FOXA1 Expression May Serve as A Prostate Cancer Biomarker

Dr. Md. Mizanur Rahman
Genetics, Innovation, Molecular Therapeutics, Patient

03

Sep 24

Abstract: 

Background of the study: Prostate adenocarcinoma (PRAD) ranks as the fifth most common cause of death globally and is the second most common cancer in males. Early-stage prostate cancer may not have any symptoms and early diagnosis remains challenging.

Method and materials: Here, we propose the forkhead box A1 (FOXA1) gene as a biomarker using the freely accessible public TCGA dataset and bioinformatics tools cBioportal, UALCAN, and GEPIA2.

Results: In 28% of PRAD cases, the FOXA1 gene was mutated. The overexpression of FOXA1 is dependent on PRAD patients, race, Gleason score, and molecular signature. The FOXA1 gene influences both overall and disease-free survival.

Discussion: FOXA1 could be used as diagnostic biomarker in PRAD.

Conclusion: Further research was required to validate our proposal.

Keywords: FOXA1, PRAD, TCGA, cBioportal, UALCAN, GEPIA2.

Introduction

In 2022, prostate adenocarcinoma (PRAD) was the fifth largest cause of cancer-related death in males and the second most common cancer globally with an anticipated 1.5 million new cases and 397,000 deaths worldwide.1 As per GLOBOCAN 2020, there were 4728 instances in Bangladesh, which is equivalent to 1.4/100000.2 The underlying genetic and demographic factors for such high frequency of PRAD is inclusive of gene variation, susceptibility, age, race, and family history.3 In its clinical course, PRAD exhibits significant variety. While some individual’s cancers are extremely aggressive, spreading quickly to metastases and resistant to treatment, others have slow-growing, indolent cancer that will never spread. Currently, the biopsy Gleason Score, clinical stages, and prostate-specific antigen (PSA) levels are the main markers used to classify patients into risk groups at the time of initial evaluation. However, these diagnostics have lower sensitivity and specificity, which affects the selection of an appropriate treatment.4 The heterogeneity of PRAD was discovered with the growing understanding of prostate cancer, and despite the high death rate from the disease, early diagnosis proved to be quite successful.5 Numerous functions for FOXA1 have been demonstrated in the development of organs as well as in a number of illnesses, such as prostate and breast cancer.6,7 Biomarkers are molecules or genetic markers that can be used to assess and indicate the existence or progression of a disease, biological process, or treatment response, and are essential to molecular biology. They are crucial for comprehending the molecular mechanisms behind many biological processes and can offer important insights into illness diagnosis, prognosis, and treatment.

FOXA1 also known as HNF3A; TCF3A is a Protein Coding Gene, encodes a member of the forkhead class of DNA-binding proteins, is located in chromosome 14 at the cytogenetic band: 14q21.1, and consists of 473 amino acids.8  It is a pioneer transcription factor that opens sites in the compacted chromatin for other transcription factors by interacting with the nucleosomal histones.9

It regulates tissue-specific gene expression and is involved in embryonic development.9 Prostate, breast, bladder, and salivary gland tumors that are driven by hormone receptors frequently have mutations in FOXA1.10

Figure 1: Chromosomal location of FOXA1 gene. [Red line denotes FOXA1 location] 8

Condensed chromatin is unbound by FOXA1, which facilitates the recruitment of additional transcription factors to the DNA. In order to modify cell proliferation and response to endocrine treatments or phosphoinositide 3-kinase (PI3K) inhibitors, FOXA1 reprogrammed ER recruitment at cis regulatory sites.11 In addition to increasing gene expression capacity, FOXA1 is essential for several biological processes, such as drug resistance, proliferation, glycolipid metabolism, organogenesis and differentiation, and migration and invasion.12Changes in the protein levels of FOXA1 are closely linked to the advancement of PRAD. Unfortunately, due to differences in FOXA1 protein levels, increasing FOXA1 mutations at different stages of PRAD, and mysterious post-translational FOXA1 regulatory mechanisms, direct targeting of FOXA1 in progressive PRAD remains difficult.13

Figure 2: Protein assembly and schematic structures of FOXA1 gene. [TA: Transactivation domain]13

Numerous investigations have demonstrated that the great majority of malignancies, including PRAD, have high levels of FOXA1 expression, which is linked to a favorable prognosis.15This study aims to investigate the function of FOXA1 as a novel PRAD biomarker by examining its role in the development and prognosis of PRAD.

