Local estrogen biosynthesis and the roles of SLC and ABC gene families of transporter proteins in endometrial and ovarian cancers

Ozadje Rak endometrija in rak jajčnikov sta pogosti hormonsko-odvisni ginekološki bolezni, ki sta v svetovnem merilu v letu 2020 prizadeli več kot 700 000 žensk in povzročili več kot 300 000 smrti. Razvoj obeh bolezni je pogostejši pri ženskah po menopavzi, ko se sinteza estrogenov iz jajčnikov preusmeri v lokalna tkiva. Sinteza estrogenov v lokalnih tkivih poteka iz dehidroepiandrosteron sulfata (DHEA-S) po aromatazni poti, ali iz estron sulfata (E1-S) po sulfatazni poti. Uspešnost sinteze je odvisna od delovanja številnih encimov sinteze in presnove estrogenov ter od prenašalcev, ki omogočajo prenos DHEA-S in E1-S skozi celično membrano. Pri vnosu v celico sodelujejo prenašalci organskih anionov OATP (SLCO) in OAT (SLC), prenos iz celice pa omogočajo prenašalci z ATP-vezavno kaseto (ABC) in prenašalec organskih topljencev alfa beta (OSTab, SLC51). Razmerje med prenašalci in encimi sinteze in presnove vpliva na znotrajcelično raven estrogenov in posledično na raven njihovega protumorskega delovanja. Kljub številnim študijam, ki potrjujejo vlogo estrogenov v patogenezi raka endometrija in raka jajčnikov, njihova vloga pri posameznih podtipih teh bolezni še ni sistematično raziskana. Metode V doktorski disertaciji smo uporabili nesmrtne humane celične linije normalnega proliferativnega endometrija, normalnega površinskega epitelija jajčnikov, rakov endometrija različnih gradusov in seroznih rakov jajčnikov visokih gradusov (HGSOC), ki so se razlikovali v rezistenci proti kemoterapevtikom. Poleg tega smo uporabili tudi tkivo raka endometrija in okolnega kontrolnega endometrija ter tkivo HGSOC in kontrolnega tkiva jajcevodov. V modelnih sistemih smo proučevali: i) ravni mRNA in proteinov prenašalcev, encimov sinteze in presnove estrogenov in receptorjev za estrogene s qPCR, imunocitokemijo in imunohistokemijo, ii) celični vnos DHEA-S in E1-S v prisotnosti in odsotnosti inhibitorjev prenašalcev ter po utišanju genov prenašalcev s pristopom siRNA s tekočinskim scintilacijskim štetjem, iii) presnovo izhodnih spojin sinteze estrogenov v prisotnosti ali odsotnosti inhibitorja steroid sulfataze (STS), STX64, s tekočinsko kromatografijo in masno spektrometrijo, iv) vpliv estrogenov na proliferacijo celic z uporabo metod alamarBlue in xCelligence Rezultati Študije presnove E1-S in DHEA-S so razkrile, da se pri raku endometrija estrogeni sintetizirajo le preko sulfatazne poti iz E1-S. Nadaljnje študije so vključevale proučevanje razlik med rakom endometrija nizkega gradusa pred menopavzo in po njej ter med rakom endometrija nizkega in visokega gradusa. Pri raku endometrija nizkega gradusa po menopavzi (celice HEC-1-A) je bila sinteza estrogenov iz E1-S višja kot pri raku pred menopavzo (celice Ishikawa). Nadaljnji rezultati so pokazali, da je višja pretvorba E1-S do aktivnih estrogenov po menopavzi povezana z višjim izražanjem STS, višjim izražanjem in delovanjem prenašalcev za vnos OATP, predvsem OATP1B3 in OATP2B1, ter nižjim izražanjem prenašalca ABCG2. Analiza izražanja genov prenašalcev v tkivu je še pokazala, da sta pri bolnicah z rakom endometrija po menopavzi ABCG2 in SLC51B manj izražena v tumorskem kot v okolnem kontrolnem tkivu. Z imunohistokemijskim barvanjem smo potrdili nizke proteinske ravni ABCG2 in OSTbeta (SLC51B) v tkivu raka endometrija v primerjavi z okolnim kontrolnim endometrijem. Študije presnove E1-S pri raku endometrija visokega gradusa (celice KLE) v primerjavi z rakom endometrija nizkega gradusa (celice RL95-2) so pokazale, da se E1-S v višji meri pretvarja do aktivnih estrogenov pri raku nizkega gradusa. Razlog je lahko v višji ravni STS pri celicah nizkega gradusa RL95-2, kar smo potrdili z imunodetekcijo po prenosu western in z znižanjem metabolizma E1-S do aktivnih estrogenov v prisotnosti inhibitorja STX64. Po drugi strani je bil pri raku endometrija visokega gradusa celični vnos E1-S višji, kar je nakazovalo, da se E1-S v teh celicah najverjetneje pretvarja do drugih metabolitov estrogenov, ki jih nismo proučevali. Študije vpliva estrogenov na proliferacijo celic so pokazale največji vpliv na proliferacijo celic nizkega gradusa Ishikawa, kar je povezano z visokim izražanjem ERalfa v teh celicah. Pri najpogostejšem raku jajčnikov, seroznem karcinomu visokega gradusa (HGSOC), so rezultati izražanja genov v celicah in tkivu razkrili visoko izražanje STS in nizko izražanje aromataze (CYP19A1), kar je nakazovalo, da se tudi pri tej patologiji estrogeni sintetizirajo iz E1-S. Nadaljnje analize pretvorb E1-S do aktivnih estrogenov v celičnih linijah HGSOC so pokazale najvišjo raven sinteze estradiola v najmanj kemorezistentnih celicah OVSAHO in najnižjo v najbolj kemorezistentnih celicah COV362. Analiza izražanja genov v tkivih raka jajčnikov je še pokazala, da je kemorezistenca s sintezo estrogenov lahko povezana preko vpliva na izražanje HSD17B14 in CYP1A2. Raven mRNA HSD17B14 in CYP1A2 je bila višja pri kemorezistentnih tkivih, v primerjavi s kemosenzitivnimi tkivi raka jajčnikov. Tkiva raka jajčnikov smo razdelili glede na molekularni podtip HGSOC. Rezultati so pokazali, da se štirje podtipi (imunoreaktivni, proliferativni, diferencirani in mezenhimski) razlikujejo v proteinskih ravneh HSD17B10, SULT1E1, CYP1B1 in NQO1, kar nakazuje razlike v sintezi in presnovi estrogenov. Zaključek Z rezultati doktorskega dela smo pokazali razlike v lokalni sintezi in presnovi estrogenov med različnimi podtipi raka endometrija in raka jajčnikov. Identificirali smo posamezne prenašalce in encime, ki imajo velik potencial za nadaljnje študije, ki bi lahko vodile do validacije novih biooznačevalcev ali do razvoja novih načinov zdravljenja.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

