Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe.

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Down-regulation of ABCB1 in Everolimus-resistant Renal Cell Carcinoma Cells.

BACKGROUND/AIM: Everolimus-resistant Caki/EV and 786/EV cells have been established from human derived renal cell carcinoma cells, Caki-2 and 786-O, respectively. These cells exhibit resistance to everolimus and to other mTOR inhibitors and erlotinib. However, the sensitivity of these resistant cells to classical and cytotoxic anticancer drugs remain unclear. The aim of the study was to examine sensitivity of Caki/EV and 786/EV cells to classical and cytotoxic anticancer drugs. MATERIALS AND METHODS: Sensitivity to classical and cytotoxic anticancer drugs in Caki/EV and 786/EV cells was evaluated using the WST-1 (tetrazolium salts) colorimetric assay and was compared to those of the corresponding parental cells. The mRNA expression levels were measured using SYBR® green based quantitative reverse transcription-polymerase chain reaction. RESULTS: Sensitivity to vinblastine, vincristine, paclitaxel, doxorubicin, etoposide, SN-38 (active metabolite of irinotecan), 5-fluorouracil, cisplatin, and carboplatin varied in the resistant cells. Sensitivity to carboplatin and SN-38 was comparable between resistant cells and their parental cells, whereas sensitivity to vinca alkaloids, etoposide, 5-fluorouracil, and cisplatin decreased in the resistant cells. However, sensitivity to paclitaxel and doxorubicin was remarkably enhanced in both resistant cells compared to that of parental cells, this could be partially explained by down-regulation of ABCB1 mRNA expression. CONCLUSION: The everolimus-resistant Caki/EV and 786/EV cells showed cross-resistance to classical and cytotoxic anticancer drugs. However, Caki/EV and 786/EV cells exhibited a remarkable increase in sensitivity to paclitaxel and doxorubicin, and ABCB1 mRNA was down-regulated in response to long-term exposure to everolimus.

Comparison of the effect of Everolimus, Prednisolone, and a combination of both on experimentally induced peritoneal adhesions in rats.

Postoperative intra-abdominal adhesions represent a significant post-surgical problem. Its complications can cause a considerable clinical and cost burden. Herein, our study aimed to investigate the effect of Everolimus on peritoneal adhesion formation after inducing adhesions in rats. In this experimental study, adhesion bands were induced by intraperitoneal injection of 3 ml of 10% sterile talc solution in 64 male albino rats. The first group served as the control group. The second one received oral Prednisolone (1 mg/kg/day), the third received Everolimus (0.1 mg/kg/day), and group four received both drugs with similar dosages for four consecutive weeks. The formation of adhesion bands was qualitatively graded according to the Nair classification. The rats in the control group had extensive adhesions between the abdominal wall and the organs. Regarding substantial adhesion formation, 50% (8/16) of animals in the control group had substantial adhesions, while this rate in the groups receiving Prednisolone, Everolimus, and combination treatment was 31%, 31%, and 31%, respectively. Also, 68.75% (5/11) of the Prednisolone recipients had insubstantial adhesions, the same as Everolimus recipients, while in the combination group, 66.66% (10/15) rats had insubstantial adhesions. Everolimus demonstrated satisfactory results in reducing the rates of induced peritoneal adhesion in an experimental model, similar to Prednisolone and superior to a combination regime.

Primary prophylaxis with mTOR inhibitor enhances T cell effector function and prevents heart transplant rejection during talimogene laherparepvec therapy of squamous cell carcinoma.

