Adrenocortical carcinoma is a heterogeneous and aggressive cancer that originates from steroidogenic cells within the adrenal cortex. In this study, we have assessed for the preclinical gold standard NCI-H295 in direct comparison with the more recently established MUC-1 and a here newly reported ACC cell line (TVBF-7) the mutational status of important driver genes (TP53, MEN1, PRKAR1A, CTNNB1, APC, ZNRF-3, IGF-2, EGFR, RB1, BRCA1, BRCA2, RET, GNAS and PTEN), Wnt-signaling specificities (CTNNB1 mutation vs. APC mutation vs. wildtype), steroidogenic-(CYP11A1, CYP17A1, HSD3B2, HSD17B4, CYP21A2, CYP11B1, CYP11B2, MC2R, AT1R) and nuclear-receptor-signaling (AR, ER, GCR), varying electrophysiological potentials as well as highly individual hormone secretion profiles (Cortisol, Aldosterone, DHEA, DHEAS, Testosterone, 17-OH Progesterone, among others) which were investigated under basal and stimulated conditions (ACTH, AngII, FSK). Our findings reveal important genetic and pathophysiological characteristics for these three cell lines and reveal the importance of such cell-line panels reflecting differential endocrine functionalities to thereby better reflect clinically well-known ACC patient heterogeneities in preclinical studies.

Surgery, followed or not by adjuvant mitotane, is the current mainstay of therapy for patients with early-stage adrenocortical carcinoma (ACC). Mitotane, either alone or in association with EDP (Et...

Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). The regimen to be added to mitotane is a chemotherapy including etoposide, doxorubicin, and cisplatin. This pharmacological approach, however, has a limited efficacy and significant toxicity. Evidence indicates that ACC seems to be sensitive to alkylating agents. Trabectedin is an anti-tumor drug that acts as an alkylating agent with a complex mechanism of action. Here, we investigated whether trabectedin could exert a cytotoxic activity in in vitro cell models of ACC. Cell viability was evaluated by MTT assay on ACC cell lines and primary cell cultures. The gene expression was evaluated by q-RT-PCR, while protein expression and localization were studied by Western blot and immunocytochemistry. Combination experiments were performed to evaluate their interaction on ACC cell line viability. Trabectedin demonstrated high cytotoxicity at sub-nanomolar concentrations in ACC cell lines and patient-derived primary cell cultures. The drug was able to reduce /β catenin nuclear localization, although it is unclear whether this effect is involved in the observed cytotoxicity. Trabectedin/mitotane combination exerted a synergic cytotoxic effect in NCI-H295R cells. Trabectedin has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of trabectedin with mitotane provides the rationale for testing this combination in a clinical study.

The pharmacological approach to adrenocortical carcinoma (ACC) is based on mitotane with/without etoposide, doxorubicin, and cisplatin, according to the disease stage. Considering the limited efficacy and toxicity of this treatment, new strategies are required. Trabectedin is a marine-derivated antitumoral agent that inhibits oncogenic transcription. We have already demonstrated trabectedin cytotoxic activity at sub-nanomolar concentrations in ACC cells. Here, we expanded the investigation of trabectedin effect on ACC preclinical models, evaluating whether trabectedin could affect ACC cells’ invasiveness and metastasis formation. NCI-H295R, MUC-1, and TVBF-7 cell lines were used. Cell tumor xenografts in Danio rerio embryos were performed. The tumor mass areas and the number of embryos with metastasis were evaluated. The in vitro invasiveness of cells was evaluated. Effects of trabectedin of MMP2, TIMP1, and TIMP2 were evaluated at gene level qRT-PCR. MMP2 secreted in the cell medium was evaluated by Western blot and by zymography. Xenograft experiments demonstrated that trabectedin significantly reduced the tumor area in each ACC cell model and metastasis formation in embryos injected with metastasis-derived cell lines. Trabectedin treatment reduced the invasiveness of ACC cells across the matrix, which was greater at baseline for the metastatic models. In metastatic cell models, protein analysis demonstrated a reduction of MMP2 secretion and activity in the culture medium after treatment. Our results indicate that trabectedin interferes with invasiveness and metastasis processes, both dramatic features of ACC. Furthermore, these results support those previously published in providing the rationale for a clinical evaluation of the efficacy of trabectedin in ACC patients.

Mitotane is the only approved drug for treating adrenocortical carcinoma (ACC). The regimen added to mitotane is chemotherapy with etoposide, doxorubicin, and cisplatin. This pharmacological approach, however, has a limited efficacy and significant toxicity. Target-therapy agents represent a new promising approach to cancer therapy. Among these, a preeminent role is played by agents that interfere with cell-cycle progression, such as CDK4/6-inhibitors. Here, we investigate whether ribociclib could induce a cytotoxic effect both in ACC cell line and patient-derived primary cell cultures, alone or in combined settings. Cell viability was determined by 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide assay, whereas cell proliferation was evaluated by direct count. Binary combination experiments were performed using Chou and Talalay method. Gene expression was analyzed by quantitative RT-PCR, whereas protein expression was evaluated by immunofluorescence. A double staining assay revealed that ribociclib induced a prevalent apoptotic cell death. Cell-cycle analysis was performed to evaluate the effect of ribociclib treatment on cell-cycle progression in ACC cell models. Our results indicate that ribociclib was cytotoxic and reduced the cell proliferation rate. The effect on cell viability was enhanced when ribociclib was combined with progesterone and/or mitotane. The effect of ribociclib on cell-cycle progression revealed a drug-induced cell accumulation in G2 phase. The positive relationship underlined by our results between ribociclib, progesterone, and mitotane strengthen the clinical potential of this combination.