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Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancer diagnoses across the world. Despite recent appreciable improvements in treatment plans for patients with NSCLC, the prognosis for those with the cancer still remains poor. Recently, a growing number of studies have shown that N-myristoyltransferases (NMTs) may be critical in carcinogenesis, however, the functional and clinical significance of this pathway in NSCLC remains unclear and requires further research.Initially, we evaluated the expression levels of NMT1 or NMT2 in a clinical cohort comprising of 303 paired primary NSCLC tissues and matched normal mucosae by using ELISA. We subsequently performed a tissue microarray analysis (TMA) to confirm its expression pattern in an independent validation cohort (n = 78). Then, we used a publicly available KM plotter database (n = 1921) to evaluate the prognostic impact of NMT1 and NMT2 in NSCLC. Lastly, a series of in-vitro molecular/cellular and animal experiments were performed for mechanistic understanding of the role of N-myristoyltransferases in NSCLC.Our ELISA data revealed that the expression level of NMT1 and NMT2 was down-regulated in tumor tissues (n = 303, P 0.0001), which was confirmed in an independent validation cohort by TMA (n = 78, P = 0.014 for NMT1 and P 0.0001 for NMT2). On the other hand, patients with low expression of NMT1 or NMT2 had shorter overall survival (P = 0.013, HR = 0.85 for NMT1; P = 0.00059, HR = 0.8, for NMT2). Mechanistically, we revealed that the interaction and co-localization of NMT1 and NMT2 in NSCLC, and N-terminus of NMT1 and NMT2 was observed to be crucial for their interaction as well as for their catalytic activity. Moreover, we found that NMT1 can significantly promote the expression of NMT2 by enhancing its stability. We corroborated these findings by performing functional assays in which the knockout of NMT1 and NMT2 resulted in enhanced cell proliferation, migration and invasion as well as increased tumorxenograftgrowth. In addition, we identified miR-182 as a novel regulator of both NMT1 and NMT2. More specifically, the overexpression or inhibition of miR-182 modulated globe N-myristoylation level, contributed to phenotypic alterations in NSCLS cells.NMT1 and NMT2 can act as potential tumor suppressors in NSCLC, and the inhibition of miR-182 expression or therapeutic NMTs replenishment may be a promising treatment option for patients with NSCLC.

The aim of this study was to build up a novel chiral mesoporous silica called PEIs@TA-CMS through a facile biomimetic strategy and to explore its potential to serve as a drug carrier for improving the delivery efficiency of poorly water-soluble drug. PEIs@TA-CMS was synthesized by using a chiral crystalline complex associated of tartaric acid and polyethyleneimine (PEIs) as templates, scaffolds and catalysts. The structural features including morphology, size, pore structure and texture properties were systematacially studied. The results showed that PEIs@TA-CMS was monodispersed spherical nanoparticles in a uniformed diameter of 120–130 nm with well-developed pore structure (SBET: 1009.94 m2/g, pore size

Background/Aims: Recent studies have demonstrated that the manipulation of the gut microbiome represents a promising treatment for inflammatory bowel disease (IBD). We previously identified micro integral membrane protein (MIMP) as the smallest domain of surface layer protein from Lactobacillus Plantarum. However, the therapeutic relevance of MIMP in IBD remains unknown. Methods: We initially employed a dextran sodium sulphate (DSS)-induced colitis model and evaluated the effect of MIMP on the inflammation response, intestinal barrier and gut microbiota using histological examination, Fluorescein isothiocyanate-Dextran detection and pyrosequencing analysis respectively. We then established peripheral blood mononuclear cells (PBMCs) and an epithelial CaCO-2 co-culture model to investigate the regulatory role of MIMP in inflammatory cytokines. The level changes of inflammatory cytokines were detected using Enzyme-linked immunosorbent and real-time polymerase chain reaction assay. The involved regulatory mechanisms were investigated mainly using dual luciferase reporter and chromatin immunoprecipitation assay. Results: In the DSS-induced colitis model, we observed that MIMP intervention effectively improved the body weight loss, increased the colon length and decreased disease activity index. Consistently, the inflammation scores in the MIMP treatment group were significantly lower than those in the DSS treatment group. Furthermore, MIMP intervention was found to successfully neutralize DSS treatment by decreasing the expression of pro-inflammatory cytokines (IFN-γ, IL-17 and IL-23) and increasing the expression of anti-inflammatory cytokines (IL-4 and IL-10). Notably, the permeability assay demonstrated that the MIMP treatment group was remarkably lower than that in the DSS treatment group. We also showed that MIMP improved gut microbiota dysbiosis caused by DSS-induced inflammation. Additionally, in PBMCs and the CaCO-2 co-culture model, MIMP showed an obvious suppressive effect on lipopolysaccharide-induced inflammation in a time- and dose-dependent manner. Furthermore, we revealed that MIMP could modulate inflammatory cytokine expression through the toll-like receptor 4 pathway and histone acetylation. Conclusions: Our results suggested that MIMP showed a significant anti-inflammatory effect through regulating the gut barrier, microbiota and inflammatory cytokines. MIMP may have translational relevance as clinically relevant therapy for IBD patients.