The application of mammalian target of rapamycin inhibition (mTORi) as primary prophylactic therapy to optimize T cell effector function while preserving allograft tolerance remains challenging. Here, we present a comprehensive two-step therapeutic approach in a male patient with metastatic cutaneous squamous cell carcinoma and heart transplantation followed with concomitant longitudinal analysis of systemic immunologic changes. In the first step, calcineurin inhibitor/ mycophenolic acid is replaced by the mTORi everolimus to achieve an improved effector T cell status with increased cytotoxic activity (perforin, granzyme), enhanced proliferation (Ki67) and upregulated activation markers (CD38, CD69). In the second step, talimogene laherparepvec (T-VEC) injection further enhances effector function by switching CD4 and CD8 cells from central memory to effector memory profiles, enhancing Th1 responses, and boosting cytotoxic and proliferative activities. In addition, cytokine release (IL-6, IL-18, sCD25, CCL-2, CCL-4) is enhanced and the frequency of circulating regulatory T cells is increased. Notably, no histologic signs of allograft rejection are observed in consecutive end-myocardial biopsies. These findings provide valuable insights into the dynamics of T cell activation and differentiation and suggest that timely initiation of mTORi-based primary prophylaxis may provide a dual benefit of revitalizing T cell function while maintaining allograft tolerance.

Prognostic value of EndoPredict test in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative primary breast cancer screened for the randomized, double-blind, phase III UNIRAD trial.

BACKGROUND: The purpose of this study was to evaluate the prognostic value of the multigene EndoPredict test in prospectively collected data of patients screened for the randomized, double-blind, phase III UNIRAD trial, which evaluated the addition of everolimus to adjuvant endocrine therapy in high-risk, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative early breast cancer. PATIENTS AND METHODS: Patients were classified into low or high risk according to the EPclin score, consisting of a 12-gene molecular score combined with tumor size and nodal status. Association of the EPclin score with disease-free survival (DFS) and distant metastasis-free survival (DMFS) was evaluated using Kaplan-Meier estimates. The independent prognostic added value of EPclin score was tested in a multivariate Cox model after adjusting on tumor characteristics. RESULTS: EndoPredict test results were available for 768 patients: 663 patients classified as EPclin high risk (EPCH) and 105 patients as EPclin low risk (EPCL). Median follow-up was 70 months (range 1-172 months). For the 429 EPCH randomized patients, there was no significant difference in DFS between treatment arms. The 60-month relapse rate for patients in the EPCL and EPCH groups was 0% and 7%, respectively. Hazard ratio (HR) supposing continuous EPclin score was 1.87 [95% confidence interval (CI) 1.4-2.5, P < 0.0001]. This prognostic effect remained significant when assessed in a Cox model adjusting on tumor size, number of positive nodes and tumor grade (HR 1.52, 95% CI 1.09-2.13, P = 0.0141). The 60-month DMFS for patients in the EPCL and EPCH groups was 100% and 94%, respectively (adjusted HR 8.10, 95% CI 1.1-59.1, P < 0.0001). CONCLUSIONS: The results confirm the value of EPclin score as an independent prognostic parameter in node-positive, hormone receptor-positive, HER2-negative early breast cancer patients receiving standard adjuvant treatment. EPclin score can be used to identify patients at higher risk of recurrence who may warrant additional systemic treatments.

Discovery of a Long Half-Life AURKA Inhibitor to Treat MYC-Amplified Solid Tumors as a Monotherapy and in Combination with Everolimus.

Aurora kinase inhibitors, such as alisertib, can destabilize MYC-family oncoproteins and have demonstrated compelling antitumor efficacy. In this study, we report 6K465, a novel pyrimidine-based Aurora A inhibitor, that reduces levels of c-MYC and N-MYC oncoproteins more potently than alisertib. In an analysis of the antiproliferative effect of 6K465, the sensitivities of small cell lung cancer (SCLC) and breast cancer cell lines to 6K465 were strongly associated with the protein levels of c-MYC and/or N-MYC. We also report DBPR728, an acyl-based prodrug of 6K465 bearing fewer hydrogen-bond donors, that exhibited 10-fold improved oral bioavailability. DBPR728 induced durable tumor regression of c-MYC- and/or N-MYC-overexpressing xenografts including SCLC, triple-negative breast cancer, hepatocellular carcinoma, and medulloblastoma using a 5-on-2-off or once-a-week dosing regimen on a 21-day cycle. A single oral dose of DBPR728 at 300 mg/kg induced c-MYC reduction and cell apoptosis in the tumor xenografts for more than 7 days. The inhibitory effect of DBPR728 at a reduced dosing frequency was attributed to its uniquely high tumor/plasma ratio (3.6-fold within 7 days) and the long tumor half-life of active moiety 6K465. Furthermore, DBPR728 was found to synergize with the mTOR inhibitor everolimus to suppress c-MYC- or N-MYC-driven SCLC. Collectively, these results suggest DBPR728 has the potential to treat cancers overexpressing c-MYC and/or N-MYC.

