HIF3A: A Potent Prognostic Biomarker in Different Kinds of Cancer

Background: Hypoxia-inducible factors (HIFs) are transcription factors that get activated and stabilized in the heterodimerized form under hypoxic conditions. The three members of the HIF alpha factors share high structural similarity but have tissue-specific expression patterns. A majority of studies have reported the importance of the HIF1A and HIF2A activity in the survival, proliferation, metastatic potential, and metabolic regulation of hypoxic cancer cells. However, the importance of the expression pattern and activity of HIF3A in a variety of cancers remains unknown. Method and materials: The expression profile of 13 different types of The Cancer Genome Atlas (TCGA) cancer samples were downloaded, normalized and differential gene expression analysis (DGE) was performed to compare the expression pattern of HIF alpha family members in cancer and adjacent normal tissues, as well as at different stages and tumor-sizes. Receiver operating characteristic (ROC) test and survival analysis were carried out to estimate the diagnostic potential of HIF alpha isomers in different cancers, as well as the survival rate of patients with the varying expression levels of HIF alpha factors. Results: The expression status of HIF3A was notably less in all cancer samples in contrast to their adjacent normal tissues. The expression degree of HIF1A varied among distinct types of cancer and the expression degree of HIF2A was lower in nearly all types of cancers. The expression level of HIF alpha isomers did not significantly correlate with different sizes of tumor samples and stages of different tumor tissue samples. HIF3A had very weak diagnostic potential, while HIF2A had better diagnostic potential in most types of cancers compared to HIF1A. Patients who had a higher level of HIF3A had better survival, while the higher expression levels of HIF1A and HIF2A were associated with worse survival in many types of cancers. Conclusion: Our study shows the heterogenous expression pattern of HIF alpha subunits in distinctive kinds of cancers and the influence of HIF3A expression level in the survival of patients with varying types of cancers.


Introduction 71
Hypoxia refers to a state when the concentration of oxygen around the cell's microenvironment is less 72 than 2% mmHg (1). Hypoxic environment can enhance the resisting behavior of solid tumor cells against 73 drugs that are administrated for cancer treatments (2-4). Active dimer of HIF factors is generated when alpha 74 and beta subunits create a dimer whose activity and stability is tightly dependent on the status of oxygen 75 tension in cellular environment (5-7). Active HIF heterodimer is formed between HIF alpha and HIF beta 76 subunits (8,9). HIF subunits share high sequence similarity in their structure and domains. However, HIF 77 beta subunit lacks the ODD domain and is not sensitive to oxygen level, while ODD domain is present in all 78 three of HIF alpha subunits (HIF1A, HIF2A, and HIF3A) and will lead to their degradation under normoxic 79 condition by hydroxylation and ubiquination reactions mediated by PHD and VHL proteins respectively (10- HIF3A has shown tissue-specific expression patterns, but its exact expression pattern in many types of 92 cancer remains unknown. HIF3A has multiple variants(23). The long variants of HIF3A have been shown to 93 be able to heterodimerize with HIF beta subunits, bind to HRE element and perform weak transcriptional 94 activity (24). While the short variant of HIF3A, also known as IPAS (Inhibitory PAS domain) can prevent 95 the transcriptional activity of HIF1A by forming a dimer with HIF1A directly and prevents its binding on 96

HRE elements (25). 97
In order to gain better insight on the expression pattern and importance of HIF3A in cancers, we have 98 taken a bioinformatic approach and performed differential gene expression (DGE) analysis along receiver 99 operating characteristic test and survival analysis on different types of TCGA cancer samples. Our study will 100 help clarifying the expression pattern, diagnostic, and prognostic potential of HIF alpha subunits in diverse 101 kinds of cancer with different stages and sizes.

Differential gene expression analysis 111
Downloaded gene expression data of TCGA cancer samples are in single raw count form. Therefore, the 112 count data was normalized using Voom package in R program and were converted into logarithmic form 113 (log2 ratio). Limma and EdgeR packages were utilized for differential gene expression analysis (DGE). 114 Missing values from gene expression data were removed before DGE analysis. Cut-off of 0.01 was applied 115 for calculation of p-value by t-test for measuring differential expression level of HIF1A, HIF2A, and HIF3A 116 between tumor and normal paired tissue samples along different stages and tumor. sizes of cancer samples. 117

Receiver operating characteristic (ROC) test 118
ROC test is useful for measuring the performance of an interest biomarker in the classification of tumor 119 phenotype from the normal phenotype. To measure and compare the diagnostic potential of HIF1A, HIF2A, 120 and HIF3A in normalized gene expression data of different types of TCGA cancer, the receiver operating 121 characteristic test was performed using GraphPad Prism software (version 8.4) was utilized and ROC curves 122 were generated. 123

Survival analysis 124
In order to reveal the influence of HIF alpha members expression status on the survival rate of patients 125 diagnosed with various kinds of cancer, the median of the gene expression values of each HIF alpha isomer 126 was selected as a cut-off value to group the samples of patients based on their gene expression level. Patients 127 whose gene expression level of different HIF alpha subunits was superior than the median value were 128 considered as 'Higher than median ' class and samples with gene expression status was less than the cut off 129 were considered as 'Lower than the median' class. Survival analysis was operated employing the R tool 130       By DGE analysis, we have shown that the mRNA ratio of the third subunit of HIF alpha is lesser in nearly 204 many kinds of cancers compared to their paired normal tissues. Only one published study has shown that the 205 expression status of HIF3A was great in ovarian cancer tissues (36), a tissue that was not included in the 206 present analysis. Low expression level of HIF3A in prostate adenocarcinoma cells highly correlated with 207 high methylation level in the promoter region of HIF3A gene (37) ( Table 1)

Conclusion 234
In our study, we made an overall new comparison of the expression patterns of all three members of the 235 HIF alpha factors. The expression ratio of HIF3A was little in varying models of cancers and its expression 236 level significantly correlated with better survival of affected patients. However, its diagnostic potential was 237 weaker compared to HIF1A and HIF2A heterodimers. As earlier studies have established a positive 238 association between the expression level of HIF3A with metastatic potential of ovarian cancer and pancreatic 239 cancer cells and the progression of colorectal cancer cells, more extended investigations are desired to define 240 these differences and, more in general, the importance of HIF3A expression and function in distinctive groups 241 of cancer. 242

Ethical Approval and Consent form 243
As the TCGA data base shares its data in public form, approval from the ethics committee was not 244 necessary for this study.

Consent for publication 246
All authors agreed on the submission of the present research to this journal.

Availability of supporting data 248
Supporting and raw data are available upon a reasonable request to the corresponding author.

Competing Interest 250
All the authors affirm that they have no competing financial desire that could influence the work 251 announced in this paper.