CI, 1.30–1.90, mentary Fig. S6B) of OS, demonstrating that the results of this P < 0.001), shorter follow-up time (n < 100; HR, 1.80; 95% CI, meta-analysis were statistically stable. In addition, no evidence 1.48–2.20, P < 0.001), longer follow-up time (n 100; HR, 1.36; for significant publication bias was found for OS with univar- 95% CI, 1.10–1.67, P ¼ 0.004), high quality (HR, 1.70; 95% CI, iate analysis (Fig. 4A; Begg P ¼ 0.293 and Egger P ¼ 0.345) 1.40–2.07, P < 0.001), and low quality (HR, 1.54; 95% CI, 1.18– or multivariate analysis (Fig. 4B; Begg P ¼ 0.059 and Egger 2.02, P ¼ 0.002). P ¼ 0.060). In addition, to demonstrate the predictive role of HIF2a in multivariate analysis, subgroup analysis was conducted based on patients' region, histologic type, number of cases, months of Discussion follow-up, quality of the included study, and expression type of Mammalian cells need oxygen to maintain efficient energy HIF2a (Table 4; Supplementary Fig. S5A–S5F). After stratifying supply, and lack of oxygen can eventually lead to cell death due by histologic type, significantly poor OS were obtained in HCC to impaired energy-requiring processes (57). A hypoxic micro- (HR, 3.35; 95% CI, 1.32–8.50, P ¼ 0.011), lung cancer (HR, environment in tumors represents one of the major obstacles 3.94; 95% CI, 1.47–10.58, P ¼ 0.006), breast cancer (HR, 1.78; for effective cancer therapies (58). HIF2a is a type of HIF that 95% CI, 1.19–2.67, P ¼ 0.005), colorectal cancer (HR, 2.78; 95% mediates transcriptional response to hypoxia stress. Many lines CI, 1.23–6.31, P ¼ 0.014), HNSCC (HR, 1.85; 95% CI, 1.10–3.11, of evidence suggest that the expression level of HIF2a was P ¼ 0.021), and sarcomas (HR, 2.42; 95% CI, 1.14–5.14, P ¼ enhanced and predicted poor survival in a variety of cancers,

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Table 4. Subgroup analysis for HIF2a and OS on multivariate analysis in cancer patients Test for Number of Pooled data heterogeneity Subgroups studies (n) Case (n) HIF2aþ (n) HIF2aþ (%) HR 95% CI P value P value I2 (%) Region Asian 17 2020 1,139 56.4 2.46 1.68–3.62 <0.001 <0.001 80.7 Non-Asian 8 1,093 356 32.6 1.72 1.40–2.12 <0.001 0.532 0 Histologic type HCC 5 826 529 64.0 3.35 1.32–8.50 0.011 <0.001 91.6 RCC 4 426 84 19.7 1.19 0.56–2.50 0.654 0.012 72.4 Lung cancer 3 297 128 43.1 3.94 1.47–10.58 0.006 0.410 0 Pancreatic cancer 3 443 264 59.6 2.34 0.87–6.28 0.091 0.110 60.9 Breast cancer 2 241 107 44.4 1.78 1.19–2.67 0.005 0.280 14.2 CRC 2 292 128 43.8 2.78 1.23–6.31 0.014 0.177 45.2 HNSCC 2 337 137 40.7 1.85 1.10–3.11 0.021 0.161 45.2 Sarcomasa 2 116 60 51.7 2.42 1.14–5.14 0.022 0.016 75.8 Otherb 2 135 58 43.0 2.45 1.28–4.66 0.007 0.378 0 Expression Protein 23 2,863 1,453 50.8 2.23 1.69–2.93 <0.001 <0.001 74.0 mRNA 2 250 42 16.8 2.30 0.63–8.37 0.205 0.031 78.4 Case (n) <100 11 755 399 52.8 2.63 1.75–3.96 <0.001 0.003 61.3 100 14 2,358 1,096 46.5 1.91 1.42–2.56 <0.001 <0.001 72.0 Follow-up (months) <100 18 2,275 1,234 54.2 2.71 1.74–4.24 <0.001 0.003 63.1 100 7 838 261 31.1 1.90 1.44–2.51 <0.001 <0.001 69.9 Quality High 17 2,277 1,169 51.3 2.28 1.71–3.05 <0.001 <0.001 63.5 Low 8 836 326 39.0 1.94 1.14–3.31 0.015 <0.001 83.9 Abbreviations: CRC, colorectal cancer; HCC, hepatocellular cancer; HIF2aþ, hypoxia-inducible factor-2a–positive expression; HNSCC, head and neck squamous cell cancer; RCC, renal cell cancer. a"Sarcomas" included chondrosarcoma and osteosarcoma. b"Other" included malignant astrocytoma and diffuse large B-cell lymphoma (DLBCL). such as lung cancer (16), colorectal cancer (26), breast cancer (59), and osteosarcoma (32). Conversely, some studies have indicated that HIF2a have a significant impact on the development of tumors, and increased HIF2a expression could be a favorable prognostic factor in RCC (43) and HCC (21). Moreover, clinicopathologic significance of HIF2a in cancer patients remains inconclusive. Some studies indicated that HIF2a overexpression might predict worse tumor differentiation, higher TNM stage, and higher lymph metastasis (17, 25, 54). However, some reports found that there was no obvious association between HIF2a and the above clinicopathologic features (16, 27, 36). Although one meta-analysis has reported on the prognostic role of HIF2a in cancer, it lacks test of heterogeneity, publication bias, and correlation analysis between HIF2a and clinicopathologic features. In this meta-analysis, a total of 854 studies with 4,345 patients were obtained. Our data indicated that the overexpression of HIF2a was significantly correlated with the prognosis of cancer patients. High expression of HIF2a could predict unfavorable OS of cancer patients on both univariate and multivariate analyses (P < 0.001 and P < 0.001, respectively). Furthermore, significant results were observed in the subgroup analyses of OS on both univariate and multivariate analyses by region, histologic type, number of cases, months of follow-up, quality of the included study, and expression type of HIF2a. Meanwhile, we investigated the relationship between high HIF2a expression and clinicopathologic features. The results indicated that HIF2a overexpression was associated closely with worse tumor differentiation, higher TNM stage, and higher lymph metastasis. In addition, there was Figure 4. no obvious evidence for significant publication bias on OS with Begg funnel plot of the effect of HIF2a expression on OS with univariate both univariate and multivariate analyses. analysis (A) and multivariate (B) analysis.

