sirolimus and Adrenocortical-Carcinoma

Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy without an available effective systemic chemotherapy. Insulin growth factor 2 (IGF-2) overexpression leading to the activation of the IGF-1 receptor (IGF-1R)/mammalian target of rapamycin (mTOR) pathway is well described in ACC. Cixutumumab, a fully human IgG1 monoclonal antibody directed at IGF-1R was combined with temsirolimus on the basis of preclinical data.. Patients received cixutumumab, 3-6 mg kg(-1) intravenously (IV) weekly, and temsirolimus, 25-37.5 mg IV weekly (4-week cycles), with restaging after 8 weeks.. Twenty-six patients were enrolled (13 (50%) men); median age, 47 years; median number of prior therapies, 4. Five patients previously received an IGF-1R inhibitor and one, temsirolimus. The most frequent toxicities, at least possibly drug related, were grade 1-2 thrombocytopenia (38%), mucositis (58%), hypercholesterolaemia (31%), hypertriglyceridemia (35%), and hyperglycaemia (31%). In all, 11 of 26 patients (42%) achieved stable disease (SD) >6 months (duration range=6-21 months) with 3 of the 11 having received a prior IGF-1R inhibitor.. Cixutumumab combined with temsirolimus was well tolerated and >40% of patients achieved prolonged SD.

Topics: Adrenocortical Carcinoma; Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Disease-Free Survival; Female; Humans; Male; Middle Aged; Sirolimus; Young Adult

Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined.. HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA.. The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production.. mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Aldosterone; Angiotensin II; Humans; Hydrocortisone; RNA, Guide, Kinetoplastida; Sirolimus; TOR Serine-Threonine Kinases

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Pan-genomic analyses identified p53/Rb and WNT/β-catenin signaling pathways as main contributors to the disease. However, isolated β-catenin constitutive activation failed to induce malignant progression in mouse adrenocortical tumors. Therefore, there still was a need for a relevant animal model to study ACC pathogenesis and to test new therapeutic approaches. Here, we have developed a transgenic mice model with adrenocortical specific expression of SV40 large T-antigen (AdTAg mice), to test the oncogenic potential of p53/Rb inhibition in the adrenal gland. All AdTAg mice develop large adrenal carcinomas that eventually metastasize to the liver and lungs, resulting in decreased overall survival. Consistent with ACC in patients, adrenal tumors in AdTAg mice autonomously produce large amounts of glucocorticoids and spontaneously activate WNT/β-catenin signaling pathway during malignant progression. We show that this activation is associated with downregulation of secreted frizzled related proteins (Sfrp) and Znrf3 that act as inhibitors of the WNT signaling. We also show that mTORC1 pathway activation is an early event during neoplasia expansion and further demonstrate that mTORC1 pathway is activated in ACC patients. Preclinical inhibition of mTORC1 activity induces a marked reduction in tumor size, associated with induction of apoptosis and inhibition of proliferation that results in normalization of corticosterone plasma levels in AdTAg mice. Altogether, these data establish AdTAg mice as the first preclinical model for metastatic ACC.

Topics: Adrenocortical Carcinoma; Animals; Antigens, Polyomavirus Transforming; beta Catenin; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Multiprotein Complexes; Neoplasm Metastasis; Retinoblastoma Protein; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Wnt Signaling Pathway

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Antineoplastic Agents, Hormonal; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Mitotane; Sirolimus

The mTOR pathway has recently been suggested as a new potential target for therapy in adrenocortical carcinomas (ACCs). The aim of the current study is to describe the expression of the mTOR pathway in normal adrenals (NAs) and pathological adrenals and to explore whether there are correlation between the expression of these proteins and the in vitro response to sirolimus. For this purpose, the MTOR, S6K1 (RPS6KB1), and 4EBP1 (EIF4EBP1) mRNA expression were evaluated in ten NAs, ten adrenal hyperplasias (AHs), 17 adrenocortical adenomas (ACAs), and 17 ACCs by qPCR, whereas total(t)/phospho(p)-MTOR, t/p-S6K, and t/p-4EBP1 protein expression were assessed in three NAs, three AHs, six ACAs, and 20 ACCs by immunohistochemistry. The effects of sirolimus on cell survival and/or cortisol secretion in 12 human primary cultures of adrenocortical tumors (ATs) were also evaluated. In NAs and AHs, layer-specific expression of evaluated proteins was observed. S6K1 mRNA levels were lower in ACCs compared with NAs, AHs, and ACAs (P<0.01). A subset of ATs presented a moderate to high staining of the evaluated proteins. Median t-S6K1 protein expression in ACCs was lower than that in ACAs (P<0.01). Moderate to high staining of p-S6K1 and/or p-4EBP1 was observed in most ATs. A subset of ACCs not having moderate to high staining had a higher Weiss score than others (P<0.029). In primary AT cultures, sirolimus significantly reduced cell survival or cortisol secretion only in sporadic cases. In conclusion, these data suggest the presence of an activated mTOR pathway in a subset of ATs and a possible response to sirolimus only in certain ACC cases.

