Germline HOXB13 mutation p.G84E do not confer an increased bladder or kidney cancer risk in polish population

Introduction

In 2017, cancer caused 26% of deaths among men and 23% among women in Poland. In men one of the most common cancers of the urinary tract is bladder cancer - 7% and kidney cancer - 4%. In women, bladder cancer accounts for less than 1% and kidney cancer for 2.5% of all cancer cases [1].

The exact molecular mechanisms underlying the initiation and progression of bladder or kidney cancer remain largely unknown. High hereditary predisposition for bladder cancer is presents in 4% and for kidney cancer 5% [2, 3]. A number of mechanisms play a large role in the initiation of neoplastic transformation of bladder or kidney cancer, including: mutation of suppressor genes, activation of proto-oncogenes, abnormal and over-expression of oncogenes through amplification and deletion of some regions of chromosomes, and methylation. Thus the genetic basis leading to a better or worse prognosis for survival in cancer patients may be dependent on functional polymorphisms in genes such as genes responsible for tumor transformation, xenobiotic metabolism, oxidative stress, detoxification and DNA repair. Genes whose expression leads to the development of bladder and kidney cancer are being searched for. These are the so-called high penetration genes or so-called low penetration genes whose expression disturbs metabolism and may initiate the process of neoplastic transformation of various organs. To date, no genes with high penetration into bladder cancer or kidney cancer have been identified. The low penetration genes responsible for the metabolism of carcinogenic chemicals, repair and removal of DNA damage include: XRCC1, XRCC3, NAT2, XPD, RAG1, hOGG1 GSTM, NAT2, XRCC3, GSTP1, RAD23B, ERCC2, ERCC5, ERCC1, XPC, GSTT1, ADPRT. The genes encoding enzymes involved in the process of metabolic activation, detoxification and DNA repair are polymorphic and there are some structural variants of genes in the population associated with different activity of the enzyme they encode.

The HOX gene family belongs to the homeobox superfamily of transcription factors. HOX consensus binding sites were found in the p53 promoter [4]. A disorder of the cell cycle and mutations in the TP53 gene were identified in 50% of patients with bladder cancer [5, 6]. A mutation in the TP53 gene overexpresses the p53 protein and increases the risk of tumor progression [7]. It is noteworthy that the HOXB13 gene is located on chromosome 17q, the loss of heterozygosity of which has been noted in cancer of the kidney, breast, ovary, colon and some haematological malignancies. In turn more copies of chromosomes have been observed in bladder cancer or neuroblastoma [8–15]. Altered expression of HOX genes may be important for oncogenesis and tumor suppression by influencing various pathways that promote tumorigenesis and metastasis. HOX and homeobox genes play an important role in the regulation of many processes including cell proliferation, differentiation, angiogenesis, receptor signaling, apoptosis and regulate transcription of target androgen receptor genes [16]. Abnormalities in the expression of the HOX gene have been reported in abnormal development and malignant neoplasms of breast, leukemia, prostate, cervix, ovary, bladder, kidney, neuroblastoma, lung and esophageal squamous cell carcinoma [17]. HOX genes showed pro-angiogenic properties through increased expression of CXCL1, FGF2, IL8 and VEGFA which suggests a role in tumor progression [18].

In Poland, mutations in HOXB13 are the cause of 0.6% unselected cases of prostate cancer [19]. The bladder and prostate derive from the same embryological structure, the urogenital sinus [20]. To determine whether the G84E mutation in the HOXB13 gene contributes to the development of bladder or kidney cancer in Poland, and to measure the impact of this variant on cancer risk and on survival, we genotyped 1418 unselected bladder patients and 813 unselected kidney cancer cases and 4497 healthy controls.

