Role of androgen hormones and 11-oxyandrogen metabolites in endometrial and ovarian cancersGjorgoska, Marija (Avtor) Lanišnik-Rižner, Tea (Mentor) gynecological cancerendometrial cancerhigh-grade serous ovarian cancerintracrinologysteroid hormonesbiomarker discoverydiagnostic biomarkersprognostic biomarkerssteroidomicstranscriptomicsmetabolomicsmachine learningliquid chromatography-tandem mass spectrometryBackground: Endometrial cancer (EC) and ovarian cancer (OC) are the sixth and seventh most diagnosed cancers in women worldwide, with incidence rates rising due to demographic changes. Both cancers mainly affect postmenopausal women, with a median diagnosis age in the early to mid-sixties. EC generally has better survival rates, whereas OC, especially its high-grade serous subtype (HGSOC), is often diagnosed at an advanced stage, resulting in poor survival and frequent chemoresistance. Currently, there is an unmet need for accurate, cost-effective triage tools, less invasive diagnostics, reliable prognostic biomarkers, and new therapeutic targets for both cancers. Steroid hormones have been poorly studied for their role in the pathophysiology of both EC and OC. They are also gaining new attention in cancer research for their role in modulating anti-tumor immunity and chemoresistance. Others and our group have shown that both ECs and HGSOCs express steroid-metabolizing enzymes and receptors, supporting intra-tumoral steroid interconversion that may influence local hormone signaling and thereby impact tumor cell behavior. In this doctoral dissertation, we focused on a specific class of steroid hormones, namely androgens and their 11-oxygenated derivatives, i.e., 11-oxyandrogens. We systematically investigated: (i) the local metabolism of classic androgen and 11-oxyandrogen precursors in well-characterized cell models; (ii) the transcriptomic and metabolomic effects of potent androgens and 11-oxyandrogens on cell models, along with the prognostic relevance of steroid-metabolizing enzymes and steroid receptors in tumor tissues; and (iii) the diagnostic potential of these steroids in distinguishing EC and OC from benign uterine conditions and non-malignant adnexal masses, respectively. Methods: We used four EC and six HGSOC cell lines, along with immortalized control cell lines derived from normal endometrial and ovarian surface epithelium. To investigate local androgen metabolism, we performed quantitative gene expression analysis of key enzymes in androgen and 11-oxyandrogen metabolism and incubated cell lines with physiologically relevant concentrations of classical androgen precursors (dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA) and androstenedione (A4)) as well as 11-oxyandrogen precursors (11β-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4)). The resulting steroid metabolites were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantitative gene expression analysis of key enzymes involved in classic and 11-oxyandrogen metabolism was performed using qPCR, and the metabolic profiles were interpreted in the context of enzyme expression levels. To assess the functional effects of androgens, selected EC and HGSOC cell lines were treated with potent classic and 11-oxyandrogens, followed by untargeted transcriptomic analysis via RNA sequencing and metabolomic profiling using nuclear magnetic resonance (NMR). Additionally, publicly available clinical datasets were analyzed to evaluate the expression of key steroid-metabolizing enzymes and steroid receptors in EC and HGSOC tumors, along with their associations with patient survival. Finally, two prospective clinical studies were conducted to evaluate the diagnostic potential of circulating steroids in EC and OC. Serum steroid levels were quantified by LC-MS/MS, and diagnostic accuracy was assessed using machine learning. Results: (i) In both EC and HGSOC cell models, classic androgen precursors did not support local production of 11-oxyandrogens, attributable to the observed absence of CYP11B1 expression across all cell lines. Furthermore, DHEAS and DHEA served as limited sources of bioactive androgens, testosterone (T) and 5α-dihydrotestosterone (DHT), primarily due to low HSD3B1/2 expression, likely diverting DHEA toward alternative metabolites such as 5α-androstenediol via reductive HSD17B enzymes or oxygenated derivatives through CYP3B isoforms. Among EC cell lines, the low-grade model RL95-2 exhibited the highest capacity to metabolize DHEAS and DHEA into T. In HGSOC, the primary tumor cell line Caov-3 and the non-cancerous control line HIO-80 showed the greatest T production from these precursors. Similarly, A4 was a poor substrate for bioactive androgen synthesis in both cancer types, suggesting that it is preferentially converted to 5α-reduced metabolites via SRD5A isoforms, which were expressed by both EC and HGSOC cells. In contrast, 11-oxyandrogen precursors, 11OHA4 and 11KA4, were efficiently converted into the potent androgen receptor (AR) agonist 11-ketotestosterone (11KT) in selected cell