Background:
Endometrial and ovarian cancers are common hormone-dependent gynecological diseases, with more than 0.7 million new cases and more than 300 000 deaths registered worldwide in 2020. The development of both cancers usually occurs after menopause and is associated with increased estrogen activity. In postmenopausal women, estrogens are formed in extragonadal tissues from dehydroepiandrosterone sulfate (DHEA-S) via the aromatase pathway or estrone-sulfate (E1-S) via the sulfatase pathway. Successful local estrogen biosynthesis depends on several enzymes for estrogen biosynthesis and metabolism in addition to transporters that transfer precursors across cellular membranes. The cellular uptake of precursors is enabled by organic anion transporting polypeptides (OATPs, SLCOs) and organic anion transporters (OATs, SLCs), whereas the efflux of precursors is enabled by ATP-binding cassette transporters (ABCs, ABCs) and organic solute transporter alfa beta (OSTab, SLC51). The balance between transporters and enzymes affects intracellular estrogen formation and thus the rate of the cancer-promoting actions of estrogens. Despite the known role of estrogens in promoting cancer, individual transporters or enzymes for estrogen biosynthesis and metabolism have not yet been systematically evaluated in different endometrial and ovarian cancer subtypes
Methodology:
We used immortalized human cell lines of normal proliferative endometrium and normal ovarian surface epithelium, cell lines of endometrial cancers of different grades, and cell lines of high-grade serous ovarian cancer (HGSOC) with different responsiveness to chemotherapeutics. Also, we used tissues of endometrial cancer, adjacent control non-cancerous endometrium, tissues of HGSOC, and control fallopian tube tissues. We aimed to evaluate i) the mRNA and protein levels of transporters, enzymes for estrogen biosynthesis and metabolism, and estrogen receptors using qPCR, immunocytochemistry, and immunohistochemistry, ii) the uptake of steroid precursors in the presence and absence of transporter-specific inhibitors or after siRNA-mediated gene silencing using liquid scintillation counting, iii) the metabolism of steroid precursors into active estrogens and their most common metabolites in the presence or absence of inhibitor of the steroid sulfatase (STS), STX64, using high-performance liquid chromatography and mass spectrometry, and iv) the effects of estrogens on cell proliferation using alamarBlue and xCelligence assays.
Results:
In endometrial cancers of different grades, active estrogens (estrone (E1) and estradiol (E2)) are formed exclusively from E1-S via the sulfatase pathway. We further investigated the differences between premenopausal and postmenopausal high-grade and low-grade endometrial cancers. In low-grade postmenopausal cancer (HEC-1-A cells), estrogen formation from E1-S was higher than that in premenopausal low-grade (Ishikawa) cells. These differences emerged from higher STS levels, higher OATP expression and activity (especially of OATP1B3 and OATP2B1), and lower ABCG2 levels in HEC-1-A cells. In addition, ABCG2 and SLC51B efflux transporters in tissues were significantly affected by menopausal status—both were less expressed in postmenopausal tumors compared to adjacent control postmenopausal endometrium. Immunohistochemistry confirmed low ABCG2 and OSTbeta (SLC51B) protein levels in endometrial cancer compared to control adjacent endometrial tissue.
The rate of E1-S transformation to active estrogens was higher in low-grade (RL95-2 cells) compared to high-grade (KLE cells) endometrial cancer. This can be explained by the higher STS levels in low-grade RL95-2 cells, as confirmed by western blot and E1-S metabolism studies in the presence of STX64. Conversely, high-grade endometrial cancer showed higher uptake of E1-S, indicating that this precursor is potentially transformed into other metabolites that were not analyzed in our studies. For endometrial cancer cells, estrogens had the highest impact on the proliferation of low-grade (grade 1) Ishikawa cells, which express relatively high ERalfa levels.
In ovarian cancer, STS expression was high, and CYP19A1 expression was low/undetected, suggesting that estrogens are formed via the sulfatase and not the aromatase pathway in this pathology. Furthermore, the analysis of E1-S metabolism in cell lines of the most common ovarian cancer, HGSOC, revealed the highest formation of E2 in the least chemoresistant (OVSAHO) cells, followed by moderately chemoresistant (Kuramochi) cells, and the lowest formation in highly chemoresistant (COV362) cells. Moreover, HSD17B14 and CYP1A2 expression was increased in chemoresistant compared to chemosensitive tissues. Ovarian cancer tissues were also divided according to the HGSOC molecular subtype. The four HGSOC subtypes (i.e., immunoreactive, proliferative, differentiated, and mesenchymal) exhibited different HSD17B10, SULT1E1, CYP1B1, and NQO1 protein levels, indicating that these subtypes differ in estrogen biosynthesis and metabolism.
Conclusions:
The results of this doctoral dissertation reveal differences in steroid precursor transport and estrogen biosynthesis and metabolism between different subtypes of endometrial and ovarian cancers. The individual transporters and enzymes highlighted in this work represent candidate diagnostic or prognostic biomarkers and drug targets, which warrant further research for the potential biomarker validation or development of new treatments.
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