Material and Methods

First, we examined FOXA1 gene expression in PRAD using the cBioportal 16 using TCGA datasets. In cBioportal, we selected PRAD and TCGA, Pancancer Atlas, where 494 samples information was deposited. Then we selected FOXA1 as query by gene. Then we searched for mutational status of FOXA1 gene. Then we use UALCAN 17 using TCGA dataset. Here we selected TCGA dataset, gene option as FOXA1 and cancer as Prostate. We extracted results by sample types, patient’s race, patient’s age, patient’s Gleason score, molecular signature and TP53 mutation status. Then we use GEPIA2,18 using TCGA datasets. Here, we analyze FOXA1 gene expression compared between normal tissue and prostate cancer tissue. Then, we evaluated FOXA1 gene expression to examine overall survival and disease-free survival using the bioinformatics tool GEPIA2.

Results

A. 28% prostate adenocarcinoma cases from cBioportal showed FOXA1 mutation:

Using TCGA datasets from cBioportal, FOXA1 was found mutated in 137 cases out of the 488 (28%) prostate adenocarcinoma (PRAD) cases. Among them, mutation occurred in 4.1% (20 cases), structural variant in 0.41% (2 cases), amplification in 1.64% (8 cases), deep deletion in 0.02% (1 cases), mRNA high in 6.97% (34 cases), mRNA low in 12.09% (59 cases), and multiple alteration occurred in 2.66% (13 cases).

Figure 3: FOXA1 gene mutated in 28% of cases.

B. FOXA1 expression analysis using UALCAN from TCGA datasets:

We explored FOXA1 expression from UALCAN-TCGA datasets and observed that FOXA1 was overexpressed in tumor tissue (n=497) than in normal tissue (n=52) where p value is very highly significant (4.9515946898282E-14) (Figure 4A). Regarding race, Caucasians samples (n=147) showed overexpression than normal (n=52) and very highly significant (<1E-12), normal versus African-American (n=6) FOXA1 overexpression was of significant level (3.263100E-03). However, expression level comparison between Caucasian and African-American was not significant (Figure 4B). Regarding age, there is no statistical significance observed (Figure 4C). Gleason score is very vital to categorize prostate cancer. FOXA1 expression comparison between normal (n=52) and Gleason score 6 (n=45) was found to be highly significant (4.02070000005139E-06). FOXA1 expression comparison between normal (n=52) and Gleason score 7 (n=247) was found to be even more significant (6.16839912481737E-13). FOXA1 expression comparison between normal (n=52) and Gleason score 8 (n=64) was also found to be highly significant (1.82080017729902E-10). FOXA1 expression comparison between normal (n=52) and Gleason score 9 (n=136) was found to be less significant (1.91158200379959E-12) than a Gleason score of 7 and 8. Finally, FOXA1 expression comparison between normal (n=52) and Gleason score 10 (n=4) was found to be non-significant (Figure 4D).

Figure 4: Expression of FOXA1 in PRAD based on different parameter using UALCAN.

Molecular signatures are important in various areas of molecular biology, such as in the diagnosis and treatment of diseases, the identification of genetic variants, and the study of biological pathways and processes. In the case of Prostate cancer, not only is FOXA1 mutated, but other genes like ERG, ETV1, ETV4, FLI1, IDH1 and SPOP are also altered. Expression comparison between normal (n=52) and ERG fusion (n=152), ETV1 fusion (n=28), ETV4 fusion (n=14), FLI1 fusion (n=4), and SPOP mutation (n=35) are respectively (1.62447832963153E-12), (1.74680270248473E-11), (4.26919999996223E-06),  (4.113600E-02), and (1.66400004886214E-10) which are all very highly significant.

