MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling.
ABSTRACT: MAPK4 is an atypical MAPK. Currently, little is known about its physiological function and involvement in diseases, including cancer. A comprehensive analysis of 8887 gene expression profiles in The Cancer Genome Atlas (TCGA) revealed that MAPK4 overexpression correlates with decreased overall survival, with particularly marked survival effects in patients with lung adenocarcinoma, bladder cancer, low-grade glioma, and thyroid carcinoma. Interestingly, human tumor MAPK4 overexpression also correlated with phosphorylation of AKT, 4E-BP1, and p70S6K, independent of the loss of PTEN or mutation of PIK3CA. This led us to examine whether MAPK4 activates the key metabolic, prosurvival, and proliferative kinase AKT and mTORC1 signaling, independent of the canonical PI3K pathway. We found that MAPK4 activated AKT via a novel, concerted mechanism independent of PI3K. Mechanistically, MAPK4 directly bound and activated AKT by phosphorylation of the activation loop at threonine 308. It also activated mTORC2 to phosphorylate AKT at serine 473 for full activation. MAPK4 overexpression induced oncogenic outcomes, including transforming prostate epithelial cells into anchorage-independent growth, and MAPK4 knockdown inhibited cancer cell proliferation, anchorage-independent growth, and xenograft growth. We concluded that MAPK4 can promote cancer by activating the AKT/mTOR signaling pathway and that targeting MAPK4 may provide a novel therapeutic approach for cancer.
Project description:BACKGROUND:Cervical cancer is one of the most common cancers among females worldwide and advanced patients have extremely poor prognosis. However, adverse reactions and accumulating resistance to radiation therapy require further investigation. METHODS:The expression levels of mitogen-activated protein kinase 4 (MAPK4) mRNA were analyzed by real-time PCR and its association with overall survival was analyzed using Kaplan-Mier method. Colony formation, immunofluorescence and western blotting were used to examine the effects of MAPK4 knockout or over-expression on cervical cancer cells after radiation treatment. Drug-sensitivity of cervical cancer cells to PARP1 inhibitors, olaparib or veliparib, was analyzed by CCK-8 cell viability assays, and the 50% inhibitory concentration (IC50) was quantified using GraphPad Prism. The functional effects of MAPK4 knockout on the sensitivity of cervical cancer to radiation treatment and PARP1 inhibitors were further examined using xenograft tumor mouse models in vivo. RESULTS:Cervical cancer patients with high MAPK4 mRNA expression have lower survival rate. After radiation treatment, the colony number of MAPK4 knockout cells was markedly reduced, and the markers for DNA double-chain breakage were significantly up-regulated. In addition, MAPK4 knockout reduced protein kinase B (AKT) phosphorylation, whereas its over-expression resulted in opposite effects. In MAPK4 KO cells with irradiation treatment, inhibition of AKT phosphorylation promoted DNA double-chain breakage. Constitutive activation of AKT (CA-AKT) increased the levels of phosphorylated-AKT (p-AKT), and DNA repair-related proteins, phosphorylated-DNA-dependent protein kinase (p-DNA-PK) and RAD51 recombinase (RAD51). Furthermore, MAPK4 knockout was found to affect the sensitivity of cervical cancer cells to poly ADP-ribose polymerase 1 (PARP1) inhibitors by activating the phosphorylation of AKT. Moreover, in vivo results demonstrated that MAPK4 knockout enhanced the sensitivity of cervical cancer to radiation and PARP1 inhibitors in mouse xenograft models. CONCLUSIONS:Collectively, our data suggest that combined application of MAPK4 knockout and PARP1 inhibition can be used as therapeutic strategy in radiation treatment for advanced cervical carcinoma.