Topics: Adaptor Proteins, Signal Transducing; Adrenal Cortex; Adrenal Cortex Neoplasms; Adrenocortical Adenoma; Adrenocortical Carcinoma; Adult; Antibiotics, Antineoplastic; Cell Cycle Proteins; Cell Line, Tumor; Child; Child, Preschool; Humans; Hyperplasia; Phosphoproteins; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Adult; Antineoplastic Agents; Everolimus; Female; Humans; Hypoglycemia; Insulin-Like Growth Factor II; Sirolimus; Tomography, X-Ray Computed

Patients with advanced adrenocortical carcinoma (ACC) have limited treatment options after failure of chemotherapy. Tumor IGF2 expression has been shown to be amplified in the majority of cases of ACC and autocrine/paracrine activation of the IGF receptor (IGF-R) is thought to play a major role in the pathogenesis of ACC. It has been shown in vitro that inhibition of the IGF-R inhibits ACC cell proliferation. mTOR is a downstream effector of the IGFR signaling pathway; therefore, the rapamycin analog everolimus could prove to be useful for treatment of patients with ACC. Four women with ACC (ages 25-60 years) developed stage IV disease after surgery. All had progressive disease (PD) despite treatment with mitotane and other treatment modalities (etoposide, doxorubicin, cis-platinum in 3/4 patients, further streptozotocin + 5-FU in 1/4 patients, further thalidomide therapy in 2/4 patients; 1 patient progressed on an IGF-R antagonist). The patients were started on everolimus 10 mg/day orally and 2/4 patients also continued mitotane. Disease progression was monitored monthly by CT in 3/4 and after 3 months in 1/4. In all patients everolimus was well tolerated. In the three patients monitored monthly, PD was evident after 1, 3, and 4 months; in the patient evaluated after 3 months PD was also evident. In this small exploratory study, no clinically meaningful response was observed with everolimus in four patients with advanced ACC. The failure of efficacy could be related to an interaction with mitotane, multiple signaling pathways, and/or other downstream IGF-R effectors operative in the pathogenesis of ACC.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Adult; Antineoplastic Agents; Everolimus; Female; Humans; Middle Aged; Sirolimus; Treatment Outcome

Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 could have greater effects than mTOR inhibitors alone.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Hydrocortisone; Insulin-Like Growth Factor II; Protein Kinase Inhibitors; Receptor, IGF Type 1; Sirolimus; TOR Serine-Threonine Kinases

Adrenocortical cancer is a rare malignancy for which current pharmacological therapies are still insufficient. We tested the effect of a novel PI3 kinase - mammalian target of rapamycin dual inhibitor (NVP-BEZ235) on proliferation of the H295R adrenocortical cancer cell line in vitro and grown as xenografts in immunodeficient mice. NVP-BEZ235 was able to significantly inhibit phosporylation of Akt kinase and S6 ribosomal protein in H295R cells and to significantly reduce their proliferation in vitro and xenograft growth in vivo. The drug also induced activation of Erk phosphorylation, which could be inhibited by simultaneous treatment with the Erk inhibitor FR180204. This latter drug synergized with NVP-BEZ235 in the inhibition of H295R proliferation in vitro. Our data suggest that dual PI3K/mTOR inhibitors may represent a useful pharmacological tool in the therapy of advanced adrenocortical cancer and that simultaneous inhibition of both Erk and PI3K - mTOR pathways may be required to obtain a higher antiproliferative effect in this type of tumor.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Evaluation, Preclinical; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Mice; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyridazines; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Xenograft Model Antitumor Assays

MicroRNAs (miRNAs) act at the posttranscriptional level to control gene expression in virtually every biological process, including oncogenesis. Here, we report the identification of a set of miRNAs that are differentially regulated in childhood adrenocortical tumors (ACT), including miR-99a and miR-100. Functional analysis of these miRNAs in ACT cell lines showed that they coordinately regulate expression of the insulin-like growth factor-mammalian target of rapamycin (mTOR)-raptor signaling pathway through binding sites in their 3'-untranslated regions. In these cells, the active Ser(2448)-phosphorylated form of mTOR is present only in mitotic cells in association with the mitotic spindle and midbody in the G(2)-M phases of the cell cycle. Pharmacologic inhibition of mTOR signaling by everolimus greatly reduces tumor cell growth in vitro and in vivo. Our results reveal a novel mechanism of regulation of mTOR signaling by miRNAs, and they lay the groundwork for clinical evaluation of drugs inhibiting the mTOR pathway for treatment of adrenocortical cancer.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Adenoma; Adrenocortical Carcinoma; Animals; Cell Growth Processes; Everolimus; Female; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Protein Serine-Threonine Kinases; Sirolimus; Somatomedins; TOR Serine-Threonine Kinases; Transplantation, Heterologous