lines. This conversion, catalyzed by the AKR1C3 enzyme, was most pronounced in low-grade EC models (Ishikawa, HEC-1-A, RL95-2) and chemo-sensitive HGSOC lines (OVSAHO, Kuramochi). Notably, the amount of 11KT formed from 11-oxyandrogen precursors was several-fold higher than T produced from classic precursors, underscoring intra-tumoral 11-oxyandrogen metabolism as significant sources of androgen receptor (AR)-activating metabolites in low-grade ECs and chemo-sensitive HGSOCs. (ii) In EC cell models, treatment with DHT and 11-keto-dihydrotestosterone (11KDHT) induced modest transcriptomic changes after 48 hours of incubation. In AR-expressing Ishikawa cells, androgen exposure upregulated genes such as MYO1D and LAMC3, whereas no significant transcriptomic effects were observed in RL95-2 cells, which express low AR levels. In contrast, the AR-positive HGSOC model OVSAHO exhibited pronounced transcriptomic responses to potent androgens (T, DHT) and 11-oxyandrogens (11KT, 11KDHT) following 72 hours of incubation. These responses included activation of stress response and cell cycle-related pathways. At the metabolomic level, both EC and HGSOC cell models exhibited mild intracellular alterations following 72 hours of incubation with potent androgens and 11-oxyandrogens. Specifically, in the low-grade EC model Ishikawa, DHT and 11KDHT reduced intracellular levels of amino acids and lactic acid, while in the HGSOC model OVSAHO, 11KDHT lowered intracellular amino acid and glutathione levels. Analysis of tumor tissues revealed that expression patterns of AR and steroid-metabolizing enzymes varied by tumor type and clinical characteristics. In EC, higher intra-tumoral expression of HSD11B2, SRD5A2, and AR was associated with improved survival, whereas elevated SRD5A1 expression correlated with poorer outcomes. Similarly, in HGSOC, increased expression of HSD11B2, HSD17B2, and AR was linked to better survival, while higher levels of PAPSS1, H6PD, and HSD17B4 were associated with worse prognosis. (iii) In patients with EC (n = 62), preoperative serum levels of classic androgens (A4, T), 11-oxyandrogens (11OHA4, 11OHT), and glucocorticoids (17α-hydroxyprogesterone, 11-deoxycortisol) were elevated compared to controls with benign uterine conditions (n = 70). Conversely, patients with primary or recurrent OC (n = 43) showed reduced preoperative serum levels of T and 11-oxyandrogens (11OHT, 11KT) relative to controls with non-malignant adnexal masses (n = 56). Individually, steroid hormones had limited diagnostic accuracy for both EC and OC (AUC < 0.7). However, in EC, a logistic regression model combining the androgen pool (DHEA, A4, T), tumor biomarkers (CA125, HE4), and clinical parameters (BMI, parity) achieved an AUC of 0.87 (sensitivity 74.7%, specificity 79.1%), significantly outperforming CA125, HE4, or their combination alone in differentiating EC from benign uterine conditions. For OC, two models integrating 11-oxyandrogens, age, and either CA125 or HE4 reached an AUC of 0.91, with sensitivities of 88.9% and 94.4% and specificities of 82.0% and 77.3%, respectively, surpassing CA125, HE4, and the ROMA index in differentiating OC from non-malignant adnexal masses. These findings emphasize the added diagnostic value of steroid hormone profiling in the preoperative evaluation of gynecologic tumors, however validation in independent cohorts is needed. Conclusions: This doctoral dissertation provides new insights into (i) local steroid metabolism in EC and HGSOC cell models; (ii) transcriptomic and metabolomic effects of androgen signaling in EC and HGSOC cell models; and (iii) diagnostic potential of circulating steroids in distinguishing EC and OC from their respective control counterparts. We identified distinct patterns of 11-oxyandrogen metabolism across tumor types and clinical subgroups, between low- and high-grade EC, between EC tumors and normal endometrium, and between chemo-sensitive and chemo-resistant HGSOC models, as well as between HGSOC tumors and normal ovarian epithelium. Functionally, androgen and 11-oxyandrogen exposure triggered significant transcriptomic changes in AR-expressing HGSOC cells, particularly in stress response and cell cycle pathways. In tumor tissues, we identified prognostic biomarker candidates, including steroid-metabolizing enzymes and AR, that could improve risk stratification and support personalized clinical management. Importantly, we also uncovered, for the first time, systemic alterations in circulating 11-oxyandrogens in patients with EC and OC compared to their respective control groups. By integrating steroid profiles with traditional tumor markers and clinical parameters we developed novel diagnostic models for EC and OC, which significantly outperformed tumor biomarkers CA-125 and HE4. To our knowledge, these are the first steroid-protein-based models for these cancers. If validated in larger, independent cohorts, they hold promise to improve cancer detection and ultimately improve clinical outcomes.20252025-07-22 13:32:14Neznano170940sl