Project description:Background:Acute lung injury (ALI) is a serious disease with highly morbidity and mortality that causes serious health problems worldwide. Atypical mitogen activated protein kinases (MAPKs) play critical roles in the development of tissues and have been proposed as promising therapeutic targets for various diseases. However, the potential role of atypical MAPKs in ALI remains elusive. In this study, we investigated the role of atypical MAPKs family member MAPK4 in ALI using LPS-induced murine ALI model. Results:We found that MAPK4 deficiency mice exhibited prolonged survival time after LPS challenge, accompanied by alleviated pathology in lung tissues, decreased levels of pro-inflammatory cytokines and altered composition of immune cells in BALF. Furthermore, the transduction of related signaling pathways, including MK5, AKT, JNK, and p38 MAPK pathways, was reduced obviously in LPS-treated MAPK4-/- mice. Notably, the expression of MAPK4 was up-regulated in lung tissues of ALI model, which was not related with MAPK4 promoter methylation, but negatively orchestrated by transcriptional factors NFKB1 and NR3C1. Further studies have shown that the expression of MAPK4 was also increased in LPS-treated macrophages. Meanwhile, MAPK4 deficiency reduced the expression of related pro-inflammatory cytokines in macrophage in response to LPS treatment. Finally, MAPK4 knockdown using shRNA pre-treatment could ameliorate the pathology of lung tissues and prolong the survival time of mice after LPS challenge. Conclusions:Collectively, these findings reveal an important biological function of atypical MAPK in mediating the pathology of ALI, indicating that MAPK4 might be a novel potential therapeutic target for ALI treatment.
Project description:The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchoragedependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways.
Project description:BACKGROUND:Recent evidences have shown that circular RNAs (circRNAs) are frequently dysregulated and play paramount roles in various cancers. circRNAs are abundant in central nervous system (CNS); however, few studies describe the clinical significance and role of circRNAs in gliomas, which is the most common and aggressive primary malignant tumor in the CNS. METHODS:A bioinformatics analysis was performed to profile and screen the dyregulated circRNAs during early neural development. Quantitative real-time PCR was used to detect the expression of circ-MAPK4 and target miRNAs. Glioma cells were transfected with circ-MAPK4 siRNAs, then cell proliferation, apoptosis, transwell assays, as well as tumorigenesis and TUNEL assays, were performed to examine effect of circ-MAPK4 in vitro and vivo. Biotinylated-circ-MAPK4 probe based pull-down assay was conducted to confirm the relationship between circ-MAPK4 and miR-125-3p. RESULTS:In this study, we identified a circRNA, circ-MAPK4 (has_circ_0047688), which was downregulated during early neural differentiation. In gliomas, circ-MAPK4 acted as an oncogene, was inversely upregulated and linked to clinical pathological stage of gliomas (P?<?0.05). Next, we verified that circ-MAPK4 promoted the survival and inhibited the apoptosis of glioma cells in vitro and in vivo. Furthermore, we proved that circ-MAPK4 was involved in regulating p38/MAPK pathway, which affected glioma proliferation and apoptosis. Finally, miR-125a-3p, a miRNA exhibited tumor-suppressive function through impairing p38/MAPK pathway, which was increased by inhibiting circ-MAPK4 and could be pulled down by circ-MAPK4. Inhibition of miR-125a-3p could partly rescue the increased phosphorylation levels of p38/MAPK and the elevated amount of apoptosis inducing by knockdown of circ-MAPK4. CONCLUSIONS:Our findings suggest that circ-MAPK4 is a critical player in glioma cell survival and apoptosis via p38/MAPK signaling pathway through modulation of miR-125a-3p, which can serve as a new therapeutic target for treatment of gliomas.
Project description:3-phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of the phosphatidylinositol 3 kinase (PI3K) signaling pathway because it phosphorylates Akt/PKB through interactions with phosphatidylinositol 3,4,5 phosphate. Recent data indicate that PDK1 is overexpressed in many breast carcinomas and that alterations of PDK1 are critical in the context of oncogenic PI3K activation. However, the role of PDK1 in tumor progression is still controversial. Here, we show that PDK1 is required for anchorage-independent and xenograft growth of breast cancer cells harboring either PI3KCA or KRAS mutations. In fact, PDK1 silencing leads to increased anoikis, reduced soft agar growth, and pronounced apoptosis inside tumors. Interestingly, these phenotypes are reverted by PDK1 wild-type but not kinase-dead mutant, suggesting a relevant role of PDK1 kinase activity, even if PDK1 is not relevant for Akt activation here. Indeed, the expression of constitutively active forms of Akt in PDK1 knockdown cells is unable to rescue the anchorage-independent growth. In addition, Akt down-regulation and pharmacological inhibition do not inhibit the effects of PDK1 overexpression. In summary, these results suggest that PDK1 may contribute to breast cancer, even in the absence of PI3K oncogenic mutations and through both Akt-dependent and Akt-independent mechanisms.
