The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

Activation of peroxisome proliferation–activated receptor-γ inhibits transforming growth factor-β1-induced airway smooth muscle cell proliferation by suppressing Smad–miR-21 signaling

The aims of the current study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation–activated receptor-γ (PPAR-γ) on TGF-β1-induced rat ASMC proliferation and its underlying mechanisms. TGF-β1 upregulated microRNA 21 (miR-21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF-β1–Smad–miR-21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de-repressed the PI3K–Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K–Akt pathway or proteasome function reversed PTEN knockdown-induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR-γ activator, blocked TGF-β1-induced activation of Smad2/3 and its downstream targets changes of miR-21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR-γ inhibits rat ASMC proliferation by suppressing Smad–miR-21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.     

MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.     

Regulation of Epidermal Growth Factor Receptor Signaling and Erlotinib Sensitivity in Head and Neck Cancer Cells by miR-7

Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3′-untranslated region (3′-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 target genes, providing insight into the tumor suppressor function of miR-7. Furthermore, we identified several target miR-7 mRNAs with a putative role in the sensitization of FaDu cells to erlotinib. Together, these data support the coordinate regulation of Akt signaling by miR-7 in HNC cells and suggest the therapeutic potential of miR-7 alone or in combination with EGFR TKIs in this disease.     

MicroRNA-30e-5p suppresses non-small cell lung cancer tumorigenesis by regulating USP22-mediated Sirt1/JAK/STAT3 signaling

MicroRNA-30e-5p (miR-30e-5p) is a tumor suppressor that is known to be downregulated in non-small cell lung cancer (NSCLC). However, how miR-30e-5p inhibits NSCLC tumorigenesis is not known. Ubiquitin-specific peptidase 22 (USP22) is upregulated in NSCLC and promotes tumorigenesis via a Sirt1-JAK-STAT3 pathway. In this study, we investigated whether miR-30e-5p inhibits tumor growth by targeting USP22 in NSCLC. Our results reveal that miR-30e-5p expression was correlated negatively with USP22 in NSCLC tissues. Luciferase reporter assays showed that miR-30e-5p negatively regulated USP22 expression by binding to a specific sequence in the 3?UTR. MiR-30e-5p overexpression and USP22 knockdown significantly inhibited tumor growth in vivo and induced cell cycle arrest and apoptosis in NSCLC cells in vitro. The effects of miR-30e-5p inhibition were prevented by USP22 knockdown. MiR-30e-5p inhibited SIRT1 expression and increased expression of p53 and the phosphorylated form of STAT3 (pSTAT3). Furthermore, miR-30e-5p prevented USP22-mediated regulation of SIRT1, pSTAT3, and p53 expression. Taken together, these findings suggest that miR-30e-5p suppresses NSCLC tumorigenesis by downregulatingUSP22-mediated Sirt1/JAK/STAT3 signaling. Our study has identified miR-30e-5p as a potential therapeutic target for the treatment of NSCLC.     

miR-30d负性调节HeLa细胞的增殖

有证据显示,microRNA( miRNA)可作为癌基因或抑癌基因发挥功能,参与细胞增殖和凋等 调控;PI3K/Akt通路的持续活化与肿瘤的发生、发展密切相关．在本研究中,我们通过microarray技术在HeLa细胞中检测经ly294002作用24 h阻断PI3K/Akt通路后microRNA的表达变化．Taqman 实时 PCR实验结果显示,has-miR-30d的表达在PI3K/Akt通路被阻断后明显上调．萤光素酶报告基因转染结合Taqman 实时 PCR显示,稳定转染miR- 30d过表达质粒的HeLa细胞系中miR-30d的表达量升高并且有活性．MTT及流式细胞术分析显示,过表达miR-30d的HeLa细胞其增殖受到抑制．这些结果说明,has-miR-30d可抑制HeLa细胞增殖,负性调节细胞生长,是一个潜在的抑癌基因．     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.     

Expressions of Forkhead Class Box O 3a on Crushed Rat Sciatic Nerves and Differentiated Primary Schwann cells

Forkhead box-containing protein, class O 3 a (FOXO3a), an Akt downstream target, plays an important role in peripheral nervous system. FOXO3a shares the ability to be inhibited and translocated from the nucleus on phosphorylation by proteins such as Akt/PKB in the PI3K signaling pathway. To elucidate the expression and possible function of FOXO3a in lesion and repair, we performed an acute sciatic nerve crush model and studied differential expressions of FOXO3a. We observed that expressions of FOXO3a in Schwann cells (SCs) of the peripheral nervous system and cAMP-induced differentiation were dynamically regulated. Western blot analysis showed FOXO3a level significantly decreased post injury. Moreover, Immunofluorescence double labeling suggested the changes were striking especially in SCs. In vitro, Western blot analysis showed that the expression of FOXO3a was decreased in cAMP-induced differentiated primary SCs. The FOXO3a siRNA-transfected SCs treated by cAMP promote differentiation of SCs through the PI3K/Akt pathway. The results indicate that FOXO3a plays an important role during differentiation of SCs.     

Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC₅₀ value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.     

Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3beta inhibition and nuclear beta-catenin accumulation

PI3K/Akt plays a critical role in prostate cancer cell growth and survival. Recent studies have shown that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. The PI3K inhibitor, LY294002, and a tumor suppressor, PTEN, negatively regulate the PI3K/Akt pathway and repress AR activity. However, the molecular mechanisms whereby PI3K/Akt and PTEN regulate the androgen pathway are currently unclear. Here, we demonstrate that blocking the PI3K/Akt pathway reduces the expression of an endogenous AR target gene. Moreover, we show that the repression of AR activity by LY294002 is mediated through phosphorylation and inactivation of GSK3beta, a downstream substrate of PI3K/Akt, which results in the nuclear accumulation of beta-catenin. Given the recent evidence that beta-catenin acts as a coactivator of AR, our findings suggest a novel mechanism by which PI3K/Akt modulates androgen signaling. In a PTEN-null prostate cancer cell line, we show that PTEN expression reduces beta-catenin-mediated augmentation of AR transactivation. Using the mutants of beta-catenin, we further demonstrate that the repressive effect of PTEN is mediated by a GSK3beta-regulated degradation of beta-catenin. Our results delineate a novel link among the PI3K, wnt, and androgen pathways and provide fresh insights into the mechanisms of prostate tumor development and progression.     

MicroRNA-375 inhibits colorectal cancer growth by targeting PIK3CA

Colorectal cancer (CRC) is the second most common cause of death from cancer. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by triggering RNA degradation or interfering with translation. Aberrant miRNA expression is involved in human disease including cancer. Herein, we showed that miR-375 was frequently down-regulated in human colorectal cancer cell lines and tissues when compared to normal human colon tissues. PIK3CA was identified as a potential miR-375 target by bioinformatics. Overexpression of miR-375 in SW480 and HCT15 cells reduced PIK3CA protein expression. Subsequently, using reporter constructs, we showed that the PIK3CA untranslated region (3′-UTR) carries the directly binding site of miR-375. Additionally, miR-375 suppressed CRC cell proliferation and colony formation and led to cell cycle arrest. Furthermore, miR-375 overexpression resulted in inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. SiRNA-mediated silencing of PIK3CA blocked the inhibitory effect of miR-375 on CRC cell growth. Lastly, we found overexpressed miR-375 effectively repressed tumor growth in xenograft animal experiments. Taken together, we propose that overexpression of miR-375 may provide a selective growth inhibition for CRC cells by targeting PI3K/Akt signaling pathway.     

Proteomic studies of rat tibialis anterior muscle during postnatal growth and development

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.     

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.