Targeting the EGFR pathway: An alternative strategy for the treatment of tuberous sclerosis complex?

INTRODUCTION: Tuberous sclerosis complex (TSC) is caused by variants in TSC1/TSC2, leading to constitutive activation of the mammalian target of rapamycin (mTOR) complex 1. Therapy with everolimus has been approved for TSC, but variations in success are frequent. Recently, caudal late interneuron progenitor (CLIP) cells were identified as a common origin of the TSC brain pathologies such as subependymal giant cell astrocytomas (SEGA) and cortical tubers (CT). Further, targeting the epidermal growth factor receptor (EGFR) with afatinib, which is expressed in CLIP cells, reduces cell growth in cerebral TSC organoids. However, investigation of clinical patient-derived data is lacking. AIMS: Observation of EGFR expression in SEGA, CT and focal cortical dysplasia (FCD) 2B human brain specimen and investigation of whether its inhibition could be a potential therapeutic intervention for these patients. METHODS: Brain specimens of 23 SEGAs, 6 CTs, 20 FCD2Bs and 17 controls were analysed via immunohistochemistry to characterise EGFR expression, cell proliferation (via Mib1) and mTOR signalling. In a cell-based assay using primary patient-derived cells (CT n = 1, FCD2B n = 1 and SEGA n = 4), the effects of afatinib and everolimus on cell proliferation and cell viability were observed. RESULTS: EGFR overexpression was observed in histological sections of SEGA, CT and FCD2B patients. Both everolimus and afatinib decreased the proliferation and viability in primary SEGA, tuber and FCD2B cells. CONCLUSION: Our study demonstrates that EGFR suppression might be an effective alternative treatment option for SEGAs and tubers, as well as other mTOR-associated malformations of cortical development, including FCD2B.

Everolimus decreases [U-13C]glucose utilization by pyruvate carboxylase in breast cancer cells in vitro and in vivo.

Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U-13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified.

MEK Inhibition for RASopathy-Associated Hypertrophic Cardiomyopathy: Clinical Application of a Basic Concept.

The term "RASopathies" designates a group of developmental syndromes that are caused by activating variants of the rat sarcoma virus protein (RAS)/mitogen-activated protein kinase (MAPK) cascade. The most prevalent clinical diagnosis is Noonan syndrome, and other, less prevalent conditions include Noonan syndrome with multiple lentigines, Costello syndrome, cardiofaciocutaneous syndrome, and others. Hypertrophic cardiomyopathy occurs in 10% of these patients and can be severe and life-threating. Recently, repurposing of medications inhibiting the RAS/MAPK on a compassionate use basis has emerged as a promising concept to improve the outcome of these patients. Herein, we specifically review the role of the RAS/MAPK pathway in RASopathy-associated cardiomyopathy, and discuss the role of small-molecule inhibition in the treatment of this condition. We describe how drug repurposing of trametinib (mitogen-activated protein/extracellular signal-regulated kinase inhibition) and sirolimus/everolimus (mammalian target of rapamycin inhibition) was performed, how genotype-specific therapies are chosen and followed, as well as initial outcomes from early case series. Finally, we lay out the challenges and opportunities for trials that aim to quantify the benefits of this approach.

Everolimus inhibits hepatoblastoma by inducing autophagy-dependent ferroptosis.