Background Emerging evidence indicates that small nucleolar RNAs (snoRNAs) play a central role in oncogenesis. Herein, we systematically evaluated expression profiles of snoRNAs in colorectal cancer (CRC) and investigated their clinical and functional role in this malignancy. Methods We compared expression levels of snoRNAs between cancer and normal tissues using publicly available datasets and identified the most differentially expressed and commonly upregulated snoRNAs in CRC. These results were examined in 489 colorectal tissues to assess their clinical significance, followed by a series of in vitro and in vivo experiments to evaluate the functional role of candidate snoRNAs. Results Using multiple RNA profiling datasets, we identified consistent overexpression of SNORA21 in CRC. In the clinical validation cohorts, the expression level of SNORA21 was upregulated in colorectal adenomas and cancers. Furthermore, elevated SNORA21 emerged as an independent factor for predicting poor survival. Both in vitro and in vivo experiments revealed that CRISPR/Cas9-mediated inhibition of SNORA21 expression resulted in decreased cell proliferation and invasion through modulation of multiple cancer related pathways. Conclusions We systematically identified SNORA21 as a key oncogenic snoRNA in CRC, which plays an important role in cancer progression, and might serve as an important prognostic biomarker in CRC., Highlights • Systematic and comprehensive analysis revealed that SNORA21 was significantly upregulated in CRC. • Elevated SNONA21 expression was related to metastasis and predicted poor prognosis in CRC patients. • SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC. Emerging evidence indicates that small nucleolar RNAs (snoRNAs) might play a central role in oncogenesis; however, the clinical significance and functional roles of snoRNAs in colorectal cancer (CRC) remain unclear. Herein, using multiple publicly available datasets, we systematically assessed expression profiles of snoRNAs in CRC and elucidated that the high expression of SNORA21 was associated with distant metastasis and poor overall survival. A series of in vitro and in vivo experiments revealed oncogenic role for SNORA21. Our findings indicate that SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC.

Piwi-interacting RNAs (piRNAs) are a very recently discovered class of small non-coding RNAs (ncRNAs), with approximately 20,000 piRNA genes already identified within the human genome. These short RNAs were originally described as key functional regulators for the germline maintenance and transposon silencing. However, due to our limited knowledge regarding their function, piRNAs were for a long time assumed to be the "dark matter" of ncRNAs in our genome. However, recent evidence has now changed our viewpoint of their biological and clinical significance in various diseases, as newly emerging data reveals that aberrant expression of piRNAs is a unique and distinct feature in many diseases, including multiple human cancers. Furthermore, their altered expression in cancer patients has been significantly associated with clinical outcomes, highlighting their important biological functional role in disease progression. Functionally, piRNAs maintain genomic integrity by silencing transposable elements, and are capable of regulating the expression of specific downstream target genes in a post-transcriptional manner. Moreover, accumulating evidences demonstrates that analogous to other small ncRNAs (e.g. miRNAs) piRNAs have both oncogenic and tumor suppressive roles in cancer development. In this article, we discuss emerging insights into roles of piRNAs in a variety of cancers, reveal new findings underpinning various mechanisms of piRNAs-mediated gene regulation, and highlight their potential clinical significance in the management of cancer patients.

ABSTRACT Colorectal carcinogenesis represents a sequential progression of normal colonic mucosa from adenoma to carcinoma. It has become apparent that miRNA deregulation contributes to the initiation and progression of colorectal cancer (CRC). These oncogenic or tumor-suppressive miRNAs interact with intracellular signaling networks and lead to alteration of cell proliferation, apoptosis, metastasis and even response to chemotherapeutic treatments. This article aims to review the cutting edge progress in the discovery of the role of novel mechanisms for miRNAs in the development of CRC. We will also discuss the potential use of miRNAs as biomarkers for early diagnosis and prognosis of CRC. Furthermore, with advancements in RNA delivery technology, it is anticipated that manipulation of miRNAs may offer an alternative therapy for CRC treatment.

Emerging studies have revealed that Malat1 is overexpressed in many malignant diseases, including liver cancer, and contributes to enhancing cell migration or facilitating proliferation. However, the mechanism underlying its regulation has largely remained elusive. Here, we characterised the oncoprotein Yes-associated protein (YAP), which up-regulated metastasis-associated lung adenocarcinoma transcript 1 (Malat1) expression at both transcriptional and post-transcriptional levels, whereas serine/arginine-rich splicing factor 1 (SRSF1) played an opposing role. SRSF1 inhibited YAP activity by preventing its co-occupation with TCF/β-catenin on the Malat1 promoter. In contrast, overexpression of YAP impaired the nuclear retention of both SRSF1 and itself via an interaction with Angiomotin (AMOT). This effect removed the inhibitory role of SRSF1 on Malat1 in the nucleus. Furthermore, higher expression of YAP was consistent with a lower SRSF1 nuclear accumulation in human liver cancer tissues. We also revealed that overexpression of YAP combined with a knockdown of SRSF1 resulted in conspicuously enhanced transwell cell mobility, accelerated tumour growth rate, and loss of body weight in a tail vein-injected mouse models. Taken together, these data provided a novel mechanism underlying the balance between SRSF1, YAP and Malat1 and uncovered a new role of YAP in regulating long non-coding RNA (lncRNA). Thus, disrupting the interaction between YAP and SRSF1 may serve as a crucial therapeutic method in liver cancer.