Model Cell Lines and Tissues of Different HGSOC Subtypes Differ in Local Estrogen Biosynthesis

Ovarian cancer (OC) is highly lethal and heterogeneous. Several hormones are involved in OC etiology including estrogens; however, their role in OC is not completely understood. Here, we performed targeted transcriptomics and estrogen metabolism analyses in high-grade serous OC (HGSOC), OVSAHO, Kuramochi, COV632, and immortalized normal ovarian epithelial HIO-80 cells. We compared these data with public transcriptome and proteome data for the HGSOC tissues. In all model systems, high steroid sulfatase expression and weak/undetected aromatase (CYP19A1) expression indicated the formation of estrogens from the precursor estrone-sulfate (E1-S). In OC cells, the metabolism of E1-S to estradiol was the highest in OVSAHO, followed by Kuramochi and COV362 cells, and decreased with increasing chemoresistance. In addition, higher HSD17B14 and CYP1A2 expressions were observed in highly chemoresistant COV362 cells and platinum-resistant tissues compared to those in HIO-80 cells and platinum-sensitive tissues. The HGSOC cell models differed in HSD17B10, CYP1B1, and NQO1 expression. Proteomic data also showed different levels of HSD17B10, CYP1B1, NQO1, and SULT1E1 between the four HGSOC subtypes. These results suggest that different HGSOC subtypes form different levels of estrogens and their metabolites and that the estrogen-biosynthesis-associated targets should be further studied for the development of personalized treatment

