Antiproliferative Effects Of Everolimus Research Articles

Abstract 5488: Everolimus inhibits the proliferation of aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for estrogen receptor positive breast cancer in postmenopausal women. Unfortunately, the majority of patients treated with AIs eventually develop resistance to these drugs. The mechanism by which AI resistance occurs is still not known, however, there is strong evidence that upregulation of the PI3K/Akt/mTOR pathway might play an important role. As such, mTOR inhibition is now under study in clinical trials as a breast cancer treatment. The newest mTOR inhibitor, Everolimus (Afinitor®), has been FDA approved for the treatment of metastatic breast cancer. We have previously reported the development of two AI-resistant breast cancer cell lines (MCF-7:5C and MCF-7:2A) which were clonally derived from parental MCF-7 cells following long term estrogen deprivation. Both MCF-7:5C and MCF-7:2A cells have been shown to overexpress members of the PI3K/Akt/mTOR pathway. In this study, we examined the therapeutic benefits of everolimus in AI-resistant MCF-7:5C and MCF-7:2A cells compared to endocrine sensitive MCF-7 breast cancer cells and normal breast epithelium MCF10A cells. Results from cell viability assay showed that everolimus markedly inhibited the proliferation of endocrine sensitive MCF-7 cells and AI-resistant MCF-7:5C and MCF-7:2A cells in a time-dependent and concentration-dependent manner with IC50s of 30 nM, 40 nM and 10 nM, respectively. Notably, the MCF-7:2A cells were the most sensitive to everolimus which caused a 70% reduction in proliferation at 24 hours whereas in MCF-7 and MCF-7:5C cells only 30% inhibition was observed at the same time point. The inhibitory effect of everolimus was due primarily to G1 arrest and was associated with significant downregulation of phospho-mTOR, phospho-p70S6K, phospho-Akt, GSK3β, cyclin D1 and p21. Colony formation assay in soft agar indicated that AI-resistant MCF-7:5C and MCF-7:2A cells formed significantly more colonies than MCF-7 cells and that treatment with everolimus notably reduced the size and number of colonies for all three cell lines. Finally, we found that exposure of AI-resistant MCF-7:5C and MCF-7:2A cells to a single dose of gamma-irradiation (6 Gy) significantly enhanced the anti-proliferative effect of everolimus on these cells. In summary, these studies showed that everolimus significantly inhibited the proliferation and tumorigenicity of AI-resistant breast cancer cells and that everolimus might be an effective treatment option for patients with AI-resistant breast cancer. Citation Format: Asona Lui, Joan Lewis-Wambi. Everolimus inhibits the proliferation of aromatase inhibitor-resistant breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5488. doi:10.1158/1538-7445.AM2014-5488

Abstract 162: Everolimus Prevents Vascular Smooth Muscle Cell Proliferation by Repressing Cell Cycle-Dependent Telomerase Activation

INTRODUCTION: Everolimus is a potent antiproliferative agent that prevents growth factor-stimulated proliferation of vascular smooth muscle cells and is frequently used as platform for drug-eluting coronary stents to prevent restenosis after coronary artery angioplasty. We have previously demonstrated that telomerase plays a crucial role in vascular smooth muscle cell proliferation. However, whether telomerase is involved in the anti-proliferative activity of everolimus and the putative molecular mechanisms underlying this activity remain elusive. In this study, we assessed the hypothesis that telomerase is a key downstream target for the anti-proliferative activity of everolimus. METHODS AND RESULTS: We demonstrate that everolimus treatment resulted in a dose-dependent inhibition of mitogen-induced telomerase activity in human coronary artery VSMCs (hcVSMCs). Moreover mitogen-induced telomerase reverse transcriptase (TERT) promoter activity and protein expression were dose-dependently repressed by everolimus treatment. Everolimus inhibited mitogen-induced cell proliferation as a result of a G1 →S phase arrest of the cell cycle. In addition, everolimus repressed protein expression of cell cycle-regulated genes (MCM 7, PCNA and Cyclin A) without altering phosphorylation of the retinoblastoma (Rb) protein, being the key gatekeeper of cell cycle transition. In addition, the effects of everolimus were not related to telomere shorting and cellular senescence, supporting the concept of a noncanonical telomere length-independent mechanism. Finally, we demonstrated in transient transfection studies that TERT overexpression abolished the anti-proliferative effect of everolimus in hcVSMC, pointing to a key role of telomerase as a target for the anti-proliferative effects of everolimus. CONCLUSIONS: These results indicate that telomerase constitutes a key target for the anti-proliferative activity of everolimus and identify a novel mechanism for the inhibition of hcVSMC proliferation.