Material

Methods

DNA was extracted from peripheral blood for all cases and controls. The presence of the p.G84E was genotyped as described previously [19]. In brief, we assessed this variant by genotyping using a TaqMan assay (Life Technologies, Carlsbad, CA) in a LightCycler Real-Time PCR 480 System (Roche Life Science, Mannheim, Germany). All mutations were confirmed by Sanger sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies), according to the manufacturer’s protocol. In all reaction sets, positive and negative controls (without DNA) were used.

Results

Discussion

In this study, we found no impact of the p.G84E mutation in the HOXB13 gene on bladder or kidney cancer. We examined an unselected cohort of 1418 bladder cancer, 813 kidney cancer cases and 4497 controls and the p.G84E mutation was rare in the general population (0.1%). In the present study we noticed that mutation in HOXB13 gene were observed in three (0.2%) unselected bladder cancer patients and three (0.4%) unselected renal cancer patients. The statistical power of the study is 35,5% for bladder cancer and 93,2% for kidney cancer.

Based on a review of the evidence in the literature, there are several studies that investigate the effect of the p.G84E mutation in the HOXB13 gene in unselected bladder and kidney cancer cases, but they are based on small study cohorts. The investigators Beebe-Dimmer et al. genotyped 208 men with bladder and 137 men with kidney cancer patients and suggested the association between p.G84E and bladder cancer. The risk of a p.G84E mutation in bladder cancer was almost significant (p = 0.06). Their studies excluded women [22]. In another study by Hoffmann et al. followed 335 patients with bladder and 303 cases with kidney cancer. They observed similar to Beebe-Dimmer et al. that in bladder cancer the mutation was almost statistical significant (p = 0.06). Hoffmann et al. found an increased risk of p.G84E mutation in kidney cancer (p = 0.03) [23]. Okuda et al demonstrated that HOXB13, like the tumor suppressor gene, plays a role in tumorigenesis of human renal cell carcinoma and progression by participating in the apoptotic pathway [24]. Marra et al. in a small group of 86 bladder cancer samples over expression and more aggressive phenotype of HOXB13 was observed in the muscle invasive with non-muscle invasive tumors [25].

In the literature there are several studies on mutation in the HOXB13 gene and association with different cancers. Some studies have confirmed that mutation in the HOXB13 gene was associated with early-onset prostate cancer men, cases with significantly elevated PSA and family history of prostate cancer [18, 26–28]. Additionally, abnormal expression of HOXB13 has been detected in the development of leukemia, non-Hodkin’s Lymphoma, oral squamous cell carcinoma, glioma, colon, stomach and genitourinary cancers [17, 29–36]. Ovarian, cervical and endometrial cancers overexpress HOXB13 [29, 37]. Moreover a link with melanoma cancer has been suggested and it predicts breast cancer recurrence and response to tamoxifen treatment [23, 37–39].

There are several strengths of our study including the large number of patients and controls, and the sampling of incident cases, unselected for age or family history. Cases and controls are all residents of Szczecin. There is no reason to believe that age, gender or smoking behavior are important confounders of the observed association.

In conclusion, this study reveals that the p.G84E mutation in the HOXB13 gene does not seem to play a role in development of bladder or kidney cancer. Our results indicate that testing p.G84E mutations is unlikely to be relevant for the identification of individuals at risk of bladder or kidney cancer, at least in the Polish population.

Authors’ contributions

EZ-P - wrote the manuscript, carried out the molecular genetic studies, performed the statistical analysis; AT-G - enrolled the patients into the study group; JL - dcritically revised the manuscript and approved its final version. All authors read and approved the final version of the manuscript.

Funding

This study was funded by Pomeranian Medical University, Szczecin. These authors are employees of Pomeranian Medical University, Szczecin: EZP, ATG, JL. The specific roles of these authors are articulated in the ‘author contributions’ section. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

Our data contain potentially sensitive information therefore we have not included it with our manuscript. Those who would like to request access to data may contact Melissa Sidhu at the Research Ethics Board of Women’s College Hospital by calling (416) 351–3732 × 2723 or email ac.latipsohcw@uhdis.assilem.

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