A mechanism for synergy with combined mTOR and PI3 kinase inhibitors

Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.     

AIF inhibits tumor metastasis by protecting PTEN from oxidation

Apoptosis-inducing factor (AIF) exerts dual roles on cell death and survival, but its substrates as a putative oxidoreductase and roles in tumorigenesis remain elusive. Here, we report that AIF physically interacts with and inhibits the oxidation of phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor susceptible for oxidation-mediated inactivation. More intriguingly, we also identify PTEN as a mitochondrial protein and the ectopic expression of mitochondrial targeting sequence-carrying PTEN almost completely inhibits Akt phosphorylation in PTEN-deficient cells. AIF knockdown causes oxidation-mediated inactivation of the lipid phosphatase activity of PTEN, with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK-3β, and activation of β-catenin signaling in cancer cells. Through its effect on β-catenin signaling, AIF inhibits epithelial–mesenchymal transition (EMT) and metastasis of cancer cells in vitro and in orthotopically implanted xenografts. Accordingly, the expression of AIF is correlated with the survival of human patients with cancers of multiple origins. These results identify PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis.     

Inhibition of mycobacterial infection by the tumor suppressor PTEN

The tumor suppressor PTEN is a lipid phosphatase that is frequently mutated in various human cancers. PTEN suppresses tumor cell proliferation, survival, and growth mainly by inhibiting the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. In addition to it role in tumor suppression, the PTEN-PI3K pathway controls many cellular functions, some of which may be important for cellular resistance to infection. Currently, the intersection between tumorigenic signaling pathways and cellular susceptibility to infection is not well defined. In this study we report that PTEN signaling regulates infection of both noncancerous and cancerous cells by multiple intracellular mycobacterial pathogens and that pharmacological modulation of PTEN signaling can affect mycobacterial infection. We found that PTEN deficiency renders multiple types of cells hyper-susceptible to infection by Mycoplasma and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The lipid phosphatase activity of PTEN is required for attenuating infection. Furthermore, we found mycobacterial infection activates host cell Akt phosphorylation, and pharmacological inhibition of Akt or PI3K activity reduced levels of intracellular infection. Intriguingly, inhibition of mTOR, one of the downstream components of the Akt signaling and a promising cancer therapeutic target, also lowered intracellular Bacillus Calmette-Guérin levels in mammary epithelial cancer MCF-7 cells. These findings demonstrate a critical role of PTEN-regulated pathways in pathogen infection. The relationship of PTEN-PI3K-Akt mTOR status and susceptibility to mycobacterial infection suggests that the interaction of mycobacterial pathogens with cancer cells may be influenced by genetic alterations in the tumor cells.     

神经前体细胞表达发育性下调蛋白4在消化系统肿瘤发生中的作用

神经前体细胞表达发育性下调蛋白4(neural precursor cell expressed,developmentally down-regulated protein 4,NEDD4-1,部分文章也称NEDD4)是近年来才备受关注的肿瘤相关基因,属于E3 HECT(homologous to E6 associated protein C terminus,E6蛋白c端同源基因)泛素连接酶NEDD4样家族成员。泛素连接酶,能够参与多种蛋白质的泛素化、溶酶体及蛋白酶体的降解、胞核-胞质转位等,间接影响不同恶性肿瘤的多种信号通路。随着大量NEDD4-1与肿瘤相关实验的不断深入,目前已发现其可通过调控细胞周期、癌细胞侵袭转移、拮抗耐药性等许多途径影响肿瘤的生物学行为。在消化系统肿瘤中,NEDD4-1主要通过PTEN/PI3K/AKT、TGF-β、Hippo、LDLRAD4等多条通路促进肝细胞癌的增殖、侵袭和迁移能力;在胰腺癌中发现,NEDD4-1在PI3K/AKT信号通路中发挥癌基因作用,但在与Myc-SIRT2所形成的信号环路中,却发挥抑癌基因的作用;在胃癌和结直肠癌中,NEDD4-1所参与的信号通路与其他消化系统肿瘤均不相同,NEDD4-1能独立于PTEN/PI3K/AKT通路而发挥促进胃癌恶化、转移(EGFR信号通路)和抑制结直肠癌肿瘤生长(WNT信号通路)的作用。NEDD4-1已经成为人们治愈肿瘤的热门研究方向。本文通过系统总结NEDD4-1在不同消化系统肿瘤中的功能、信号通路和潜在抑制剂等,进行探讨NEDD4-1与不同信号通路的关系,旨为临床在癌症治疗领域提供重要的参考数据。     