Recommendations on the use of everolimus in lung transplantation

The antiproliferative effect of everolimus provides a therapeutic option in the immunosuppression therapy of lung transplantation, by reducing both the risk of acute rejection and the process of progressive fibrosis that determines chronic graft rejection. However, few data on the use of everolimus in lung transplantation have been published to date, and the specific indications of the drug, along with the most adequate time for its introduction or dosing, have not been defined yet. The aim of this article is to propose recommendations for the use of everolimus in lung transplant recipients, including indications, dosing schedules and the use of concomitant immunosuppression. This consensus document has been developed by experts of all the Spanish lung transplant groups from the review of the existing literature and the clinical experience.

Incidence of Wound Events and Lymphocele Formation with De Novo Everolimus Use: The Results of 3 Randomized Clinical Trials

Introduction: The antiproliferative effect of everolimus (EVR) is associated with influencing rates of wound events. Targeting lower exposure of EVR with low exposure to CNIs can preserve efficacy while minimizing events, but other factors that impact events and timing is not well-described. Data was pooled across 3 RCTs in de novo renal transplantation to understand these associations. Methods: Data was pooled from 3 Randomized Clnical Trials: Two compared EVR 1.5 or 3.0 mg/d (levels collected) vs MPA with standard dose cyclosporine (CsA). The other compared the two controlled doses of EVR (0.75 mg bid, targeted to 3-8 ng/ml or 1.5 mg bid, goal 6-12 ng/ml) with reduced-dose CsA vs MPA with standard CsA. Investigatorreported rates of wound events were compared at 30, 60, and 90 days post-operatively with subgroup analysis of those with baseline diabetes and increased BMI, and those requiring a bedside or surgical intervention. The association between EVR level and wound risk in a time dependent model for EVR 1.5 was also evaluated. Results: Of the 1996 patients, 16.6, 21.8,and 14.3% of the EVR 1.5, EVR 3.0,and MPA patients had a reported wound event at 90 days, respectively. There was no difference in patient or graft survival among groups at 1 and 2 years.Table: [Wound Healing Events]EVR targeted to 3-8 ng/ml with reduced exposure to CsA allows for similar rates and timing of wound events compared to MPA. The timedependent analysis showed that diabetes and high BMI were associated with higher risk(HR=1.58 & 1.05; p=0.06 & 0.01, respectively), but EVR levels were not associated with risk of wound events. Conclusion: This is the most comprehensive evaluation of de novo use of EVR in kidney transplantation in the development of wound ‘events’. At the recommended EVR goal of 3-8 ng/ml, the overall incidence of wound events was not increased compared to MPA. The results of this review prompt the discussion of further de novo use of EVR in kidney transplantation to improve long-term graft survival and minimize immunosuppressive toxicity.

Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury

Inhibitors of mammalian target of rapamycin (mTOR) have immunosuppressive and anti-cancer effects, but their effects on the progression of kidney disease are not fully understood. Using cells from normal kidney epithelial cell lines, we found that the antiproliferative effects of mTOR inhibitor everolimus accompanied the accumulation of a marker for cellular autophagic activity, the phosphatidylethanolamine-conjugated form of microtubule-associated protein 1 light chain 3 (LC3-II) in cells. We also showed that the primary autophagy factor UNC-51-like kinase 1 was involved in the antiproliferative effects of everolimus. Levels of LC3-II decreased in the kidneys of rats treated with ischemia-reperfusion or cisplatin; however, renal LC3-II levels increased after administration of everolimus to rats subjected to ischemia-reperfusion or cisplatin treatment. Simultaneously, increased signals for kidney injury molecule-1 and single-stranded DNA and decreased signals for Ki-67 in the proximal tubules were observed after treatment with everolimus, indicating that everolimus diminished renal function after acute tubular injury. We also found leakage of LC3 protein into rat urine after treatment with everolimus, and urinary LC3 protein was successfully measured between 0.1 and 500ng/mL by using an enzyme-linked immunosorbent assay. Urinary LC3 levels were increased after administration of everolimus to rats subjected to ischemia-reperfusion or cisplatin treatment, suggesting that renal LC3-II and urinary LC3 protein are new biomarkers for autophagy in acute kidney injury. Taken together, our results demonstrated that the induction of autophagy by everolimus aggravates tubular dysfunction during recovery from kidney injury.

Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids

Bronchial carcinoids (BCs) are rare tumors originating from endocrine cells dispersed in the respiratory epithelium. It has been previously demonstrated that everolimus, or RAD001, an mTOR inhibitor, has potent antiproliferative effects in human endocrine tumors. Our aim was to evaluate the possible antiproliferative effects of everolimus in human BCs in primary culture. We collected 24 BCs that were dispersed in primary cultures, treated without or with 1 nM-1 muM everolimus, 10 nM SOM230 (pasireotide, a somatostatin receptor multiligand), and/or 50 nM IGF1. Cell viability was evaluated after 48 h, and chromogranin A (CgA) as well as vascular endothelial growth factor (VEGF) secretion was assessed after 8 h incubation. Somatostatin receptors, mTOR, and AKT expression were investigated by quantitative PCR. We found that in 15 cultures (67.5%), everolimus significantly reduced cell viability (by approximately 30%; P<0.05 versus control), inhibited p70S6K activity (-30%), and blocked IGF1 proliferative effects. Everolimus also significantly reduced CgA (by approximately 20%) and VEGF (by approximately 15%) secretion. Cotreatment with SOM230 did not exert additive effects on cell viability and secretory activity. AKT expression was similar in responder and nonresponder tissues, while mTOR expression was significantly higher in the responder group, which was characterized by higher CgA plasma levels and bigger tumors with higher mitotic index and angiogenesis. Our data demonstrate that everolimus reduces VEGF secretion and cell viability in BCs with a mechanism likely involving IGF1 signaling, suggesting that it might represent a possible medical treatment for BCs.

Correction to “Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27Kip1/Cyclin E Expression”

Correction to “Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27<i>^Kip1</i>/Cyclin E Expression”

Everolimus: an immunosuppressive agent in transplantation

Everolimus is a novel immunosuppressive agent related to sirolimus. It is a proliferation signal inhibitor with an improved pharmacokinetic profile and bioavailability compared with sirolimus. Everolimus has been shown to be as effective as mycophenolate mofetil in reducing acute rejection in renal transplantation. In cardiac transplant recipients, it is superior to azathioprine in reducing acute rejection and cardiac allograft vasculopathy. Its use is also associated with a decrease in cytomegalovirus infection. However, coadministration with calcineurin inhibitors requires careful dose adjustment to prevent renal toxicity. Antiproliferative effects of everolimus may abrogate the increased risk of malignancy seen in solid organ transplantation.

Experience with everolimus

Everolimus is a novel macrolide immunosuppressant that acts as a T-lymphocyte proliferation signal inhibitor. Its actions are complementary to and synergistic with those of the calcineurin inhibitors. Compared with sirolimus, everolimus has unique pharmacokinetic characteristics including greater bioavailability and a shorter half-life, allowing more rapid achievement of a steady state. Clinical experience to date, largely limited to use in kidney transplant patients receiving cyclosporine-based immunosuppression, indicates that administration of everolimus is associated with low rates of acute rejection and a tolerable safety profile. Recent observations in heart transplant patients suggest that the antiproliferative effects of everolimus may prevent allograft vasculopathy.