Predicted mechanisms of resistance to mTOR inhibitors

The serine/threonine kinase, mTOR (mammalian Target of Rapamycin) has become a focus for cancer drug development. Rapamycins are highly specific inhibitors of mTOR and potently suppress tumour cell growth by retarding cells in G1 phase or potentially inducing apoptosis. Currently, both rapamycin and several analogues are being evaluated as anticancer agents in clinical trials. Results indicate that many human cancers have intrinsic resistance and tumours initially sensitive to rapamycins become refractory, demonstrating acquired resistance. Here, we consider mechanisms of resistance to inhibitors of mTOR.     

Overcoming endocrine resistance in breast cancer: importance of mTOR inhibition

Approaches to treatment for many patients with advanced breast cancer are based on the expression of specific receptors. Treatments targeting the hormone receptor (typically the estrogen receptor) are used to reduce signaling through these receptors and thereby inhibit proliferation of breast cancer cells expressing these receptors. Although these treatments are effective for many patients, resistance to treatment is common. Recent clinical trials suggest that using multiple agents targeting the same pathway is not sufficient to overcome resistance. New treatment approaches are needed for these patients. Inhibition of the mTOR signaling pathway, a key point of confluence for multiple signaling cascades, offers a promising approach to restoring sensitivity to endocrine therapy in breast cancer. This article reviews the current data from studies of mTOR inhibitors everolimus and temsirolimus in combination with endocrine therapies to overcome treatment resistance in patients with advanced breast cancer.     

Molecular characterization of anastrozole resistance in breast cancer: Pivotal role of the Akt/mTOR pathway in the emergence of de novo or acquired resistance and importance of combining the allosteric Akt inhibitor MK‐2206 with an aromatase inhibitor

Acquisition of resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of estrogen receptor alpha (ERα)‐positive breast cancers. The Res‐Ana cells, a new model of acquired resistance to anastrozole, were established by long‐term exposure of aromatase‐overexpressing MCF‐7 cells to this drug. These resistant cells developed ER‐independent mechanisms of resistance and decreased sensitivity to the AI letrozole or to ERα antagonists. They also displayed a constitutive activation of the PI3K/Akt/mTOR pathway and a deregulated expression of several ErbB receptors. An observed increase in the phospho‐Akt/Akt ratio between primary and matched recurrent breast tumors of patients who relapsed under anastrozole adjuvant therapy also argued for a pivotal role of the Akt pathway in acquired resistance to anastrozole. Ectopic overexpression of constitutively active Akt1 in control cells was sufficient to induce de novo resistance to anastrozole. Strikingly, combining anastrozole with the highly selective and allosteric Akt inhibitor MK‐2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings lead to us proposing a model of anastrozole‐acquired resistance based on the selection of cancer‐initiating‐like cells possessing self‐renewing properties, intrinsic resistance to anastrozole and sensitivity to MK‐2206. Altogether, our work demonstrated that the Akt/mTOR pathway plays a key role in resistance to anastrozole and that combining anastrozole with Akt/mTOR pathway inhibitors represents a promising strategy in the clinical management of hormone‐dependent breast cancer patients.     

Current development of the second generation of mTOR inhibitors as anticancer agents

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a "master switch" for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-interacting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.     

Antihormonal therapy in breast cancer and mTOR inhibitors

Hormonal dependence of breast cancer has been known for a long time, yet about half of breast cancers with estrogen receptor will not respond to antihormonal therapy. Now, we know that this resistance may be related to a dysfunction of the estrogen pathway, or that of growth factors and particularly the pathway of cell activation PI3K/Akt/mTOR. Prevention of these different mechanisms of resistance could involve combination therapies such as anti-estrogens (SERMs, aromatase inhibitors) with inhibitors of the activity of growth factors that are particularly temsirolimus and everolimus for the activation pathway cell PI3K/Akt/mTOR.     

mTOR as a therapeutic target in patients with gastric cancer

The poor long-term outcomes associated with current chemotherapy treatment of patients with advanced gastric cancer suggest a need for novel targeted agents that may confer a better survival benefit. Evidence of mammalian target of rapamycin (mTOR) activation has been demonstrated in patient-derived gastric cancer cells and tumors. This review explores the relevance of the mTOR pathway to gastric cancer pathogenesis and its potential as a therapeutic target in patients with gastric cancer as well as presenting the first available clinical data on mTOR inhibitors in this disease setting. Preclinical data suggest that suppression of the mTOR pathway inhibited the proliferation of gastric cancer cells and delayed tumor progression in in vitro and animal models. In the clinical setting, the mTOR inhibitor everolimus has been active and well tolerated in phase I/II studies of patients with chemotherapy-refractory metastatic gastric cancer. Based on these promising results, everolimus currently is being investigated as a monotherapy or in combination with chemotherapeutic agents in ongoing phase II/III clinical studies.     

Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs

Inhibition of a single transduction pathway is often inefficient due to activation of alternative signalling. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation, survival and angiogenic pathways and has been implicated in the resistance to EGFR inhibitors. Thus, mTOR blockade is pursued to interfere at multiple levels with tumour growth. We used everolimus (RAD001) to inhibit mTOR, alone or in combination with anti-EGFR drugs gefitinib or cetuximab, on human cancer cell lines sensitive and resistant to EGFR inhibitors, both in vitro and in vivo. We demonstrated that everolimus is active against EGFR-resistant cancer cell lines and partially restores the ability of EGFR inhibitors to inhibit growth and survival. Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib. Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion. Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.     

Advances in mechanisms of resistance to aromatase inhibitors

Clinically, there are two distinct types of aromatase inhibitor (AI) resistance, namely acquired and innate resistance. Because the underlying mechanisms of these two types of resistance may not be mutually exclusive, strategies to tackle these resistances may not be effective when used interchangeably. Activation of growth factor receptor pathways is the hallmark of acquired AI resistance. These pathways can be targeted either at the cell surface receptor level or their downstream signaling cascades. Currently, everolimus in combination with exemestane represents a new standard of care for patients progressing on non-steroidal AIs. HDAC inhibitors have also shown promising results For innate resistance, the combination of fulvestrant and AI in the front line setting represents a new treatment option, particularly for patients who present with de novo metastatic disease. A Phase III trial is currently ongoing to evaluate the benefit of CDK 4/6 inhibitor, palbociclib, in the first line setting in combination with AI.     

mTOR inhibitors in cancer therapy

The mammalian target of rapamycin (mTOR) plays a key role in regulation of cellular metabolism, growth, and proliferation. The frequent hyperactivation of mTOR signaling makes it an attractive target for therapeutic intervention and has driven the development of a number of mTOR inhibitors. Encouraging data from preclinical studies have resulted in initiation of multiple clinical trials. Furthermore, combinational strategies are being studied in an effort to overcome resistance and enhance efficacy. Although additional studies are required to determine their specific role in the clinical setting, mTOR inhibitors remain a promising therapeutic option for the treatment of cancer.     

PI3K/Akt/mTOR通路抑制剂对白血病细胞株增殖及其PHLPP表达的影响

目的:观察PI3K/Akt/mTOR通路抑制剂wortmannin或rapamycin对白血病细胞株增殖及其PHLPP(PH domainleucine-rich repeat protein phosphatase)蛋白表达的影响。方法:以不同浓度的wortmannin或rapamycin分别作用于人类髓细胞白血病细胞系K562、HL-60,采用WST-1法检测细胞的增殖活性,AnnexinⅤ-FITC双染流式细胞术检测细胞凋亡,Westernblotting法检测细胞中p-Akt、Akt、PHLPP蛋白的表达。结果:Wortmannin以时间以及剂量依赖方式抑制K562、HL-60细胞的增殖(P<0.05),48 h的IC50值分别为(187.6±48.4)、(185.5±48.1)nmol/L。100 nmol/L wortmannin作用于K562细胞、50nmol/L wortmannin作用于HL-60细胞12和24 h后,细胞凋亡率均较对照细胞显著升高[(12.4±0.7)%、(17.6±2.3)%vs(5.0±0.6)%,P<0.05;(11.0±0.2)%、(17.9±1.6)%vs(6.8±0.4)%,P<0.05]。Wortmannin分别作用于K562、HL-60细胞12、24、36 h后,p-Akt、PHLPP的蛋白表达水平明显降低;rapamycin同样可使K562、HL-60细胞中PHLPP蛋白的表达水平降低。结论:PI3K/Akt/mTOR信号通路抑制剂抑制白血病细胞株增殖的同时降低其PHLPP蛋白的表达。     