Akt-mTOR的互动与癌症的发生

P13K的下游效应蛋白Akt,在人癌中经常处于高度激活状态。mTOR作为Akt下游的重要效应子在肿瘤发生中扮演重要角色。在P13K-Akt—mTOR这条信号通路中,Akt所产生的效应受到两个肿瘤抑制基因的负调控：PTEN,处于Akt的上游：TSC1／TSC2,位于AKT的下游和mTOR的上游。新的研究结果表明,当缺少TSC1／TSC2的负性调节时,mTOR则通过两种复合物的平衡移动来反馈抑制Akt活性。利用小鼠遗传学手段研究PTEN和TSC2在癌症发生和进展中的角色,也证明AKT—mTOR的互相作用在癌症发展与治疗中的重要性。     

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.     

Role of PTEN protein in multidrug resistance of prostate cancer cells

In a past decade became evident that phosphatidylinositol-3-kinase controlled signal transduction cascade (PI3K/Akt/PTEN/mTOR) is implicated in resistance of tumor cells to anticancer drugs. Another well studied mechanism of multidrug resistance is associated with the activity of drug transporters of ABC superfamily (first of all P-glycoprotein (Pgp), MRP1, BCRP). Several mechanisms of cell defense can be turned on in one cell. The interconnections between different mechanisms involved in drug resistance are poorly studied. In the present study we used PC3 and DU145 human prostate cell lines to show that PTEN functional status determines level of cell resistance to some drugs, it correlates with expression level of MRP1 and BCRP proteins. We showed that Pgp is not involved in development of drug resistance in these cells. Transfection of PTEN into PTEN-deficient PC3 as well as rapamycin treatment caused the inhibition of PI3K/Akt/mTOR signaling and resulted in cell sensitization to the action of doxorubicin and vinblastine. We showed that PTEN transfection leads to the change in expression of MRP1 and BCRP. Our results show that in prostate cancer cells at least two mechanisms of drug resistance are interconnected. PTEN and mTOR signaling were shown: to be involved into regulation of MRP1 and BCRP.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

Role of the PTEN protein in the multidrug resistance of prostate cancer cells

At present, there is no doubt that the signal transduction pathway P13K/Akt/PTEN/mTOR, controlled by phosphatidylinositol-3-kinase, is involved in tumor cell resistance to a number of drugs. Another well-known mechanism determining drug resistance in tumors is associated with the activity of drug transporters of the ABC superfamily (first of all, P-glycoprotein (Pgp), MRP1, BCRP, and LRP). Several mechanisms of cell defense can simultaneously operate in one cell. The interplay of different mechanisms involved in drug resistance is poorly understood. The PC3 and DU145 human prostate cell lines were used to show that the PTEN functional status determined the cell resistance to some drugs and that correlated with the levels of MRP1 and BCRP. Pgp was not involved in drug resistance of these cells. Introduction of PTEN into PTEN-deficient PC3 cells, as well as rapamycin treatment, inhibited Akt and mTOR and sensitized cells to doxorubicin and vinblastine. Exogenous PTEN altered the MRP1 and BCRP expression. The results indicate that at least two mechanisms of drug resistance operate in prostate cancer cells: the PI3K/Akt/PTEN/mTOR pathway and an elevated MRP1 expression. The mechanisms are interconnected: PTEN and mTOR signaling is involved in MRP1 and BCRP expression regulation.     

