HNF1B, EZH2 and ECI2 in prostate carcinoma. Molecular, immunohistochemical and clinico-pathological study

Abstract

Hepatocyte nuclear factor 1 beta (HNF1B) is a tissue specific transcription factor, which seems to play an important role in the carcinogenesis of several tumors. In our study we focused on analyzing HNF1B in prostate carcinoma (PC) and adenomyomatous hyperplasia (AH), as well as its possible relation to the upstream gene EZH2 and downstream gene ECI2. The results of our study showed that on an immunohistochemical level, the expression of HNF1B was low in PC, did not differ between PC and AH, and did not correlate with any clinical outcomes. In PC, mutations of HNF1B gene were rare, but the methylation of its promotor was a common finding and was positively correlated with Gleason score and stage. The relationship between HNF1B and EZH2/ECI2 was equivocal, but EZH2 and ECI2 were positively correlated on both mRNA and protein level. The expression of EZH2 was associated with poor prognosis. ECI2 did not correlate with any clinical outcomes. Our results support the oncosuppressive role of HNF1B in PC, which may be silenced by promotor methylation and other mechanisms, but not by gene mutation. The high expression of EZH2 (especially) and ECI2 in PC seems to be a potential therapeutic target. Hepatocyte nuclear factor 1 beta (HNF1B) is an important tissue specific transcription factor, which is essential for the embryonic development of several organs including the kidney, pancreas, liver, biliary tract, genital tract, and gastrointestinal system 1,2. Heterozygous germline mutations are associated with several congenital diseases of the kidney, pancreas, and urogenital tract, and the role of HNF1B in these diseases is well known 3-5. However, despite the increased awareness that HNF1B plays an important role in the carcinogenesis of several solid tumors, the exact significance of HNF1B in carcinogenesis is not yet fully understood 6-9. The regulatory mechanisms and pathways in which HNF1B is involved in the process of carcinogenesis are not clear, but it appears that HNF1B may act as either oncogene or oncosuppressor gene based on the type of tumor and its histogenesis 6,7,10. In the prostate, an increased expression of HNF1B seems to be protective against prostate cancer, and HNF1B therefore has been attributed an oncosuppressive role 10. However, the levels of protein expression of HNF1B in prostate cancer are not well known, and the mechanisms playing role in its regulation are of interest. According to current knowledge, HNF1B is commonly inactivated in prostate cancer, especially due to HNF1B promoter methylation, which occurs in about 50% of cases 11. Another recently suggested possible mechanisms of HNF1B inactivation is the effect of the enhancer of zeste homolog 2 (EZH2), the overexpression of which has been suggested to downregulate the expression of HNF1B 12,13. EZH2 is a gene belonging to the category of Polycomb

Comprehensive quantitative analysis of alternative splicing variants reveals the HNF1B mRNA splicing pattern in various tumour and non-tumour tissues

“…3 ). A similar analysis was published previously for the same sample set of prostate carcinomas, where no correlation between mRNA and protein expression was observed 17 .

Figure 3 Correlation of the overall HNF1B mRNA and protein expression in 78 large intestine carcinoma samples showed weak positive correlation (R = 0.39; p < 0.001).

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“…The HNF1B DNA mutation analysis of this sample set was done and published previously 15 – 17 and did not reveal any variant located in canonical or potential cryptic splice site of T or NT samples in the coding HNF1B regions with flanking intronic sequences (+ − 20 bp). Overall, few different variants were detected in our dataset.…”

“…There are studies suggesting that HNF1B may act as an oncogene in specific cancer types, such as ovarian clear cell carcinoma 5 , 6 , 21 and papillary renal cell carcinoma 16 . Other studies suggested that HNF1B acts mainly as a tumour suppressor in colorectal, prostate, ovarian, and some other types of solid tumours 3 , 15 , 17 , 18 , 22 . One of the possible explanations for this ambivalent character is the existence of one or more alternative splicing variants with dysregulated expression in tumour tissues which have either a regulatory role or code for HNF1B protein isoforms with different function 19 .…”

“…Our previous work showed a loss of the HNF1B protein expression in several solid tumours, and thus suggests that HNF1B may act in a tumour suppressive fashion in colorectal carcinoma (CRC) 15 , clear cell renal carcinoma (ccRCC) and chromophobe renal cell carcinoma 16 , prostate carcinoma (PC) 17 , and high-grade serous carcinoma (HGSC) 18 . The HNF1B gene promoter methylation (as a typical factor leading to a loss of protein expression) was observed in 55% PC and 38% HGSC samples.…”

A comprehensive analysis of the expression, epigenetic and genetic changes of HNF1B and ECI2 in 122 cases of high‑grade serous ovarian carcinoma

“…Those RNA samples with an RQN lower than 7.5 were removed from further analysis (tissue samples RQN mean=9.3; range 5–10). Otherwise, 3.75 µg of total RNA of each sample (where available) was treated by DNase I (Thermo Fisher Scientific, Inc.), and cDNA was synthetized in a 40 µl reaction using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.) with random hexamers (Roche) as described in Dundr et al ( 23 ).…”