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It is well known that oxygen is important for tumor cell survival Although we executed exhaustive meta-analysis, our study had and lack of oxygen would cause cell death. Tumor cell was able to some limitations. First, all the studies included were retrospective adapt to the hypoxia environment through reducing energy articles, and randomized controlled trials were not found. Second, consumption and increasing anaerobic metabolism. Hypoxia- although being modified for heterogeneity by the application of inducible transcription factors, especially HIF2a, play a critical random-effect model, subgroup analyses, and sensitivity analyses, role in the adaptation process. Tumor hypoxia and high HIF2a there was still some heterogeneity in some subgroups. Third, the protein levels are frequently related with aggressive disease and studies included in this meta-analysis were reported only in the represent major obstacles to effective cancer therapies (60). A English and Chinese languages, and studies reported in other previous study has shown that targeting HIF2a had great clinical languages were omitted. potential by affecting tumor spread and transition into advanced In summary, this meta-analysis revealed that overexpression clinical stages (61). of HIF2a was positively associated with clinicopathologic HIF2a is known to regulate proteins involved in cell prolifer- features and poor OS of cancer patients, suggesting that HIF2a ation, cell growth, cellular glucose transport, and angiogenesis. might be a significant biomarker for cancer diagnosis and Evidence indicates that HIF2a is involved mainly in metastasis prognosis. and chemoresistance in advanced cancers (61). Recently, we have a seen great progress being made in understanding HIF2 -medi- Disclosure of Potential Conflicts of Interest ated cancer pathways. Overexpression of angiogenic VEGF and No potential conflicts of interest were disclosed. EphA2 markers, which were mediated by HIF2a, is well correlated with tumor recurrence and progression in patients with HCC. Acknowledgments a Moreover, activation of HIF2 promoted PKC-mediated tumor This work was supported by the Natural Science Foundation of Zhangzhou, cell migration (62). HIF2a could promote epithelial–mesenchy- Fujian, China (ZZ2017J36 to H.Y. Ren) and the Youth Nursery Foundation of mal transition by regulating the expression of N-cadherin and the Affiliated Southeast Hospital of Xiamen University, Zhangzhou, Fujian, E-cadherin via activation of the MEK/ERK pathway in gastric China (16Y012 to D.Q. Luo). cancer (63). Furthermore, HIF2a increased multidrug resistance of stomach cancer cells by directly activating the pregnane X The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked receptor (PXR) signaling pathway (64). In addition, HIF2a pro- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate moted colon cancer growth by potentiating Yes-associated pro- this fact. tein 1 (YAP1) activity, suggesting that this pathway might be targeted in potential anticancer approaches for treating colorectal Received August 8, 2018; revised October 12, 2018; accepted December 4, cancer patients (65). 2018; published first December 27, 2018.