Project description:The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3? overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3?-overexpression-mediated Akt phosphorylation, and its potential impact on breast cancer progression. We found that 14-3-3? overexpression was significantly (P=0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3? overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3? expression by small interfering RNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3? enhanced Akt phosphorylation through activation of phosphoinositide 3-kinase (PI3K). Mechanistically, 14-3-3? bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3-binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3?-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3? binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage-independent growth and enhanced stress-induced apoptosis. These findings revealed a novel mechanism by which 14-3-3? overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.
Project description:Dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway occurs frequently in human cancer. PTEN tumor suppressor or PIK3CA oncogene mutations both direct PI3K-dependent tumorigenesis largely through activation of the AKT/PKB kinase. However, here we show through phosphoprotein profiling and functional genomic studies that many PIK3CA mutant cancer cell lines and human breast tumors exhibit only minimal AKT activation and a diminished reliance on AKT for anchorage-independent growth. Instead, these cells retain robust PDK1 activation and membrane localization and exhibit dependency on the PDK1 substrate SGK3. SGK3 undergoes PI3K- and PDK1-dependent activation in PIK3CA mutant cancer cells. Thus, PI3K may promote cancer through both AKT-dependent and AKT-independent mechanisms. Knowledge of differential PI3K/PDK1 signaling could inform rational therapeutics in cancers harboring PIK3CA mutations.
Project description:The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110? was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110? expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110?/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110? activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110? or PI3K p110? also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110? did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110? can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110?/p-S6.
Project description:PI3K Interacting Protein 1 (PIK3IP1) is a suppressor of the PI3K/Akt/mTOR pathway. We previously reported that activated Ras suppresses PIK3IP1 expression to positively regulate the PI3K pathway in cancer cells. Using doxycycline-inducible PIK3IP1, here we confirm that reversing the effect of Ras by inducing expression of PIK3IP1 suppresses Ras-induced anchorage-independent growth, supporting the central role of PIK3IP1 in transformation. However, the molecular mechanisms by which Ras-activation that causes loss of PIK3IP1 expression are unknown. We find that Ras activity represses PIK3IP1 expression via the recruitment of lysine-specific demethylase 1 (LSD1) to the PIK3IP1 gene promoter and enhancer, resulting in erasure of active histone marks. These studies demonstrate cross-activation of Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways, where Ras decommissions PIK3IP1 gene expression by enhancing LSD1 and its corepressor activities to suppress PIK3IP1 transcription.
Project description:The Akt activation inhibitor triciribine and the farnesyltransferase inhibitor tipifarnib have modest to little activity in clinical trials when used as single agents. In this article, preclinical data show that the combination is more effective than single agents both in cultured cells and in vivo. Combination index data analysis shows that this combination is highly synergistic at inhibiting anchorage-dependent growth of breast cancer cells. This synergistic interaction is also observed with structurally unrelated inhibitors of Akt (MK-2206) and farnesyltransferase (FTI-2153). The triciribine/tipifarnib synergistic effects are seen with several cancer cell lines including those from breast, leukemia, multiple myeloma and lung tumors with different genetic alterations such as K-Ras, B-Raf, PI3K (phosphoinositide 3-kinase), p53 and pRb mutations, PTEN, pRB and Ink4a deletions, and ErbB receptor overexpression. Furthermore, the combination is synergistic at inhibiting anchorage-independent growth and at inducing apoptosis in breast cancer cells. The combination is also more effective at inhibiting the Akt/mTOR/S6 kinase pathway. In an ErbB2-driven breast tumor transgenic mouse model, the combination, but not single agent, treatment with triciribine and tipifarnib induces significant breast tumor regression. Our findings warrant further investigation of the combination of farnesyltransferase and Akt inhibitors.