Differentiating mTOR inhibitors in renal cell carcinoma

PI3K/Akt/mTOR signalling is dysregulated in many cancers, including renal cell carcinoma (RCC), and activation of this pathway has been suggested to correlate with aggressive behavior and poor prognosis in RCC tumors. mTOR inhibition plays a principal role in the targeted treatment of many cancer types, including RCC. Although mTOR inhibitors share the same mechanism of action, differences in metabolism, formulation and dosing schedule underpin distinct PK/PD profiles such that they may be differentiated for use in a variety of treatment niches. Approved mTOR inhibitors temsirolimus and everolimus serve as important therapeutic options within the current RCC treatment paradigm, although their recommended applications differ in setting and patient population characteristics. Clinical practice guidelines recommend temsirolimus for use in treatment-naive patients with poor-prognosis metastatic RCC of any histology (predominant clear cell or non-clear cell histology). Everolimus provides a standard-of-care therapy for patients with metastatic RCC whose disease has progressed after previous vascular endothelial growth factor receptor-tyrosine kinase inhibitor therapy. As therapeutic failure impacts the vast majority of patients with RCC, sequencing strategies of available agents or simultaneous targeting of multiple members of the PI3K/Akt/mTOR pathway may provide additional clinical benefit. Various classes of agents targeting the PI3K/Akt/mTOR pathway are currently being investigated, including mTORC1/mTORC2 kinase domain inhibitors, mTOR/PI3K dual inhibitors, PI3K-selective inhibitors, and programmed cell death 6 modulators. Clinical trials of mTOR inhibitors in a variety of tumor types are ongoing, and the role of mTOR inhibitors continues to evolve across the RCC treatment landscape.     

Use of APO2L/TRAIL with mTOR inhibitors in the treatment of glioblastoma multiforme

The mammalian target of rapamycin (mTOR) plays a critical role in the regulation of cell growth, proliferation and survival. Components of the mTOR pathway are activated in a variety of tumors, including glioblastoma multiforme (GBM), and we have found that one surprising consequence of mTOR pathway activation is resistance of GBMs to the proapoptotic effects of agents such as APO2L/TRAIL. mTOR inhibition has become feasible following the development of rapamycin and comparable analogs with improved pharmacological properties, including CCI-779, RAD001 and AP23573. Numerous studies have also demonstrated promising proapoptotic activity, with relatively mild side effects, using rapamycin analogs in vitro and in vivo in conjunction with APO2L/TRAIL. These studies suggest that mTOR inhibitors can be combined with APO2L/TRAIL as a potential tumor-selective therapy.     

mTOR通路激活后上调SNCG表达水平抑制 乳腺癌细胞辐射 敏感性的研究

目的：探讨乳腺癌T47D细胞通过激活mTOR通路调控SNCG表达水平,从而抑制乳腺癌细胞辐射敏感性的分子机制。方法：检测不同剂量γ射线照射后的T47D乳腺癌细胞中mTOR蛋白表达水平。在细胞培养液中加入不用浓度磷脂酸（PA,mTOR通路激活剂）进行培养,以常规培养细胞为对照组,采用Western blot法检测对照组和激活剂组细胞SNCG蛋白的表达。对照组和激活组细胞采用4 Gy γ射线照射24 h,检测照射后SNCG mRNA和蛋白的表达情况,并采用平板细胞克隆形成实验检测克隆形成率。同时,将转染SNCG siRNA的乳腺癌T47D细胞株分成激活组和对照组,验证SNCG在乳腺癌细胞辐射敏感性抵抗中的生物学功能。结果：不同剂量射线照射后,mTOR蛋白表达水平显著升高。mTOR激活剂PA处理后的细胞对乳腺癌细胞放射敏感性具有明显的抑制作用,同时Western blot显示γ射线照射处理后的乳腺癌细胞中SNCG蛋白的表达水平异常。Western blot和qPCR方法检测发现,T47D对照组和干扰SNCG基因的T47D细胞实验组中,激活mTOR或γ射线照射均能引起SNCG蛋白和mRNA表达增加。克隆形成实验进一步证明,降低SNCG的表达可显著抑制T47D细胞克隆形成能力。结论：在乳腺癌细胞中,mTOR介导的SNCG表达调控对乳腺癌细胞抗辐射起着重要作用,降低SNCG的表达可提高辐射敏感性,提示在临床治疗中有可能通过使用SNCG抑制剂或mTOR抑制剂提高乳腺癌细胞在放化疗中的敏感性。     