Ruthenium complexes show potent inhibition of AKR1C1, AKR1C2, and AKR1C3 enzymes and anti-proliferative action against chemoresistant ovarian cancer cell line

In this study, we present the synthesis, kinetic studies of inhibitory activity toward aldo-keto reductase 1C (AKR1C) enzymes, and anticancer potential toward chemoresistant ovarian cancer of 10 organoruthenium compounds bearing diketonate (1–6) and hydroxyquinolinate (7–10) chelating ligands with the general formula [(η$^6$-p-cymene)Ru(chel)(X)]$^{n+}$ where chel represents the chelating ligand and X the chlorido or pta ligand. Our studies show that these compounds are potent inhibitors of the AKR enzymes with an uncommon inhibitory mechanism, where two inhibitor molecules bind to the enzyme in a first fast and reversible step and a second slower and irreversible step. The binding potency of each step is dependent on the chemical structure of the monodentate ligands in the metalloinhibitors with the chlorido complexes generally acting as reversible inhibitors and pta complexes as irreversible inhibitors. Our study also shows that compounds 1–9 have a moderate yet better anti-proliferative and anti-migration action on the chemoresistant ovarian cancer cell line COV362 compared to carboplatin and similar effects to cisplatin

Altered profile of E1-S transporters in endometrial cancer

Endometrial cancer (EC) is associated with increased estrogen actions. Locally, estrogens can be formed from estrone-sulphate (E1-S) after cellular uptake by organic anion-transporting polypeptides (OATP) or organic anion transporters (OAT). Efflux of E1-S is enabled by ATP Binding Cassette transporters (ABC) and organic solute transporter (OST)αβ. Currently, 19 E1-S transporters are known but their roles in EC are not yet understood. Here, we analysed levels of E1-S transporters in Ishikawa (premenopausal EC), HEC-1-A (postmenopausal EC), HIEEC (control) cell lines, in EC tissue, examined metabolism of steroid precursor E1-S, studied effects of OATPs’ inhibition and gene-silencing on E1-S uptake, and assessed associations between transporters and histopathological data. Results revealed enhanced E1-S metabolism in HEC-1-A versus Ishikawa which could be explained by higher levels of OATPs in HEC-1-A versus Ishikawa, especially 6.3-fold up-regulation of OATP1B3 (SLCO1B3), as also confirmed by immunocytochemical staining and gene silencing studies, lower ABCG2 expression and higher levels of sulfatase (STS). In EC versus adjacent control tissue the highest differences were seen for ABCG2 and SLC51B (OSTβ) which were 3.0-fold and 2.1-fold down-regulated, respectively. Immunohistochemistry confirmed lower levels of these two transporters in EC versus adjacent control tissue. Further analysis of histopathological data indicated that SLCO1B3 might be important for uptake of E1-S in tumours without lymphovascular invasion where it was 15.6-fold up-regulated as compared to adjacent control tissue. Our results clearly indicate the importance of E1-S transporters in EC pathophysiology and provide a base for further studies towards development of targeted treatment

Metabolism of estrogens

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary responsehowever, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC$_{50}$ value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, there by allowing more efficient interventions

Abstracts of the 4th Annual Graduate Entry Research in Medicine Conference

This book contains the abstracts of the papers presented at The 4th Annual Graduate Entry Research in Medicine Conference (GERMCON 2021) Organized by Warwick Medical School, University of Warwick in collaboration with Swansea University Medical School, Swansea University, Wales, UK held on 11th September 2021. This event has been designed with the intention of supporting students to develop their interests and skills within academic medicine. This was especially important for Graduate Entry Medical (GEM) students, who have less opportunity and time to engage in research due to their accelerated medical degree. Conference Title: 4th Annual Graduate Entry Research in Medicine ConferenceConference Acronym: GERMCON 2021Conference Date: 11 September 2021Conference Location: Scarman Conference Centre, The University of Warwick, England, CV4 7SH, UK.Conference Organizer: Warwick Medical School, University of Warwick, UKCo-organizer: Swansea University Medical School, Swansea University, Wales, UK Other Abstract Book of GERMCON: Abstracts of the 3rd Annual Graduate Entry Research in Medicine Conferenc