Background: Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers. Methods: NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry. Results: Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells. Conclusion: Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study.

Background: Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). Methods: The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. Results: Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. Conclusions: Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP.

(1) Background: The standard first-line therapy for advanced adrenocortical carcinoma (ACC) is represented by EDP-M (etoposide, doxorubicin, cisplatin + mitotane). Progestins have shown cytotoxic activity both in vitro and in vivo on ACC; better EDP-M tolerability and efficacy have been hypnotized due to the association with progestins. (2) Methods: The feasibility and tolerability of EDP-M combined with oral megestrol acetate (EDP-MM) were tested in 24 patients (pts) affected by metastatic ACC with a low performance status (PS); the case group was compared with a 48 pts control group according to the propensity score. The secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). (3) Results: Thirteen pts (54.2%) in the EDP-MM population experienced progestin-related toxicities; in particular, five pts experienced vaginal bleeding (20.8%); four pts experienced weight gain (16.7%); and thromboembolic events, worsening of hypertension, skin rashes, and hyperglycemia were registered in one patient each (4.2%). This led to the discontinuation of megestrol acetate in four pts (16.7%). EDP-M-related toxicities were similar in both groups. No differences in PFS and OS curves were observed; the CBR was 75.0% and 60.4%, respectively. (4) Conclusions: The association of EDP-M + megestrol acetate in ACC pts with a low PS is feasible and well tolerated; its efficacy appeared to be non-inferior to EDP-M administered to pts with a good PS.

Testicular germ cell tumors are the most common solid malignancy in young adult males with non-seminomatous representing a clinically aggressive subtype. Although cisplatin (CP)-based chemotherapy is highly effective, a subset of patients develop resistance. This study explored a potential new treatment by targeting WEE1, a key cell-cycle regulator, using the drug adavosertib in non-seminoma cell models, aiming to overcome CP resistance. Two non-seminoma cell lines, NCCIT and NT2/D1, and their CP-resistant subclones (NCCIT-R and NT2/D1-R) were used. The effects of adavosertib and CP, alone or in combination, on cell viability, cell-cycle progression, apoptosis, and DNA damage markers was assessed. WEE1 was expressed in non-seminoma cell models. Adavosertib reduced cell viability in a dose-dependent manner across all cell models in both 2D and 3D cultures. IC₅₀ values were in low micromolar range (NCCIT: 0.550 µM; NCCIT-R: 0.630 µM; NT2/D1: 0.415 µM; NT2/D1-R: 0.630 µM). Adavosertib altered cell-cycle distribution, increasing S-phase population. Western blot analysis revealed that inhibiting WEE1 increases CDK1 activity and mitotic marker pH3, indicating disrupted cell cycle control. This leads to replication stress and forces cells with DNA damage into early mitosis, causing mitotic catastrophe. The treatment also triggered higher levels of DNA damage and cell death, as shown by increased caspase activity and apoptosis markers (cleaved-PARP and cleaved-Caspase 3). In combination treatments, adavosertib enhanced CP efficacy across all models, including resistant lines. Overall, our results provide compelling preclinical evidence supporting the combination of WEE1 inhibition with standard chemotherapy as a promising strategy to overcome CP-resistance in non-seminoma testicular cancer.

The heterogeneity of the adrenocortical carcinoma (ACC), pheochromocytoma and paraganglioma cell phenotypes requires different experimental preclinical models to reproduce the peculiarity of these adrenal malignancies. Different models have been generated, either in vitro or in vivo, as cell lines growing in both two-dimensional or in three-dimensional settings; however, in particular for pheochromocytoma/paraganglioma (PPGL), new cell models are urgently needed. As in in vivo models, ACC cells or tumor tissue xenografts have been subcutaneously administered to immunocompromised mice for localized propagation and tumor growth. For ACC and PPGL, multiple transgenic mouse models are now available, obtained by genetic modifications which may recapitulate the heterogeneity of human disease. Interestingly, given the physiological relevance of the functional interaction occurring between the steroidogenic-cortical and the chromaffin-medullary components of the adrenal gland, it is likely that the adrenal/medulla crosstalk may also affect the pathogenesis and progression of adrenal tumors. The peculiar organization of mammalian adrenals with a portal system connecting cortex and medulla strongly supports the evolutionary importance of the crosstalk inside the same gland.