Expression comparison between normal (n=52) and FOXA1 mutation (n=9) shows very highly significant (2.53069999955891E-07). However, Expression comparison between normal (n=52) and IDH1 mutation (n=3) shows non-significant (Figure 4E).

One important tumor suppressor gene that halts the growth of cancer is the TP53 gene, commonly referred to as the tumor protein p53. A poor prognosis and resistance to therapy are sometimes linked to TP53 mutations, which are prevalent in a variety of cancer types. In Prostate cancer, TP53 mutant (n=38) expression compared to normal (n=52) is very highly significant (3.30702132345095E-12). On the other hand TP53 non-mutant (n=295) compared to normal (N=52) is also very highly significant (2.2992718839987E-13). Interestingly, TP53 mutant and TP53 non-mutant comparison are also statistically significant (9.380100E-04). (Figure 4F).

C. FOXA1 expression analysis using GEPIA2:

To investigate FOXA1 expression in their dataset, we employ GEPIA2 (Gene Expression Profiling Interactive Analysis). Overexpression of FOXA1 was detected in tumors (492) out of 152 normal tissues in the GEPIA2 dataset; the expression value was log2(TPM+1).  Gene expression level is calculated using Log2(TPM 1) in GEPIA2, which is a logarithmic transformation of transcripts per million (TPM). Transcript per kilobase million, or TPM, is the amount of RNA in a sample divided by the RNA sequence by expectation-maximization method, or RSEM. Prostate cancer (red box) has higher levels of FOXA1 expression than normal tissue (grey box); the number of occurrences is indicated by a dot in Figure 1. In this case, the p-value cutoff was 0.01 and the Log2FC (how one sample differs from another) cutoff value was 1. This thorough investigation demonstrates the diagnostic and prognostic importance of TPM in cancer, indicating its potential utility as a biomarker for illness identification and patient outcome prediction (Figure 5).

Figure 5: FOXA1 overexpression has been linked to prostate cancer. Prostate cancer tissue (red) has higher levels of FOXA1 expression than normal tissue (grey), and the dot indicates the incidence rate.

D. Analysis of Overall Survival and Disease-free survival

Using GEPIA2, we classified 492 prostate cancer patients into two groups: 246 patients with high FOXA1 expression and 246 patients with low FOXA1 expression. The default median cutoff value for each group was 50%. The log rank p value was 0.29 for the overall survival hazard ratio (HR) of 2, which indicates a larger risk in relation to the normal group, although there was no discernible difference in survival (log rank p value was 0.65) (Figure 6A). Conversely, the hazard ratio for disease-free survival is 0.83, with a log rank p value of 0.36. Accordingly, a reduced probability of disease recurrence, or a better prognosis, is indicated by a hazard ratio of 0.83, and no significant difference is suggested by a Log rank p value of 0.36 (Figure 6B).

Figure 6: Comparing groups with high and low expression levels of FOXA1 in terms of overall and disease-free survival.  

Discussion

Early disease detection with the use of biomarkers allows for prompt and efficient therapy. Additionally, they can assist in tracking the course of an illness and offer information on its prognosis. Because they offer important data on illness diagnosis, prognosis, treatment, medication development, research, and public health, biomarkers are essential to molecular biology. Prostate cancer is a worldwide health issue, and its incidence varies greatly by region. Although prostate cancer incidence and mortality have been steadily rising throughout Asia, the continent has historically been thought to have low incidence rates.19 Genetic analyses is becoming a more potent technique for finding new possible medication targets and biomarkers. Investigating genetic targets is vital because it would help us understand the mechanisms underlying the development of Prostate cancer and the poor prognosis. In the end, identifying these possible targets will result in a successful treatment strategy. In this article, we will be able to propose that overexpression of FOXA1 may be used as a biomarker in patients with PRAD due to its reduced risk of disease recurrence and mortality. In order to validate our proposal, more investigation was and in future is necessary.

Author of this article

Dr. Md. Mizanur Rahman, MBBS, M.Phil., PhD, Associate Professor, Department of Biochemistry, Rangamati Medical College, Rangamati, Bangladesh.

Phone: +8801819334298. Email: mizan2011bio@gmail.com

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