Everolimus, a known inhibitor of the mammalian target of rapamycin (mTOR), has shown uncertain efficacy in treating hepatoblastoma. This study delves into the potential anti-hepatoblastoma properties of everolimus and its intricate relationship with autophagy and ferroptosis, both in vitro and in vivo. In vivo, tumor tissue from hepatoblastoma patient and human hepatoblastoma cell line HuH-6 were xenografted into nude mice to establish xenograft models for observing the effect of everolimus on tumor growth. In vitro, HuH-6 cells were cultured to evaluate the anti-hepatoblastoma activity of everolimus. Transmission electron microscopy and microtubule-associated proteins 1 light chain 3 (LC3), beclin 1, and p62 protein expressions were employed to investigate autophagy. Additionally, indicators of cell apoptosis, reactive oxygen species (ROS) and proteins associated with ferroptosis were measured to evaluate ferroptosis. The results demonstrate that everolimus treatment effectively induced the formation of autophagosomes in hepatoblastoma cells, upregulated the LC3II/I ratio and beclin 1 expression, and downregulated p62 expression, indicating an enhanced autophagy level both in vitro and in vivo. Furthermore, everolimus treatment induced cell apoptosis, increased ROS level, elevated concentrations of malondialdehyde, 4-hydroxynonenal, and iron content, while reducing the ratio of glutathione/oxidized glutathione, and downregulating the protein expression of glutathione peroxidase 4 and solute carrier family 7 member 11, suggesting its ability to induce ferroptosis in hepatoblastoma cells. Importantly, the induction of ferroptosis by everolimus was significantly reversed in the presence of autophinib, an autophagy inhibitor, indicating the autophagy-dependent of everolimus-induced ferroptosis. Taken together, these findings suggest that everolimus holds promise as an effective anti-hepatoblastoma drug, with its mechanism of action potentially involving the induction of autophagy-dependent ferroptosis in hepatoblastoma cells.

Everolimus exerts anticancer effects through inhibiting the interaction of matrix metalloproteinase-7 with syndecan-2 in colon cancer cells.

Previous work showed that matrix metalloproteinase-7 (MMP-7) regulates colon cancer activities through an interaction with syndecan-2 (SDC-2) and SDC-2-derived peptide that disrupts this interaction and exhibits anticancer activity in colon cancer. Here, to identify potential anticancer agents, a library of 1,379 Food and Drug Administration (FDA)-approved drugs that interact with the MMP-7 prodomain were virtually screened by protein-ligand docking score analysis using the GalaxyDock3 program. Among five candidates selected based on their structures and total energy values for interacting with the MMP-7 prodomain, the known mechanistic target of rapamycin kinase (mTOR) inhibitor, everolimus, showed the highest binding affinity and the strongest ability to disrupt the interaction of the MMP-7 prodomain with the SDC-2 extracellular domain in vitro. Everolimus treatment of the HCT116 human colon cancer cell line did not affect the mRNA expression levels of MMP-7 and SDC-2 but reduced the adhesion of cells to MMP-7 prodomain-coated plates and the cell-surface localization of MMP-7. Thus, everolimus appears to inhibit the interaction between MMP-7 and SDC-2. Everolimus treatment of HCT116 cells also reduced their gelatin-degradation activity and anticancer activities, including colony formation. Interestingly, cells treated with sirolimus, another mTOR inhibitor, triggered less gelatin-degradation activity, suggesting that this inhibitory effect of everolimus was not due to inhibition of the mTOR pathway. Consistently, everolimus inhibited the colony-forming ability of mTOR-resistant HT29 cells. Together, these data suggest that, in addition to inhibiting mTOR signaling, everolimus exerts anticancer activity by interfering with the interaction of MMP-7 and SDC-2, and could be a useful therapeutic anticancer drug for colon cancer.NEW & NOTEWORTHY The utility of cancer therapeutics targeting the proteolytic activities of MMPs is limited because MMPs are widely distributed throughout the body and involved in many different aspects of cell functions. This work specifically targets the activation of MMP-7 through its interaction with syndecan-2. Notably, everolimus, a known mTOR inhibitor, blocked this interaction, demonstrating a novel role for everolimus in inhibiting mTOR signaling and impairing the interaction of MMP-7 with syndecan-2 in colon cancer.

Enhanced Antipediatric Sarcoma Effect of Everolimus with Secukinumab by Targeting IL17A.