Enhancement of mTOR signaling contributes to acquired X‐ray and C‐ion resistance in mouse squamous carcinoma cell line

Our aim was to evaluate whether repetition of C‐ion (carbon ion beam) irradiation induces radioresistance as well as repeated X‐ray irradiation in cancer cell lines, and to find the key molecular pathway for radioresistance by comparing radioresistant cancer cells with their parental cells. A mouse squamous cell carcinoma cell line, NR‐S1, and radioresistant cancer cells, NR‐S1‐C30 (C30) and NR‐S1‐X60 (X60), established by repetition of C‐ion and X‐ray irradiation, respectively, were used. X‐ray and C‐ion sensitivity, changes in lysosome, mitochondria, intracellular ATP and reactive oxygen species (ROS) level, and mechanistic target of rapamycin (mTOR) signaling were evaluated. Moreover, the effect of rapamycin on radioresistance was also assessed. X‐ray and C‐ion resistance of C30 cells was moderate, and the resistance of X60 cells was the highest in this study. In X60 cells, the amount of lysosome, mitochondria, intracellular ATP and ROS level were significantly increased, and mTOR and p70S6K (ribosomal protein S6 kinase p70) phosphorylation were enhanced compared with C30 and NR‐S1 cells. The inhibition of mTOR signaling was effective for X‐ray and C‐ion radiosensitization in both cell lines, especially in X60 cells in which X‐ray and C‐ion resistance was decreased to the same level as that in NR‐S1 cells. Our results indicated that the contribution to generate X‐ray and C‐ion resistance was less for repeated C‐ion irradiations compared with repeated X‐ray irradiation. Moreover, we found that activated mTOR signaling contributes to X‐ray and C‐ion resistance in the X60 cancer cells.     

Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers

Recent clinical trials with selective inhibitors of the BRAF and MEK kinases have shown promising results in patients with tumors harboring BRAF V600 mutations. However, as has been observed previously with similarly successful targeted therapies, acquired resistance to these agents is an emerging problem that limits their clinical benefit. Several recent studies from our laboratory and others have investigated the causes of acquired resistance to BRAF and MEK inhibitors, and multiple resistance mechanisms have been identified. Here, we review these mechanisms and suggest that they can be broadly grouped into two main classes: ERK-dependent and ERK-independent. We also propose distinct therapeutic strategies that might be employed to overcome each class of acquired resistance..     

Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors

Dual PI3K/mTOR(phosphatidylinositol 3-kinase/mammalian target of rapamycin) inhibitors are being evaluated clinically for the treatment of tumors with a hyperactivated PI3K/mTOR pathway. However, unexpected outcomes were obtained in clinical studies of cancer patients with an aberrant PI3K pathway. In clinical trials, applicable combination regimens are not yet available. In this study, using an integrated analysis of acquired BEZ235-resistant nasopharyngeal carcinoma cells, we demonstrate that DNA methyltransferase is a key modulator and a common node upstream of the AKT/mTOR and PDK1/MYC pathways, which are activated in cancer cells with acquired BEZ235 resistance. DNA methyltransferases were upregulated and induced PTEN and PPP2R2B gene hypermethylation, which downregulated their expression in BEZ235-resistant cancer cells. Reduced PTEN and PPP2R2B expression correlated with activated AKT/mTOR and PDK1/MYC pathways and conferred considerable BEZ235 resistance in nasopharyngeal carcinoma. Targeting methyltransferases in combination with BEZ235 sensitized BEZ235-resistant cells to BEZ235 in vitro and in vivo, suggesting the potential clinical application of this strategy to overcome BEZ235 resistance.     

Involvement of the Akt/mTOR pathway on EGF-induced cell transformation

Our previous study demonstrated that phosphatidylinositol 3-kinase (PI3K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells.