Ribosomal S6 kinase 2 (RSK2), regulated by Ras/Raf/MEKs/ERKs, transmits upstream activation signals to downstream substrates including kinases and transcription and epigenetic factors. We observed that ELK members, including ELK1, 3, and 4, highly interacted with RSK2. We further observed that the RSK2-ELK3 interaction was mediated by N-terminal kinase and linker domains of RSK2, and the D and C domains of ELK3, resulting in the phosphorylation of ELK3. Importantly, RSK2-mediated ELK3 enhanced

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BACKGROUND: Ionizing radiation (IR) therapy is the standard first-line treatment for newly diagnosed patients with glioblastoma (GBM), the most common and malignant primary brain tumor. However, the effects of IR are limited due to the aberrant radioresistance of GBM.
METHODS: Transcriptome analysis was performed using RNA-seq in radioresistant patient-derived glioma stem-like cells (GSCs). Survival of glioma patient and mice bearing-brain tumors was analyzed by Kaplan-Meier survival analysis. Lipid droplet and γ-H2AX foci-positive cells were evaluated using immunofluorescence staining.
RESULTS: Lipolytic inhibitor G0/G1 switch gene 2 (G0S2) is upregulated in radioresistant GSCs and elevated in clinical GBM. GBM patients with high G0S2 expression had significantly shorter overall survival compared with those with low expression of G0S2. Using genetic approaches targeting G0S2 in glioma cells and GSCs, we found that knockdown of G0S2 promoted lipid droplet turnover, inhibited GSC radioresistance, and extended survival of xenograft tumor mice with or without IR. In contrast, overexpression of G0S2 promoted glioma cell radiation resistance. Mechanistically, high expression of G0S2 reduced lipid droplet turnover and thereby attenuated E3 ligase RNF168-mediated 53BP1 ubiquitination through activated the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) signaling and increased 53BP1 protein stability in response to IR, leading to enhanced DNA repair and glioma radioresistance.
CONCLUSIONS: Our findings uncover a new function for lipolytic inhibitor G0S2 as an important regulator for GSC radioresistance, suggesting G0S2 as a potential therapeutic target for treating gliomas.

BACKGROUND: Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.
METHODS: Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition.
RESULTS: We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy.
CONCLUSIONS: Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered.

Colorectal carcinoma (CRC) is one of the most common malignancies worldwide and the second leading cause of cancer‑related deaths in the US. Recently, Rab1A has been reported to be an activator of mTORC1 and p‑S6K1, which is downstream of mTORC1. However, the association between Rab1A and p‑S6K1 in CRC remains elusive. In the present study, we first demonstrated that Rab1A was overexpressed in CRC tissues and Rab1A overexpression was positively related to lymph node invasion, degree of differentiation, venous invasion and tumor‑node‑metastasis (TNM) stage. In both TNM stage I‑II and III‑IV patients, Rab1A‑positive patients had a shorter survival time than Rab1A‑negative patients. Furthermore, in univariate and multivariate analyses, only Rab1A expression was verified as an independent prognostic factor for survival in CRC patients. The level of p‑S6K1 was markedly high in CRC tissues and Rab1A expression level had a positive association with p‑S6K1 level. In addition, high levels of both Rab1A and p‑S6K1 were associated with a poorer prognosis compared with low expression of either Rab1A or p‑S6K1 level. Moreover, high levels of both Rab1A and p‑S6K1 were associated with a poorer prognosis than patients with high levels of either Rab1A or p‑S6K1 alone. Finally, knockdown of Rab1A expression inhibited migration and proliferation of SW480 and HCT116 cell lines by targeting regulation of p‑S6K1. Thus, our findings indicate that Rab1A plays an important role in CRC and may provide a therapeutic target for CRC, particularly for mTORC1‑targeted therapy‑resistant cancers.

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD-L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD-L1 expression regulation in non-small-cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD-L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho-IκBα (p-IκBα) and hypoxia-inducible factor-1α (HIF-1α). Flow cytometry results showed stronger membrane co-expression of EGFR and PD-L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF-1α indicated strong accordance among protein levels of PD-L1, p-IκBα, and HIF-1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p-IκBα, HIF-1α, and PD-L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p-IκBα, HIF-1α, and PD-L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non-small-cell lung cancer tissues with either p-IκBα or HIF-1α positive staining were more likely to possess elevated PD-L1 expression compared with tissues scored negative for both p-IκBα and HIF-1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF-κB and HIF-1α in PD-L1 expression regulation by EGFR mutants in NSCLC.

Overexpression of Pim kinases has an oncogenic/pro-survival role in many hematological and solid cancers. AZD1208 is a pan-Pim kinase inhibitor that has anti-cancer and anti-adipogenic actions. Here, we investigated the effects of AZD1208 on the growth of 93T449 cells, a differentiated human liposarcoma cell line. At 20 µM, AZD1208 was cytotoxic (cytostatic) but not apoptotic, reducing cell survival without DNA fragmentation, caspase activation or increasing cells in the sub G1 phase; known apoptotic parameters. Notably, AZD1208 reduced phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in 93T449 cells. STAT-3 inhibition by AG490, a JAK2/STAT-3 inhibitor similarly reduced cell survival. AZD1208 down-regulated phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal S6 while up-regulated eukaryotic initiation factor-2α (eIF-2α). In addition, AZD1208 induced a LKB-1-independent AMPK activation, which was crucial for its cytostatic effect, as knock-down of AMPK greatly blocked AZD1208s ability to reduce cell survival. AZD1208 had no effect on expression of two members of Pim kinase family (Pim-1 and Pim-3) but inhibited phosphorylation of 4EBP-1, a downstream effector of Pim kinases. Importantly, a central role for Pim-3 in the actions of AZD1208 was confirmed by knock-down, which not only reduced 93T449 cell survival but also led to the inhibition of 4EBP-1, mTOR, eIF-2α and STAT-3, along with the activation of AMPK. In summary, this is the first report demonstrating that AZD1208 inhibits growth of liposarcoma cells and that this activity is mediated through Pim-3 kinase, STAT-3, mTOR, S6 and AMPK expression and phosphorylation pathways.

PURPOSE: Cervical cancer is one of the prevalently diagnosed cancers in women worldwide. In this study the antiproliferative and anticancer effects of Salviolone were evaluated against HeLa cervical cancer cell line along with determining the anticancer mode of action.
METHODS: MTT assay was employed to examine the proliferation rate of HeLa cells. Transmission electron microscopy (TEM) was used for the detection of the autophagic cell death. The cell invasion and migration were investigated by transwell assay and the expression of the proteins was estimated by western blotting.
RESULTS: The results revealed that Salviolone exerts anticancer effects on the HeLa cells with an IC50 of 20 µM. The effect of Salviolone on the viability of normal FR-2 cells was very low. TEM analysis showed that Salviolone triggers autophagic cell death in HeLa cells. Salviolone could also cause arrest of the HeLa cervical cancer cells in the G2/M phase of the cell cycle and suppress their ability to migrate and invade. Western blotting analysis revealed that Salviolone could inhibit the of Nf-kB/m-TOR/PI3K/AKT signalling pathway in HeLa cells.
CONCLUSION: Taken all together, it is concluded that Salviolone could prove to be an important lead molecule for the treatment of cervical cancer.

PURPOSE: To investigate the influence of carboplatin on the proliferation and apoptosis of ovarian cancer cells through mTOR/P70S6K signaling pathway.
METHODS: The mRNA and protein expressions were detected via Western blotting and RT-PCR to study whether the mTOR/p70S6K signaling pathway was activated in OVCAR-3 and Caov-3 ovarian cancer cell lines. After cells were treated with different concentrations of carboplatin, the mRNA and protein expressions of mTOR, p70S6K and 4E-BP1 were detected via RT-PCR and Western blotting. OVCAR-3 cells were treated with 20 and 50 μM carboplatin for 4 hrs, and then apoptosis was analyzed and assessed. OVCAR-3 cells were treated with different concentrations of carboplatin (20, 50, 100, 150 and 200 μM) for 24 and 48 hrs, respectively.
RESULTS: The mTOR signaling pathway was activated in OVCAR-3 and Caov-3 ovarian cancer cell lines. The mRNA level of mTOR in Caov-3 cells was higher, but that of p70S6K was lower. Carboplatin significantly reduced the mRNA expression of mTOR (p<0.01), whereas the mRNA expressions of p70S6K and 4E-BP1 in carboplatin-treated cells were increased in a dose-dependent manner (p<0.01). Carboplatin inhibited the mTOR protein expression in a dose-dependent manner (p<0.01). The proliferation of OVCAR-3 cells exposed to carboplatin was reduced compared with that of untreated cells (p<0.01), and the inhibitory effect of carboplatin on the proliferation of OVCAR-3 cells was time- and dose-dependent.
CONCLUSION: The mTOR/p70S6K pathway was activated in ovarian cancer. Carboplatin could rapidly inhibit the expression of mTOR, and the phosphorylation of its major downstream effectors p70S6K and 4E-binding protein 1 (4E-BP1) arrested cells in G0/G1 phase and induced ovarian cancer cell apoptosis.

TIMM50 (Translocase of the inner mitochondrial membrane 50), also called TIM50, plays an essential role in mitochondrial membrane transportation. The existing literature suggests that TIMM50 may perform as an oncogenetic protein in breast cancer. However, the molecular mechanism, especially in human non-small cell lung cancer (NSCLC), is uncertain to date. In the present study, using immunohistochemistry, we found that TIMM50 expression significantly correlated with larger tumor size (P = 0.049), advanced TNM stage (P = 0.001), positive regional lymph node metastasis (P = 0.007), and poor overall survival (P = 0.001). Proliferation and invasion assay showed that TIMM50 dramatically promoted the ability of proliferation and invasion of NSCLC cells. Subsequent Western blotting results revealed that TIMM50 enhanced the expression of Cyclin D1 and Snail, and inhibited the expression of E-cadherin. Moreover, TIMM50 facilitated the expression of phosphorylated ERK and P90RSK. Incorporation of ERK inhibitor counteracted the upregulating expression of CyclinD1, and Snail, and downregulating expression of E-cadherin expression induced by TIMM50 overexpression. In conclusion, our data indicated that TIMM50 facilitated tumor proliferation and invasion of NSCLC through enhancing phosphorylation of its downstream ERK/P90RSK signaling pathway. We speculated that TIMM50 might be a useful prognosis marker of NSCLC patients.

BACKGROUND: This study aimed to investigate the expression of P90 Ribosomal Protein S6 kinase 4 (RSK4) in colorectal cancer cells and its biological function.
METHODS: We selected early SW480 and HCT116 colorectal cancer cell lines, using Lipofectamine™ 2000 transfection reagent carrying RSK4 gene transfected into cells to establish the colorectal cancer cell lines with high expression of RSK4. RT-PCR and western blot (WB) analysis confirmed RSK4 expression in SW480 and HCT116 cancer cell lines. We used methylthiazoltetrazolium (MTT) assay and flow cytometry to detect the proliferation of colorectal cancer cells. After transfection of RSK4, the effect of RSK4 on the RNA levels associated with epithelial-mesenchymal transition (EMT) of colorectal cancer cells was analyzed by real-time fluorescence quantitative PCR and the expression of EMT-related protein was detected by WB analysis.
RESULTS: After transfection of RSK4 overexpression, the MTT assay detected that RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro; flow cytometry detected that S-phase cells decreased significantly, and G0/1 cells increased significantly (P < 0.05). The invasion ability of SW480 and HCT116 cells transfected with RSK4 was markedly lower than that in the control group, and the difference was statistically significant (P < 0.05). Fluorescent quantitative PCR and WB analysis showed that the expression of EMT-associated molecular E-cadherin was remarkably increased and the expression of Snail was significantly decreased (P < 0.01).
CONCLUSION: RSK4 gene in colorectal cancer cell lines with low expression of RSK4 after transfection can inhibit the growth and invasion of tumor cells. RSK4 gene may inhibit EMT and inhibit metastasis of colorectal cancer cells, may be a potential tumor suppressor gene and inhibit tumor distant metastasis, and may provide the biological basis for new therapeutic targets.

The microbiota and bacterial metabolites in the colon are regarded as alternative targets for colon cancer prevention and therapy. Among these metabolites, short-chain fatty acids (SCFAs) exhibit anticancer effects and suppress inflammation in the colon. However, the molecular mechanisms and target development of SCFAs require additional study. In the present study, using RNA-seq results from colon cancer samples derived from the Cancer Genome Atlas (TCGA) portal, overexpressed epigenetic modifiers were identified and RT-PCR and qRT-PCR analysis was performed to select target genes that responded to treatment with propionate in HCT116 cells. Downregulation of protein arginine methyltransferase 1 (PRMT1), a histone arginine methyltransferase, was observed after sodium propionate (SP) treatment. Moreover, phospho-array analysis demonstrated that the mTOR pathway was involved in propionate and siPRMT1 treatment, and regulation of this pathway was associated with apoptosis in HCT116 cells. The present study, to the best of our knowledge, was the first to demonstrate that PRMT1 levels were reduced by propionate treatment in HCT116 cells and that downregulation of PRMT1 induced cell apoptosis. Thus, this novel mechanism of sodium propionate treatment for colon cancer therapy may indicate more effective approaches, such as dietary therapy, for CRC patients.

The fibroblast growth factor 19 gene FGF19 has previously been reported to be amplified in several cancer types and encodes for a key autocrine signaler known to promote tumorigenic growth. Thus, it is imperative to understand which cancers are oncogenically addicted to FGF19 amplification as well as the role it serves in these cancer types. We report for the first time high FGF19 amplification in head and neck squamous cell carcinomas (HNSCC), which is associated with increased autocrine secretion of FGF19 and poor patient outcome in HNSCC. FGF19 amplification corresponded with constitutive activation of FGF receptor 4 (FGFR4)-dependent ERK/AKT-p70S6K-S6 signaling activation in HNSCC cells, and addition of human recombinant FGF19 could promote cell proliferation and soft agar colony formation in HNSCC cells with low FGF19 expression through activation of FGFR4 and downstream signaling cascades. In contrast, FGF19 knockout counteracts the observed effects in HNSCC cells carrying high endogenous FGF19, with knockout of FGF19 significantly suppressing tumor growth in an orthotopic mouse model of HNSCC. Collectively, this study demonstrates that FGF19 gene amplification corresponds with an increased dependency upon FGF19/FGFR4 autocrine signaling in HNSCC, revealing a therapeutic target for this cancer type.

The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.

NVP-BEZ235 or BEZ235 is a dual inhibitor of adenosine triphosphate (ATP)-competitive phosphoinositide 3-kinase (PI3K)/mammalian-target-of-rapamycin (mTOR) and is promising for cancer treatment. Because it targets more than one downstream effector, a dual approach is promising for cancer treatment. The aim of this study was to evaluate the efficacy of NVP-BEZ235 in treating oral cavity squamous cell carcinoma (OSCC). Two human OSCC cell lines, SCC-4 and SCC-25, were used in this study. PI3K-AKT signaling, proliferation, and cell migratory and invasion capabilities of OSCC cells were examined. In NVP-BEZ235-treated SCC-4 and SCC-25 cells, the phosphorylation of 70-kDa ribosomal S6 kinase (p70S6K), but not mTOR, decreased within 24 h. NVP-BEZ235 inhibited OSCC-cell proliferation, migration, and invasion possibly by directly deregulating the phosphorylation of p70S6K. The phospho-p70S6K inhibitor mimicked the effects of NVP-BEZ235 for preventing proliferation and weakening the migratory and invasion abilities of SCC-4 and SCC-25 cells. This study further confirmed the effect of NVP-BEZ235 on OSCC cells and provided a new strategy for controlling the proliferation, migration, and invasion of OSCC cells using the phopho-p70S6K inhibitor.

BACKGROUND The FOLR2 gene encodes folate receptor-beta (FR-beta), which is expressed by tumor-associated macrophages. The effects of FOLR2 gene expression in non-small cell lung cancer (NSCLC) remains unknown. This study aimed to investigate the effects of FOLR2 gene expression and gene silencing in human NSCLC cell lines and normal human bronchial epithelial (HBE) cells in vitro. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to detect the expression of the FOLR2 gene, cell cycle and apoptosis-associated genes in normal HBE cells and the NSCLC cell lines, A549, NCI-H1299, NCI-H1650, and NCI-H460. Using small interfering RNA (siRNA), or silencing RNA, FOLR2 gene silencing was performed for NCI-H1650 cells. Cell counting kit-8 (CCK-8) was used to measure cell viability. Cell cycle and apoptosis were determined using flow cytometry. Western blot evaluated the expression of Akt, mTOR, and S6K1 signaling. RESULTS Expression of the FOLR2 gene was increased in NSCLC cells compared with normal HBE cells. Silencing of the expression of the FOLR2 gene in NCI-H1650 cells reduced cell viability, increased cell apoptosis, and arrested cells in the G1 phase of the cell cycle, decreased the expression of cyclin D1, upregulated expression of cell cycle inhibitors, p21 and p27, upregulated the expression of Bax/Bcl-2, and inhibited phosphorylation of AKT, mTOR, and S6K1. CONCLUSIONS Silencing of the FOLR2 gene inhibited phosphorylation of AKT, mTOR, and S6K1, inhibited cell proliferation and increased apoptosis in the NCI-H1650 human NSCLC cell line.

BACKGROUND/AIMS: MiR-19b has been reported to be involved in several malignancies, but its role in multiple myeloma (MM) is still unknown. The objective of this study was to explore the biological mechanism of miR-19b in the progression of MM.
METHODS: First, we performed real-time polymerase chain reaction (PCR) and Western blot to study the expression of miR-19b, tuberous sclerosis 1 (TSC1), and caspase-3 in different groups. MTT assay was performed to explore the effect of miR-19b on survival and apoptosis of cancer stem cells (CSCs). Computation analysis and luciferase assay were utilized to confirm the interaction between miR-19b and TSC1.
RESULTS: A total of 38 participants comprising 20 subjects with MM and 18 healthy subjects as normal controls were enrolled in our study. Real-time PCR showed dramatic upregulation of miR-19b, but TSC1 was evidently suppressed in the MM group. MiR-19b overexpression substantially promoted clonogenicity and cell viability, and further inhibited apoptosis of CSCs in vitro. Furthermore, miR-19b overexpression downregulated the expression of caspase-3, which induced apoptosis. Using in silico analysis, we identified that TSC1 might be a direct downstream target of miR-19b, and this was further confirmed by luciferase assay showing that miR-19b apparently reduced the luciferase activity of wild-type TSC1 3´-UTR, but not that of mutant TSC1 3´-UTR. There was also evident decrease in TSC1 mRNA and protein in CSCs following introduction of miR-19b. Interestingly, reintroduction of TSC1 abolished the miR-19b-induced proliferation promotion and apoptosis inhibition in CSCs.
CONCLUSION: These findings collectively suggest that miR-19b promotes cell survival and suppresses apoptosis of MM CSCs via targeting TSC1 directly, indicating that miR-19b may serve as a potential and novel therapeutic target of MM based on miRNA expression.

Osteosarcoma (OS) is a rare tumor of the bone occurring mainly in young adults accounting for 5% of all childhood cancers. Because of the limited therapeutic options, there has been no survival improvement for OS patients in the past 40 years. The epidermal growth factor receptor (EGFR) is highly expressed in OS; however, its clinical relevance is unclear. Here, we employed an autochthonous c-Fos-dependent OS mouse model (H2

BACKGROUND: The ubiquitin-proteasome pathway, mediated in part, by ubiquitin E3 ligases, is critical in regulating cellular processes such as cell proliferation, apoptosis, and migration. FBXO17 was recently identified as an F-box protein that targets glycogen synthase kinase-3β to the E3 ubiquitin ligase protein complex for polyubiquitination and proteasomal degradation. Here, we identified that in several lung adenocarcinoma cell lines, FBXO17 cellular protein was detected at relatively high levels, as was expression in a subset of lung cancers. Hence, we investigated the effects of FBXO17 on cell proliferation.
METHODS: Single cell RNA sequencing analysis was performed on a resection of a non-small cell lung carcinoma tumor to examine FBXO17 expression. Multiple lung cancer cell lines were immunoblotted, and The Cancer Genome Atlas was analyzed to determine if FBXO17 expression was amplified in a subset of lung cancers. A549 cells were transfected with empty vector or FBXO17-V5 plasmid and immunoblotted for Akt pathway mediators including PDK1, ERK1/2, ribosomal protein S6, and CREB. Cell proliferation and viability were analyzed by trypan blue exclusion, BrdU incorporation and an MTS-based fluorometric assay. Studies were also performed after transfecting with sifbxo17. Samples were used in an RNA microarray analysis to evaluate pathways affected by reduced FBXO17 gene expression.
RESULTS: We observed that overexpression of FBXO17 increased A549 cell proliferation coupled with Akt activation. Ectopically expressed FBXO17 also increased ERK1/2 kinase activation and increased phosphorylation of RPS6, a downstream target of mTOR. We also observed an increased number of cells in S-phase and increased metabolic activity of lung epithelial cells expressing FBXO17. FBXO17 knockdown reduced Akt Ser 473 phosphorylation approaching statistical significance with no effect on Thr 308. However, ERK1/2 phosphorylation, cellular metabolic activity, and overall cell numbers were reduced. When we analyzed RNA profiles of A549 cells with reduced FBXO17 expression, we observed downregulation of several genes associated with cell proliferation and metabolism.
CONCLUSIONS: These data support a role for FBXO17 abundance, when left unchecked, in regulating cell proliferation and survival through modulation of Akt and ERK kinase activation. The data raise a potential role for the F-box subunit in modulating tumorigenesis.

BACKGROUND & AIMS: Activation of KRAS signaling and overexpression of the aurora kinase A (AURKA) are often detected in luminal gastrointestinal cancers. We investigated regulation of ribosomal protein S6 kinase B1 (RPS6KB1) by AURKA and the effects of alisertib, an AURKA inhibitor, in mice xenograft tumors grown from human gastrointestinal cancer cells with mutant, activated forms of KRAS.
METHODS: We tested the effects of alisertib or AURKA overexpression or knockdown in 10 upper gastrointestinal or colon cancer cell lines with KRAS mutations or amplifications using the CellTiter-Glo luminescence and clonogenic cell survival assays. We used the proximity ligation in situ assay to evaluate protein co-localization and immunoprecipitation to study protein interactions. Nude mice with xenograft tumors grown from HCT116, SNU-601, SW480, or SNU-1 cells were given oral alisertib (40 mg/kg, 5 times/wk) for 4 weeks. Tumor samples were collected and analyzed by immunoblots and immunohistochemistry. Tissue microarrays from 151 paraffin-embedded human colon tumors, with adjacent normal and adenoma tissues, were analyzed by immunohistochemistry for levels of AURKA.
RESULTS: Alisertib reduced proliferation and survival of the cell lines tested. AURKA knockdown or inhibition with alisertib reduced levels of phosphorylated RPS6KB1 (at T389) and increased levels of proteins that induce apoptosis, including BIM, cleaved PARP, and cleaved caspase 3. AURKA co-localized and interacted with RPS6KB1, mediating RPS6KB1 phosphorylation at T389. We detected AURKA-dependent phosphorylation of RPS6KB1 in cell lines with mutations in KRAS but not in cells with wild-type KRAS. Administration of alisertib to mice with xenograft tumors significantly reduced tumor volumes (P < .001). Alisertib reduced phosphorylation of RPS6KB1 and Ki-67 and increased levels of cleaved caspase 3 in tumor tissues. In analyses of tissue microarrays, we found significant overexpression of AURKA in gastrointestinal tumor tissues compared with non-tumor tissues (P = .0003).
CONCLUSION: In studies of gastrointestinal cancer cell lines with activated KRAS, we found AURKA to phosphorylate RPS6KB1, promoting cell proliferation and survival and growth of xenograft tumors in mice. Agents that inhibit AURKA might slow the growth of gastrointestinal tumors with activation of KRAS.

PURPOSE: To assess the prognostic value of PI3K-AKT-mTOR signaling pathway up-regulation in a contemporary cohort of penile squamous-cell carcinoma (PSCC) patients.
PATIENTS AND METHODS: Tissue microarrays were constructed for 57 patients with invasive PSCC treated at our institution between 2000 and 2013. Immunohistochemical staining was performed for PTEN, AKT, and S6. Human papillomavirus (HPV) in-situ hybridization for high-risk subtypes was also performed. Biomarker expression was evaluated by a semiquantitative H score. Overall survival, disease-specific survival and recurrence-free survival stratified by biomarker expression (low vs. high) were estimated by the Kaplan-Meier method. Multivariable Cox regression models were used to determine predictors of mortality and recurrence.
RESULTS: HPV in-situ hybridization was positive in 23 patients (40%). PTEN was down-regulated in 43 patients (75%), while phosphorylated-AKT (p-AKT) and phosphorylated-S6 (p-S6) were up-regulated in 27 (47%) and 12 patients (21%), respectively. In multivariable Cox regression models, patients with low expression of p-AKT had an increased risk of recurrence (hazard ratio [HR] = 3.95; 95% confidence interval [CI], 1.47-10.59; P = .02), while those with low expression of p-S6 had an increased risk of overall mortality (HR = 6.15; 95% CI, 1.55-24.36; P = .01). HPV status was an independent predictor of overall survival (HR = 6.99; 95% CI, 2.42-20.16; P < .001) and disease-specific survival (HR = 6.74; 95% CI, 2.02-22.48; P = .002).
CONCLUSION: PI3K-AKT-mTOR signaling pathway up-regulation and HPV coinfection in PSCC are associated with favorable disease. mTOR pathway biomarkers along with HPV status may represent novel prognosticators for risk stratification of PSCC patients and may help guide treatment decisions and follow-up strategies. These findings require further investigation.

C6orf106 was highly expressed in lung and breast cancer, and proposed as clinicopathologic factor for the development of those types of cancer. However, its expression in pancreatic cancer and the mechanism that C6orf106 functions as an oncogene has not been confirmed. In the present study, we found that C6orf106 was also up-regulated in pancreatic cancer tissues and cell lines. Furthermore, C6orf106 expression was associated with advanced T stage (P = 0.010), positive regional lymph node metastasis (P = 0.012), and advanced TNM stage (P = 0.006). In vitro experiments also showed that C6orf106 served a tumor enhancer in pancreatic cancer, through increasing the expression of Snail, Cyclin D1 and Cyclin E1, and reducing the expression of E-cadherin via activating extracellular-signal-regulated kinase (ERK)- p90-kDa ribosomal S6 kinases (P90RSK) signaling pathway. The addition of ERK inhibitor PD98059 counteracted the upregulation of Snail, Cyclin D1 and Cyclin E1, and restored the expression of E-cadherin, which indicated that C6orf106 was an upstream factor of ERK signaling pathway. Taken together, the present study indicates that C6orf106 facilitates invasion and proliferation of pancreatic cancer cells, likely via activating ERK-P90RSK signaling pathway.

Breast cancer is a leading lethal gynecological cancer. Although many tumor markers and target genes have been studied in breast cancer, its incidence is increasing. Recently, the therapeutic effects of natural phytochemicals have been studied in various cancers as adjuvants. Chrysophanol is an anti-inflammatory, anti-angiogenetic, and anti-tumor anthraquinone but has not been widely studied in cancers. Here, we verified the anti-cancer effects and cellular mechanism of chrysophanol in human breast cancer cells (BT-474 and MCF-7). Chrysophanol selectively inhibited cell proliferation and induced apoptosis of breast cancer cells but not of normal mammary ductal epithelial cells, MCF-12A. Additionally, chrysophanol increased loss of mitochondrial membrane potential and cytosolic calcium levels to activate pro-apoptotic proteins, Bax, Bak, and cytochrome c, in both cell lines. Reactive oxygen species (ROS) overproduction by chrysophanol resulted in endoplasmic reticulum (ER) stress, leading to an increase in PERK, eIF2α, GADD153, and IRE1α levels in BT-474 and MCF-7 cells. These ER stress proteins increased by chrysophanol were repressed by co-treatment with N-acetyl-L-cysteine, an ROS inhibitor. Western blotting showed that chrysophanol down-regulated ERK1/2, AKT, P70S6K, and S6 in both cell lines. However, P38 and JNK activities decreased in BT-474 cells and increased in MCF-7 cells. Additionally, co-treatment with ERK1/2 (U0126) or an AKT inhibitor (LY294002) plus chrysophanol reduced cell proliferation, whereas P38 (SB203580) and a JNK inhibitor (SP600125) showed synergic effects only in BT-474 cell lines. These results show that chrysophanol has anti-cancer effects on human breast cancer cells, specifically through mitochondrial apoptosis and ER stress induction.

Malignant pleural mesothelioma (MPM) is a rare form of cancer that is associated with asbestos exposure. Unfortunately, current therapies have limited efficacy. Previous studies have indicated that curcumin exerts antiproliferative and antitumor effects, and has low toxicity. The present study aimed to evaluate the anticancer effects of curcumin on the RN5 MPM cell line. The inhibitory effects of curcumin on cell viability were determined using the sulforhodamine B assay. In addition, cell cycle progression was analyzed by propidium iodide (PI) staining and flow cytometry, and curcumin‑induced apoptosis was measured by Annexin V/PI double staining. The translocation of apoptosis-inducing factor (AIF) was assessed by western blotting and immunofluorescence, and the expression levels of the phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase (Akt)‑mammalian target of rapamycin (mTOR) signaling pathway proteins and mitochondria-associated proteins were evaluated by western blotting. In vivo antitumor effects were evaluated in a subcutaneous murine model. Briefly, tumors were harvested from the mice, and immunohistochemistry was conducted to evaluate cell proliferation, apoptosis and angiogenesis. The results indicated that curcumin inhibited RN5 cell viability and induced apoptotic cell death. In addition the findings suggested that curcumin-induced cell apoptosis occurred via the mitochondrial pathway, and caspase‑independent and AIF-dependent pathways. Further analysis revealed that curcumin may act as a PI3K-Akt-mTOR signaling pathway inhibitor by downregulating PI3K, p-Akt, p-mTOR and p-p70 ribosomal protein S6 kinase. Furthermore, curcumin inhibited tumor angiogenesis in vivo. In conclusion, curcumin may be potent enough to be developed as a novel therapeutic agent for the treatment of MPM.

Recently, sphingolipid derivatives, such as ceramide and sphingosine‑1‑phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF‑7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal‑regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF‑7 cell proliferation. This effect was partially reversed by co‑treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6‑mediated decrease in MCF‑7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF‑7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16‑ceramide, which is generated by CerS6.