In this study, we explored the therapeutic potential of everolimus, an mTOR inhibitor, in a patient-derived xenograft (PDX) of rhabdomyosarcoma, the most prevalent malignant pediatric sarcoma. In addition, rhabdoid tumor cell line A-204 and Ewings sarcoma cell line A-673 were cultured to assess the in vitro effect of everolimus. Furthermore, the cell-derived xenograft (CDX) of A-673 was established and treated with everolimus in vivo. IHC and Western blotting were performed to detect the expressions of pertinent proteins. Results showed that everolimus intervention had limited inhibitory effect on PDX tumor growth compared with cyclophosphamide. Nevertheless, everolimus treatment significantly influenced the phosphorylation levels of S6 kinase beta 1 (S6K1) and eIF4E-binding protein 1 (p-4E-BP1), resulting in the inhibition of angiogenesis in vitro and in vivo. Interestingly, everolimus led to an upregulation in the level of IL17A in sarcoma cells. Notably, when secukinumab, a mAb of IL17A, was combined with everolimus, it synergistically enhanced the inhibitory effect of everolimus on sarcoma cell proliferation in vitro and on the growth of PDX or CDX xenograft tumors in vivo. Importantly, this combination therapy did not affect the mTOR signaling. These results indicate that everolimus exerts an antipediatric sarcoma effect by inhibiting mTOR signal. However, everolimus induces sarcoma cells to produce IL17A, which promotes tumor cell survival and counteracts its antipediatric sarcoma effect. The combination of secukinumab effectively eliminates the effects of IL17A, thereby improving the therapeutic efficacy of everolimus in the context of pediatric sarcomas.

Combining MTI-31 with RAD001 inhibits tumor growth and invasion of kidney cancer by activating autophagy.

At most of the times, patients who are diagnosed with kidney cancer should be provided with systemic treatment as drug resistance is a challenging issue in the treatment of this disease. The progression of the cancer can be inhibited with the help of mTOR inhibitors namely RAD001 (everolimus) and MTI-31. In literature, it has been revealed that these mTOR inhibitors have the potential to stimulate autophagy. This degradation pathway boosts the survival rate of the cancerous cells that are subjected to anti-cancer therapy. In this study, CCK8, colony formation assays, and ethynyl deoxyuridine (EdU) analysis were conducted to detect cell proliferation. Furthermore, Transwell assays were also conducted for cell migration analysis. In addition to these, the researchers also performed the flow cytometry process to identify the cells that are undergoing apoptosis. In vivo, experiments were conducted to measure the growth of tumors and metastasis. In this study, the treatment provided through a combination of MTI-31 and RAD001 significantly inhibited the kidney cancer cells' proliferation and tumor growth. Furthermore, there was a notable reduction in the migration and invasion of kidney cancer cells upon the neighboring cells. The outcomes from the mechanistic studies infer that the combination of MTI-31 and RAD001 increases the LC3 levels, which in turn translates into the activation of autophagy. To conclude, the combination of MTI-31 and RAD001 improves the anti-cancerous impact produced by RAD001 in vivo through the promotion of autophagy.

A Stable Micellar Formulation of RAD001 for Intracerebroventricular Delivery and the Treatment of Alzheimer's Disease and Other Neurological Disorders.

A large body of evidence, replicated in many mouse models of Alzheimer's disease (AD), supports the therapeutic efficacy of the oral mammalian target of rapamycin inhibitors (mTOR-Is). Our preliminary data show that intracerebroventricular (ICV) administration of everolimus (RAD001) soon after clinical onset greatly diminished cognitive impairment and the intracellular beta amyloid and neurofibrillary tangle load. However, RAD001 shows >90% degradation after 7 days in solution at body temperature, thus hampering the development of proper therapeutic regimens for patients. To overcome such a drawback, we developed a stable, liquid formulation of mTOR-Is by loading RAD001 into distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) micelles using the thin layer evaporation method. The formulation showed efficient encapsulation of RAD001 and a homogeneous colloidal size and stabilised RAD001, with over 95% of activity preserved after 14 days at 37 °C with a total decay only occurring after 98 days. RAD001-loaded DSPE-PEG2000 micelles were unchanged when stored at 4 and 25 °C over the time period investigated. The obtained formulation may represent a suitable platform for expedited clinical translation and effective therapeutic regimens in AD and other neurological diseases.

Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

Mammalian target of rapamycin inhibition impacts energy homeostasis and induces sex-specific body weight loss in humans.

BACKGROUND: Previous data from a 2-year randomized controlled trial (CRAD001ADE12) indicated that mammalian target of rapamycin (mTOR) inhibition by everolimus slowed cyst growth in patients with autosomal-dominant polycystic kidney disease (ADPKD). During the trial, we noted body weight loss in some patients, particularly in women. We hypothesized that everolimus causes body weight reduction by reduced food intake and/or metabolic changes, which could lead to cachexia. METHODS: Within a sub-analysis of the CRAD001ADE12 trial, body weight course was investigated regarding sex-specific differences in 433 adult ADPKD patients (everolimus, n = 215; placebo, n = 218). One hundred four out of 111 patients who participated in the clinical trial centre in Berlin were evaluated under everolimus/placebo therapy (on drug: everolimus, n = 48; placebo, n = 56) and after therapy (off drug: everolimus, n = 15; placebo, n = 18). Eating habits and nutrient/caloric intake were evaluated by validated questionnaires. Systemic and local metabolism was evaluated in four patients after an oral glucose load (OGL) by using calorimetry and adipose/muscle tissue microdialysis. RESULTS: Within the 2-year CRAD001ADE12 trial, a significant body weight loss was observed in female patients on everolimus versus placebo (P = 0.0029). Data of the Berlin Cohort revealed that weight loss was greater in women on everolimus versus men (P < 0.01). After 9 months, women and men had lost 2.6 ± 3.8 and 0.8 ± 1.5 kg (P < 0.05) in body weight, respectively, and after 21 months, they had lost 4.1 ± 6.6 and 1.0 ± 3.3 kg (P < 0.05), respectively. On everolimus, caloric intake was significantly lower in women versus men (1510 ± 128 vs. 2264 ± 216 kcal/day, P < 0.05), caused mainly by a lower fat and protein intake in women versus men. Cognitive restraints, disinhibition and hunger remained unchanged. In a subgroup of patients resting metabolic rate was unchanged whereas OGL-induced thermogenesis was reduced (7 ± 2 vs. 11 ± 2 kcal, P < 0.05). Fasting and OGL-induced fat oxidation was increased (P < 0.05) on versus off everolimus. In adipose tissue, fasting lipolytic activity was increased, but lipolytic activity was inhibited similarly after the OGL on versus off everolimus, respectively. In skeletal muscle, postprandial glucose uptake and aerobic glycolysis was reduced in patients on everolimus. CONCLUSIONS: mTOR inhibition by everolimus induces body weight reduction, specifically in female patients. This effect is possibly caused by a centrally mediated reduced food (fat and protein) intake and by centrally/peripherally mediated increased fat oxidation (systemic) and mobilization (adipose tissue). Glucose uptake and oxidation might be reduced in skeletal muscle. This could lead to cachexia and, possibly, muscle wasting. Therefore, our results have important implications for patients recieving immune-suppressive mTOR inhibition therapy.

Signaling Pathways mTOR and ERK as Therapeutic Targets in Sinonasal Intestinal-Type Adenocarcinoma.

Despite advances in surgery and radiotherapy, the overall prognosis of sinonasal intestinal-type adenocarcinoma (ITAC) is poor, and new treatment options are needed. Recent studies have indicated alterations in cellular signaling pathways that may serve as targets for modern inhibitors. Our aim was to evaluate the frequency of mTOR and ERK pathway upregulation in a retrospective series of 139 ITAC and to test the efficacy and mechanism of action of candidate targeted inhibitors in cell line ITAC-3. An immunohistochemical analysis on p-AKT, p-mTOR, p-S6, p-4E-BP1, and p-ERK indicated, respectively, a 68% and 57% mTOR and ERK pathway activation. In vitro studies using low doses of mTOR inhibitor everolimus and ERK inhibitor selumetinib showed significant growth inhibition as monotherapy and especially as combined therapy. This effect was accompanied by the downregulation of mTOR and ERK protein expression. Our data open a new and promising possibility for personalized treatment of ITAC patients.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT.

