title: Endometrial cancer: involvement of androgens and metastasis inducing proteins
Creators: name: Kamal, AM
Publisher: name: University of Liverpool Repository
Year: 2017

description: The alarming escalation of endometrial cancer incidence with associated parallel rise in mortality has increased the urge to find reliable prognostic markers in order to improve risk stratification of patients and therefore promote more individualised treatment strategies. The principal goal of this thesis was to examine the prognostic role of steroid hormone receptors, particularly AR, and metastasis inducing proteins S100A4, S100P and AGR2 in endometrial cancer and to investigate the hormonal regulation of these proteins. A comprehensive expression profile for each of these target proteins was described in a total of 161 well characterised human endometrial samples, using immunohistochemistry. The transcription of the target genes was further assessed in a subset of these samples using the RT-qPCR. Four established human endometrial cancer cell lines were then characterised for the expression of the steroid receptors and the metastasis inducing proteins, in order to select the most appropriate cell line for in vitro hormone modulation. Ishikawa cell line was subsequently utilised to study the effect of the potent natural androgen, 5α-dihydrotestosterone (DHT), and oestradiol on the expression of the aforementioned target genes. An immuno-scoring system was proposed and validated for more accurate quantification of steroid hormone receptor proteins in the endometrium. The expression of AR and ERα was significantly higher in the epithelium of postmenopausal endometrium compared with that of the premenopausal proliferative phase basalis layer. This expression pattern persisted in the hyperplastic epithelium, and also in low grade cancers. In contrast, the high grade cancers showed a significant loss of AR, PR and ERβ compared with the low grade cancers, whilst maintaining weak to moderate ERα. Unlike PR, AR expression in metastatic lesions was significantly higher than that in primary tumours. AR expression correlated positively with favourable clinicopathological features and a lower proliferation index. Loss of AR with/without a simultaneous loss of PR was associated with significantly lower disease free, cancer specific and overall survivals. AR was an independent prognostic indicator for endometrial cancer progression. S100A4, S100P and AGR2 proteins were upregulated in endometrial cancer. A substantial change in the expression profile of S100A4 was observed in endometrial cancer compared with the normal postmenopausal endometrium. The increased expression of S100A4 protein was represented by a significant increase in the nuclear and cytoplasmic expression of the malignant epithelial cells with a concomitant high expression in the associated stroma. High immunoexpression of S100A4 was positively associated with deep myometrial invasion and shorter cancer specific and overall survival but was not identified as an independent prognostic indicator. The expression of S100P was generally low and limited to the cytoplasm of the normal postmenopausal glandular epithelium. Significant nuclear translocation was observed in endometrial cancer cells simultaneously with a reduction in cytoplasmic expression. The loss of cytoplasmic S100P (not nuclear) was associated with unfavourable prognostic indicators such as lymphovascular space invasion. Elevated AGR2 expression was observed in premalignant atypical hyperplasia and peaked in low grade cancers. There was a significant association between highAGR2 and positive ERα, PR, and AR in endometrial cancers. Secreted AGR2 was detected in the serum and uterine washes from endometrial cancer patients. AGR2 protein was immunoexpressed in 93/100 (93%) of the endometrial cancer samples and was associated with a longer overall survival of the patients. In vitro studies on hormonal modulation showed that a supra-physiological dose of DHT was required to induce AR expression in Ishikawa cells. At such dose, DHT had a stimulatory effect on Ishikawa cell proliferation, an action exerted via ER. Whilst S100A4 and S100P mRNA level in Ishikawa cells did not show a significant change in response to oestradiol or DHT treatments, AGR2 mRNA level was downregulated after 24-hour treatment with DHT (but not oestradiol) which was, at least partially, via AR. In conclusion, AR may be a clinically relevant prognostic indicator and a potential therapeutic target in endometrial cancer. Although S100A4 and AGR2 were not recognised as independent prognostic indicators, both have shown significant association with patient outcome. S100A4 protein can be potentially targeted by one of the newly developed S100A4 neutralising peptide to characterize its role in endometrial cancer invasion. An association between S100P and patient outcome was not identified; however, the change in the cellular localisation of S100P may have implications in endometrial carcinogenesis. DHT can modulate AR and AGR2 directly or via ER.