BACKGROUND: Lung cancer represents the leading cause of cancer deaths worldwide, with non-small cell lung carcinoma (NSCLC) comprising the most common type of lung cancer.
OBJECTIVE: The aim of this pilot study was to ascertain RSK4 expression at both the gene and protein level in normal and cancerous tissue of patients with NSCLC.
METHODS: From January 2015 to December 2015, a total of forty patients diagnosed with NSCLC who underwent surgery were recruited into this study. All NSCLC diagnoses were confirmed by pathological examination. Normal and cancerous lung tissues were collected via surgical dissection.
RESULTS: Both
CONCLUSIONS: RSK4 expression is associated with clinical and pathological staging of NSCLC. Our preliminary data from this pilot study suggest that

Hemangiopericytoma (HPC) is a rare vascular tumor, which is thought to originate from pericytes. However, no direct evidence for the cell of origin has been found, and the mechanism of HPC tumorigenesis is poorly understood. Here we report that loss of the tumor suppressor gene Tsc2 in pericytes using a FoxD1 promoter driven cre allele (Foxd1tm1(GFP/cre) Amc, FoxD1GC) leads to the formation of HPC in multiple sites. Tsc2ffFoxD1GC mice had stunted growth with seizures and tail and hind limb tremor with a median survival of 110 days. They also showed recombination in brain, spinal cord, tongue, liver, intestine and skeletal muscle. Distinctive perivascular tumors consisting of cells with oval nuclei and scant cytoplasm were identified in multiple sites in all Tsc2ffFoxD1GC mice. Immunohistochemistry staining showed a high expression of phospho-S6-S240/244, a hallmark of activated mTORC1, as well as pericyte markers NG2 and vimentin in these tumors. In summary, we demonstrate that loss of Tsc2 in pericytes generates HPC, the first mouse model of HPC reported.

Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.

Despite advances in management, bladder cancer remains a principal cause of cancer‑associated complications. Tumor necrosis factor receptor superfamily member 14 (TNFRSF14) is dysregulated in certain types of cancer; however, limited data are available on the expression and function of TNFRSF14 in bladder cancer. In the present study, the aim was to evaluate the expression and biological functions of TNFRSF14 in bladder cancer. Firstly, the expression levels of TNFRSF14 in bladder cancer tissue were examined using The Cancer Genome Atlas (TCGA) database. Secondly, reverse transcription‑quantitative polymerase chain reaction was utilized to investigate the expression levels of TNFRSF14 in the T24, SW780 and EJ‑M3 bladder cancer cell lines. Transfection and Cell Counting kit‑8 (CCK‑8) assay was used to evaluate whether TNFRSF14 overexpression or silencing would have an effect on cell proliferation of T24 and EJ‑M3 cells. In addition, TNFRSF14‑induced apoptotic cells were identified using Annexin V‑fluorescein isothiocyanate and propidium iodide staining. Western blot analysis was used to detect proteins associated with the phosphatidylinositol 3‑kinase pathway. According to the TCGA dataset, the expression levels TNFRSF14 were decreased in bladder cancer tissue compared with in normal control samples. Patients with bladder cancer exhibiting low expression levels of TNFRSF14 had a worse prognosis compared to those with high expression levels of TNFRSF14. Overexpression of TNFRSF14 in T24 cells led to increased apoptosis and inhibited cell proliferation in vitro. Western blotting demonstrated that TNFRSF14 overexpression increased the expression levels of caspase3‑p17 in T24 cells, but significantly decreased the expression levels of phosphorylated (p)‑protein kinase B (AKT) and P70 S6 kinase (P70). TNFRSF14 silencing in EJ‑M3 cells enhanced cell growth, inhibited cell apoptosis, increased the expression levels of p‑AKT and P70, and decreased the expression levels of caspase3‑p17. In conclusion, TNFRSF14 may serve a tumor suppressive role in bladder cancer by inducing apoptosis and suppressing proliferation, and act as a novel prognostic biomarker for bladder cancer.

Mitogen-activated and stress-activated protein kinase 1 (MSK1), which belongs to the subfamily of MAPK-activated protein kinase, plays an important role in cell proliferation and neoplastic transformation. It has been recently reported that MSK1 overexpression was closely related to the progression of some tumors such as colorectal cancer. However, the clinical significance of MSK1 in glioma has not been addressed. To investigate the potential role of MSK1 in glioma, we first examined the expression pattern of MSK1 in glioma tissues and normal brain tissues using quantitative RT-PCR, and the results showing that MSK1 expression was significantly elevated in glioma tissues compared with normal brain tissues. The clinical relevance of MSK1 expression level was then analyzed, and we found that high expression of MSK1 was closely related to the larger tumor size and advanced WHO grade. Univariate and multivariate analyses revealed that glioma patients with higher expression of MSK1 had poorer overall survival, and MSK1 was identified as an independent unfavorable prognosis factor. In addition, the effects of MSK1 on glioma cells were tested through cellular experiments, and we demonstrated that MSK1 can promote proliferation and invasion capacities of tumor cells. In conclusion, patients with glioma with higher MSK1 expression were more predisposed to poorer clinical outcomes and unfavorable prognosis, indicating the potential role of MSK1 as a novel clinical biomarker and therapeutic target.