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 µM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

Molecular Characteristics of Everolimus-resistant Renal Cell Carcinoma Cells Generated by Continuous Exposure to Everolimus.

BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) inhibitors represent the standard of care for metastatic renal cell carcinoma (RCC). However, treatment outcomes are relatively poor, suggesting a potential problem with tolerating mTOR inhibitors. The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. MATERIALS AND METHODS: Human-derived RCC, Caki-2, and 786-O cells were continuously exposed to everolimus at 1 µM, and the established resistant sublines were designated as Caki/EV and 786/EV, respectively. Cellular characteristics were compared between both cells. RESULTS: Caki/EV and 786/EV cells showed a decrease in sensitivity to everolimus as well as other mTOR inhibitors. Expression of mTOR and its effectors exhibited no alteration in resistant sublines and their counterparts. However, phosphorylation of S6K, an index of mTOR activity, decreased in resistant sublines. PCR array analysis of mTOR signaling pathway-related factors indicated that the expression of INSR, TP53, and IGFBP3 increased in Caki/EV cells, whereas that of TELO2, HRAS, and SGK1 was up-regulated in 786/EV cells. The levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both cell lines. CONCLUSION: The novel everolimus-resistant Caki/EV and 786/EV cells exhibited cross-resistance to other mTOR inhibitors and decreased mTOR activity. Furthermore, down-regulation of DDIT4, DEPTOR, HIF1A, and PLD1 may contribute to everolimus resistance.

ONC201/TIC10 enhances durability of mTOR inhibitor everolimus in metastatic ER+ breast cancer.

The mTOR inhibitor, everolimus, is an important clinical management component of metastatic ER+ breast cancer (BC). However, most patients develop resistance and progress on therapy, highlighting the need to discover strategies that increase mTOR inhibitor effectiveness. We developed ER+ BC cell lines, sensitive or resistant to everolimus, and discovered that combination treatment of ONC201/TIC10 with everolimus inhibited cell growth in 2D/3D in vitro studies. We confirmed increased therapeutic response in primary patient cells progressing on everolimus, supporting clinical relevance. We show that ONC201/TIC10 mechanism in metastatic ER+ BC cells involves oxidative phosphorylation inhibition and stress response activation. Transcriptomic analysis in everolimus resistant breast patient tumors and mitochondrial functional assays in resistant cell lines demonstrated increased mitochondrial respiration dependency, contributing to ONC201/TIC10 sensitivity. We propose that ONC201/TIC10 and modulation of mitochondrial function may provide an effective add-on therapy strategy for patients with metastatic ER+ BCs resistant to mTOR inhibitors.

Breast cancer is one of the most frequently diagnosed cancers globally, particularly among women. The most common type of breast cancer expresses a receptor for the hormone estrogen. Many treatments block the activity of estrogen and therefore slow or block the development and spread of this type of breast cancer. For patients with advanced breast cancer, hormone-blocking treatments work best in combination with other drugs, including one called everolimus. However, in many patients the cancer cells become resistant to these therapies, leading to disease progression and decreased survival. To explore treatment strategies that could enhance the effectiveness of existing therapies for breast cancer, Farmaki et al. studied how cancer cells which had become resistant to everolimus responded when treated with an experimental drug called ONC201/TIC10. A combination of everolimus and ONC201/TIC10 inhibited growth of resistant cancer cells that had been grown in a three-dimensional arrangement to mimic human tumors. Moreover, the drug combination effectively targeted breast cancer cells collected from patients whose cancer had progressed while being treated with everolimus, suggesting that ONC201/TIC10 could be relevant in a clinical setting. Finally, molecular and biochemical experiments revealed that the drug ONC201/TIC10 works by disrupting the pathways that everolimus-resistant cancer cells use to generate the energy required to grow and proliferate. Taken together these findings suggest that ONC201/TIC10 may provide an effective add-on therapy for patients with certain types of advanced breast cancer that are no longer responding to everolimus. Before this becomes a reality for patients, however, there will have to be more experimental testing of ONC201/TIC10 to determine optimal dosing and timing strategy for future clinical trials.