Thioredoxin-1 binds to the C2 domain of PTEN inhibiting PTEN's lipid phosphatase activity and membrane binding: a mechanism for the functional loss of PTEN's tumor suppressor activity

Thioredoxin-1 (Trx-1) is a 12 kDa redox protein that is overexpressed in a large number of human tumors. Elevated Trx-1 is associated with increased tumor cell proliferation, inhibited apoptosis, aggressive tumor growth, and decreased patient survival. The molecular mechanisms for the promotion of tumorigenesis by Trx-1 are not known. PTEN is a major tumor suppressor of human cancer that acts by hydrolyzing membrane phosphatidylinositol (PtdIns)-3-phosphates, thus, preventing the activation of the survival signaling kinase Akt by PtdIns-3-kinase. We show that Trx-1 binds in a redox dependent manner to PTEN to inhibit its PtdIns-3-phosphatase activity which results in increased Akt activation in cells. Molecular docking and site-specific mutation studies show that the binding of Trx-1 to PTEN occurs through a disulfide bond between the active site Cys(32) of Trx-1 and Cys(212) of the C2 domain of PTEN leading to steric interference by bound Trx-1 of the catalytic site of PTEN and of the C2 lipid membrane-binding domain. The results of the study suggest that the increased levels of Trx-1 in human tumors could lead to functional inhibition of PTEN tumor suppressor activity providing an additional mechanism for tumorigenesis with loss of PTEN activity.     

mTOR, translational control and human disease

Many human diseases occur when the precise regulation of cell growth (cell mass/size) and proliferation (rates of cell division) is compromised. This review highlights those human disorders that occur as a result of inappropriate cellular signal transduction through the mammalian target of rapamycin (mTOR), a major pathway that coordinates proper cell growth and proliferation by regulating ribosomal biogenesis and protein translation. Recent studies reveal that the tuberous sclerosis complex (TSC)-1/2, PTEN, and LKB1 tumor suppressor proteins tightly control mTOR. Loss of these tumor suppressors leads to an array of hamartoma syndromes as a result of heightened mTOR signaling. Since mTOR plays a pivotal role in maintaining proper cell size and growth, dysregulation of mTOR signaling results in these benign tumor syndromes and an array of other human disorders.     

USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT/mTOR Signaling

Angiotensin II type 1 receptor-associated protein (ATRAP) is widely expressed in different tissues and organs, although its mechanistic role in breast cancer remains unclear. Here, we show that ATRAP is highly expressed in breast cancer tissues. Its aberrant upregulation promotes breast cancer aggressiveness and is positively correlated with poor prognosis. Functional assays revealed that ATRAP participates in promoting cell growth, metastasis, and aerobic glycolysis, while microarray analysis showed that ATRAP can activate the AKT/mTOR signaling pathway in cancer progression. In addition, ATRAP was revealed to direct Ubiquitin-specific protease 14 (USP14)-mediated deubiquitination and stabilization of Pre-B cell leukemia homeobox 3 (PBX3). Importantly, ATRAP is a direct target of Upstream stimulatory factor 1 (USF1), and that ATRAP overexpression reverses the inhibitory effects of USF1 knockdown. Our study demonstrates the broad contribution of the USF1/ATRAP/PBX3 axis to breast cancer progression and provides a strong potential therapeutic target.     

Autophagy Signaling and the Cogwheels of Cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival, and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haplo-insufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

Autophagy signaling and the cogwheels of cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haploinsufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

Expression and significance of new tumor suppressor gene PTEN in primary liver cancer

Objective : To investigate expression and significance of PTEN gene in primary hepatocellular carcinoma (HCC). Methods: Immunohistochemical peroxidase-conjugated streptavidin (SP) method was used to detect expression of PTEN gene in 120 cases of primary HCC and its adjacent tissue 10 cases of normal liver tissue. The relationship between expression of tumor suppressor gene of PTEN and the percentage of lymph node metastasis of HCC was analyzed. Results: It was shown that PTEN gene was expressed in all 10 cases of normal liver tissues and paracancerous liver tissues. The staining was localized mainly in cytoplasm. Expression of PTEN in 120 cases of HCC were as follows: 12.5% were negative, 17.5% were weak positive, and 70% were strong positive. At time of diagnosis, 33/120 (27.5%) presented lymph node metastasis. Lymph node metastases were present in 80% (12 out of 15) PTEN negative HCC, 57.14% (12 out of 21) PTEN weak positive HCC and only 10.71% (9 out of 84) PTEN intense positive HCC, ( P <0.05). Therefore, PTEN tumor suppresor gene malfunction seems to be involed in mtastasing capacity of HCC. Conclusion: This study suggests that PTEN gene was deleted or weakly expressed in primary hepatocellar carcinoma, which is probably related to its tumorigenesis.