References 1. Patel A, Sant S. Hypoxic tumor microenvironment: opportunities to 13. Raspaglio G, Petrillo M, Martinelli E, Li Puma DD, Mariani M, De Donato develop targeted therapies. Biotechnol Adv 2016;34:803–12. M, et al. Sox9 and Hif-2a regulate TUBB3 gene expression and affect 2. Maynard MA, Ohh M. The role of hypoxia-inducible factors in cancer. ovarian cancer aggressiveness. Gene 2014;542:173–81. Cell Mol Life Sci 2007;64:2170–80. 14. Mira E, Carmona-Rodriguez L, Perez-Villamil B, Casas J, Fernandez- 3. Maes C, Carmeliet G, Schipani E. Hypoxia-driven pathways in bone Acenero MJ, Martinez-Rey D, et al. SOD3 improves the tumor response development, regeneration and disease. Nat Rev Rheumatol 2012;8: to chemotherapy by stabilizing endothelial HIF-2alpha. Nat Commun 358–66. 2018;9:575. 4. Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors and the 15. Choudhry H, Albukhari A, Morotti M, Haider S, Moralli D, Smythies J, et al. response to hypoxic stress. Mol Cell 2010;40:294–309. Tumor hypoxia induces nuclear paraspeckle formation through HIF- 5. Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry 2alpha dependent transcriptional activation of NEAT1 leading to cancer in hypoxic tumour growth and progression. Nat Rev Cancer 2011;12:9–22. cell survival. Oncogene 2015;34:4546. 6. Ratcliffe PJ. HIF-1 and HIF-2: working alone or together in hypoxia? 16. Gao ZJ, Wang Y, Yuan WD, Yuan JQ, Yuan K. HIF-2 alpha not HIF-1 alpha J Clin Invest 2007;117:862–5. overexpression confers poor prognosis in non-small cell lung cancer. 7. Han S, Huang T, Li W, Liu S, Yang W, Shi Q, et al. Association between Tumor Biol 2017;39:1–8. hypoxia-inducible factor-2alpha (HIF-2alpha) expression and colorectal 17. Tong WW, Tong GH, Chen XX, Zheng HC, Wang YZ. HIF2 alpha is cancer and its prognostic role: a systematic analysis. Cell Physiol Biochem associated with poor prognosis and affects the expression levels of 2018;48:516–27. survivin and cyclin D1 in gastric carcinoma. Int J Oncol 2015;46: 8. Li C, Lu HJ, Na FF, Deng L, Xue JX, Wang JW, et al. Prognostic role of hypoxic 233–42. inducible factor expression in non-small cell lung cancer: a meta-analysis. 18. Yang J, Zhu DM, Zhou XG, Yin N, Zhang Y, Zhang ZX, et al. HIF-2 alpha Asian Pac J Cancer Prev 2013;14:3607–12. promotes the formation of vasculogenic mimicry in pancreatic cancer by 9. Wiesener MS, Jurgensen JS, Rosenberger C, Scholze CK, Horstrup JH, regulating the binding of Twist1 to the VE-cadherin promoter. Oncotarget Warnecke C, et al. Widespread hypoxia-inducible expression of HIF-2alpha 2017;8:47801–15. in distinct cell populations of different organs. FASEB J 2003;17:271–3. 19. Wang HX, Qin C, Han FY, Wang XH, Li N. HIF-2 alpha as a prognostic 10. Koh MY, Powis G. Passing the baton: the HIF switch. Trends Biochem Sci marker for breast cancer progression and patient survival. Genet Mol Res 2012;37:364–72. 2014;13:2817–26. 11. Yang J, Zhang X, Zhang Y, Zhu D, Zhang L, Li Y, et al. HIF-2a promotes 20. Xie M, He CS, Huang JK, Lin QZ. Phase II study of pazopanib as second-line epithelial-mesenchymal transition through regulating Twist2 binding to treatment after sunitinib in patients with metastatic renal cell carcinoma: a the promoter of E-cadherin in pancreatic cancer. J Exp Clin Cancer Res Southern China Urology Cancer Consortium Trial. Eur J Cancer 2015;51: 2016;35:26. 595–603. 12. Wu L, Yu H, Zhao Y, Zhang C, Wang J, Yue X, et al. HIF-2a mediates 21. Sun HX. The mechanism and clinical signiﬁcance of hypoxia inducible hypoxia-induced LIF expression in human colorectal cancer cells. factor-2a inducing apoptosis by TFDP3. Chinese doctor's dissertation of Oncotarget 2015;6:4406–17. Fudan University; 2011.

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22. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gotzsche PC, Ioannidis JP, 41. Sathornsumetee S, Cao Y, Marcello JE, Herndon JE 2nd, McLendon RE, et al. The PRISMA statement for reporting systematic reviews and meta- Desjardins A, et al. Tumor angiogenic and hypoxic profiles predict radio- analyses of studies that evaluate health care interventions: explanation and graphic response and survival in malignant astrocytoma patients treated elaboration. J Clin Epidemiol 2009;62:e1–34. with bevacizumab and irinotecan. J Clin Oncol 2008;26:271–8. 23. Bangoura G, Liu ZS, Qian Q, Jiang CQ, Yang GF, Jing S. Prognostic 42. Sivridis E, Giatromanolaki A, Gatter KC, Harris AL, Koukourakis MI. significance of HIF-2alpha/EPAS1 expression in hepatocellular carcinoma. Association of hypoxia-inducible factors 1alpha and 2alpha with activated World J Gastroenterol 2007;13:3176–82. angiogenic pathways and prognosis in patients with endometrial carcino- 24. Biswas S, Charlesworth PJS, Turner GDH, Leek R, Thamboo TP, Campo L, ma. Cancer 2002;95:1055–63. et al. CD31 angiogenesis and combined expression of HIF-1 alpha and HIF- 43. Szendroi A, Szasz AM, Kardos M, Tokes AM, Idan R, Szucs M, et al. Opposite 2 alpha are prognostic in primary clear-cell renal cell carcinoma (CC-RCC), prognostic roles of HIF1alpha and HIF2alpha expressions in bone meta- but HIF alpha transcriptional products are not: implications for antiangio- static clear cell renal cell cancer. Oncotarget 2016;7:42086–98. genic trials and HIF alpha biomarker studies in primary CC-RCC. 44. Wang XC, Yuan ZN, Li SS, Sheng Y, Zhao TS, Gao S, et al. Expression of Carcinogenesis 2012;33:1717–25. hypoxia-inducible factor-2a in hepatocellular carcinoma and its clinical 25. Chen CB, Ma QJ, Ma XL, Liu ZJ, Liu XG. Association of elevated HIF-2 alpha significance. Chinese J Clin Oncol 2011;38:560–3. levels with low Beclin 1 expression and poor prognosis in patients with 45. Wang Q, Peng K, He L. Expression of the HIF-2a in epithelial ovarian cancer chondrosarcoma. Ann Surg Oncol 2011;18:2364–72. and clinical significance. J Cent South Univ 2014;39:889–93. 26. Cleven AH, van Engeland M, Wouters BG, de Bruine AP. Stromal expression 46. Wei LF, Liu XL, Hu CH. Correlation between expression of HIF-2a of hypoxia regulated proteins is an adverse prognostic factor in colorectal andOCT-4andprognosisofNSCLC.JCentSouthUniv2011;36: carcinomas. Cell Oncol 2007;29:229–40. 854–8. 27. Griffiths EA, Pritchard SA, McGrath SM, Valentine HR, Price PM, Welch IM, 47. Winter SC, Shah KA, Han C, Campo L, Turley H, Leek R, et al. The relation et al. Hypoxia-associated markers in gastric carcinogenesis and HIF-2alpha between hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expres- in gastric and gastro-oesophageal cancer prognosis. Br J Cancer 2008;98: sion with anemia and outcome in surgically treated head and neck cancer. 965–73. Cancer 2006;107:757–66. 28. Holmquist-Mengelbier L, Fredlund E, Lofstedt T, Noguera R, Navarro S, 48. Yang SL, Liu LP, Jiang JX, Xiong ZF, He QJ, Wu C. The correlation of Nilsson H, et al. Recruitment of HIF-1alpha and HIF-2alpha to common expression levels of HIF-1 and HIF-2 in hepatocellular carcinoma with target genes is differentially regulated in neuroblastoma: HIF-2alpha capsular invasion, portal vein tumor thrombi and patients clinical out- promotes an aggressive phenotype. Cancer Cell 2006;10:413–23. come. Jpn J Clin Oncol 2014;44:159–67. 29. Jubb AM, Turley H, Moeller HC, Steers G, Han C, Li JL, et al. Expression of 49. Yao QY, Pan TT, Xu GL, Li JS, Ma JL, Jia WD, et al. Expression of hypoxia- delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer. inducible factor-2a in hepatocellular carcinoma and its relationship with Br J Cancer 2009;101:1749–57. epithelial–mesenchymal transition and angiogenesis. Acta Univ Meds 30. Koukourakis MI, Bentzen SM, Giatromanolaki A, Wilson GD, Daley FM, Anhui 2015;491–4. Saunders MI, et al. Endogenous markers of two separate, hypoxia 50. Yoshimura H, Dhar DK, Kohno H, Kubota H, Fujii T, Ueda S, et al. response pathways (hypoxia inducible factor 2 alpha and carbonic Prognostic impact of hypoxia-inducible factors 1alpha and 2alpha in anhydrase 9) are associated with radiotherapy failure in head and neck colorectal cancer patients: correlation with tumor angiogenesis and cyclo- cancer patients recruited in the CHART randomized trial. J Clin Oncol oxygenase-2 expression. Clin Cancer Res 2004;10:8554–60. 2006;24:727–35. 51. Yuan K, Zheng RH. The correlation between the expression of HIF-2a 31. Leek RD, Talks KL, Pezzella F, Turley H, Campo L, Brown NS, et al. Relation and the proliferation of tumor and its impaction on the prognosis in of hypoxia-inducible factor-2 alpha (HIF-2 alpha) expression in tumor- human non-small cell lung cancer. Chinese J Clin Med 2009;16: infiltrative macrophages to tumor angiogenesis and the oxidative thymi- 194–7. dine phosphorylase pathway in Human breast cancer. Cancer Res 2002;62: 52. Yun F, Jia YF, Han S, Guo ZL, Yuan HW, Shi L. Expression of hypoxia- 1326–9. inducible factor-2a in human non-small cell lung cancer and its clinico- 32. Li W, He XJ, Xue RL, Zhang Y, Zhang XQ, Lu JR, et al. Combined over- pathologic feature. Chinese J Cancer Prev Treat 2016;860–5. expression of the hypoxia-inducible factor 2 alpha gene and its long non- 53. Zang L. The expression and prognostic significance of hypoxia inducible coding RNA predicts unfavorable prognosis of patients with osteosarcoma. factor -2a in diffuse large B-cell lymphoma. Chinese doctor's dissertation of Pathol Res Pract 2016;212:861–6. Tianjin Medical University; 2016. 33. Li WZ, Chen CH, Zhao XH, Ye HL, Zhao Y, Fu ZQ, et al. HIF-2 alpha 54. Zhang Q, Lou Y, Zhang JY, Fu QH, Wei T, Sun X, et al. Hypoxia-inducible regulates non-canonical glutamine metabolism via activation of PI3K/ factor-2a promotes tumor progression and has crosstalk with Wnt/beta- mTORC2 pathway in human pancreatic ductal adenocarcinoma. catenin signaling in pancreatic cancer. Mol Cancer 2017;16:119. J Cell Mol Med 2017;21:2896–908. 55. Zhou JC, Wu KJ, Gao DX, Zhu GD, Wu DP, Wang XY, et al. Reciprocal 34. Lim E, Kuo CC, Tu HF, Yang CC. The prognosis outcome of oral squamous regulation of hypoxia-inducible factor 2 alpha and GLI1 expression asso- cell carcinoma using HIF-2alpha. J Chin Med Assoc 2017;80:651–6. ciated with the radioresistance of renal cell carcinoma. Int J Radiat 35. Liu YZ. Correlation between protein expression of HIF-2a, HSP90 Oncol Biol Phys 2014;90:942–51. and VEGF in human renal clear cell carcinoma. Chinese master's disser- 56. Zhu GQ, Tang YL, Li L, Zheng M, Jiang J, Li XY, et al. Hypoxia inducible tation of Suzhou University; 2009. factor 1alpha and hypoxia inducible factor 2alpha play distinct and 36. Luan YJ, Gao CC, Miao YJ, Li YL, Wang ZY, Qiu XF. Clinicopathological and functionally overlapping roles in oral squamous cell carcinoma. prognostic significance of HIF-1 alpha and HIF-2 alpha expression in small Clin Cancer Res 2010;16:4732–41. cell lung cancer. Pathol Res Pract 2013;209:184–9. 57. Kaelin WG Jr, Ratcliffe PJ. Oxygen sensing by metazoans: the central role of 37. Pan TT. Clinical significance and molecular mechanism of hypoxia the HIF hydroxylase pathway. Mol Cell 2008;30:393–402. inducible factor 2a in hepatocellular carcinoma. Chinese doctor's disser- 58. Ramakrishnan SK, Shah YM. Role of intestinal HIF-2alpha in health and tation of Tianjin Medical University; 2015. disease. Annu Rev Physiol 2016;78:301–25. 38.QuH,ZhengL,SongH,JiaoW,LiD,FangE,etal.microRNA-558 59. Blancher C, Moore JW, Talks KL, Houlbrook S, Harris AL. Relationship of facilitates the expression of hypoxia-inducible factor 2 alpha through hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression to binding to 50-untranslated region in neuroblastoma. Oncotarget 2016; vascular endothelial growth factor induction and hypoxia survival in 7:40657–73. human breast cancer cell lines. Cancer Res 2000;60:7106–13. 39. Roh JL, Cho KJ, Kwon GY, Choi SH, Nam SY, Kim SY. Prognostic values of 60. Pietras A, Johnsson AS, Pahlman S. The HIF-2alpha-driven pseudo-hypoxic pathologic findings and hypoxia markers in 21 patients with salivary duct phenotype in tumor aggressiveness, differentiation, and vascularization. carcinoma. J Surg Oncol 2008;97:596–600. Curr Top Microbiol Immunol 2010;345:1–20. 40. Sandlund J, Ljungberg B, Wikstrom P, Grankvist K, Lindh G, Rasmuson T. 61. Murugesan T, Rajajeyabalachandran G, Kumar S, Nagaraju S, Jegatheesan Hypoxia-inducible factor-2 alpha mRNA expression in human renal cell SK. Targeting HIF-2alpha as therapy for advanced cancers. Drug Discov carcinoma. Acta Oncol (Madr) 2009;48:909–14. Today 2018;23:1444–51.

www.aacrjournals.org Cancer Epidemiol Biomarkers Prev; 28(5) May 2019 OF9

Luo et al.

62. Emerling BM, Benes CH, Poulogiannis G, Bell EL, Courtney K, Liu H, et al. 64. Zhao J, Bai Z, Feng F, Song E, Du F, Zhao J, et al. Cross-talk between EPAS-1/ Identiﬁcation of CDCP1 as a hypoxia-inducible factor 2alpha (HIF- HIF-2alpha and PXR signaling pathway regulates multi-drug resistance of 2alpha) target gene that is associated with survival in clear cell renal cell stomach cancer cell. Int J Biochem Cell Biol 2016;72:73–88. carcinoma patients. Proc Natl Acad Sci U S A 2013;110:3483–8. 65. Ma X, Zhang H, Xue X, Shah YM. Hypoxia-inducible factor 2alpha (HIF- 63. Tong WW, Tong GH, Kong H, Liu Y. The tumor promoting roles of HSP60 2alpha) promotes colon cancer growth by potentiating Yes-associated and HIF2alpha in gastric cancer cells. Tumour Biol 2016;37:9849–54. protein 1 (YAP1) activity. J Biol Chem 2017;292:17046–56.

OF10 Cancer Epidemiol Biomarkers Prev; 28(5) May 2019 Cancer Epidemiology, Biomarkers & Prevention

The Clinicopathologic and Prognostic Value of Hypoxia-Inducible Factor-2 α in Cancer Patients: A Systematic Review and Meta-Analysis

Deqing Luo, Hui Liu, Dasheng Lin, et al.

Cancer Epidemiol Biomarkers Prev Published OnlineFirst December 27, 2018.

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