Background & Aims Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice. Methods Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate-buffered saline (PBS [control]) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL adenomatous polyposis coli min/+ , C57BL miR21a −/− , and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane and dextran sodium sulfate to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by enzyme-linked immunosorbent assay. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. Fusobacterium nucleatum DNA in 90 tumor and matched nontumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times. Results Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. Adenomatous polyposis coli min/+ mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F, interleukin 21, and interleukin 22, and MIP3A). We found 50 miRNAs to be significantly up-regulated and 52 miRNAs to be significantly down-regulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (>4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a −/− mice had a later appearance of fecal blood and diarrhea after administration of azoxymethane and dextran sodium sulfate, and had longer survival times compared with control mice. The colorectum of miR21a −/− mice had fewer tumors, of smaller size, and the miR21a −/− mice survived longer than control mice. We found RASA1 , which encodes an RAS GTPase, to be one of the target genes consistently down-regulated in cells that overexpressed miR21 and up-regulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor−κB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues) compared with non-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 had shorter survival times than patients whose tumors had lower amounts. Conclusions We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. Fusobacterium nucleatum activates Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor−κB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes.

Purpose: Colorectal cancer ranks as the third most frequent cancer type, and its incidence continues to rise gradually worldwide, highlighting the need to identify previously unrecognized molecular events that propel development of this malignancy. Recent evidence suggests that dysregulated expression of FOX family of transcription factors may be critical in various genetic disorders as well as cancer; however, the functional and clinical significance of this pathway in colorectal cancer remains unclear. Experimental Design and Results: Herein, we performed a systematic and comprehensive discovery step by evaluating the expression of FOX family members, and identified that FOXM1 and FOXQ1 are frequently overexpressed in colorectal cancer. We subsequently confirmed these findings in two large testing cohorts (n = 550) and an independent clinical validation cohort (n = 134), in which high expression of FOXM1 and FOXQ1 emerged as an independent prognostic factor in colorectal cancer patients. We corroborated these findings by performing functional assays in which knockdown of FOXM1 and FOXQ1 resulted in inhibited cell proliferation and suppressed migration and invasion in colorectal cancer cells. Furthermore, using bioinformatic approaches, we identified miR-342 as a novel regulator of both FOXM1 and FOXQ1. Overexpression or inhibition of miR-342 modulated the expression of both genes and contributed to phenotypic alterations in colorectal cancer cells, which was subsequently validated in a xenograft animal model. Conclusions: Collectively, we have firstly identified FOXM1 and FOXQ1 as promising prognostic biomarkers in colorectal cancer patients, and provided novel evidence that therapeutic targeting of these genes or miR-342 may be a potential treatment approach in colorectal cancer patients. Clin Cancer Res; 22(19); 4947–57. ©2016 AACR.

Yes-associated protein (YAP) is overexpressed and has an oncogenic role in hepatocellular carcinoma (HCC). However, whether membrane protein can serve not only as a tumor marker that reflects YAP function but also as a therapeutic target that stimulates tumorigenesis in HCC remains unknown. Here we report that the membrane protein melanoma cell adhesion molecule (MCAM) was under positive regulation by YAP and was highly elevated in HCC cells. Within the MCAM promoter, we found the presence of a cAMP Response Element (CRE; -32 to -25 nt), which is conserved among species and is essential for YAP- and CREB-dependent regulation. Moreover, the interaction between CREB and YAP at the CRE site was dependent on PTPIY-WW domain interactions. However, MCAM expression was low and could not be regulated by YAP in breast and colon cancer cells because of the low levels of the acetyltransferase p300. In HCC cells, high levels of p300 facilitated the binding of YAP to the MCAM promoter, which in turn enhanced histone acetylation and polymerase II recruitment through the dissociation of the deacetylase Sirt1. These results suggest that MCAM is an HCC-specific target of YAP. In clinical serum samples, we found that the serum levels of MCAM were highly elevated in patients with HCC compared with healthy controls and with patients with cirrhosis, hepatitis, colon cancer and breast cancer. MCAM levels were shown to be a slightly better indicator than serum alpha-fetoprotein for predicting HCC. We further demonstrated that MCAM is essential for the survival and transformation of HCC. Mechanistically, MCAM induced translation initiation and the transcriptional activities of c-Jun/c-Fos. In addition, AKT activation had an essential role in the MCAM-promoted binding of eukaryotic initiation factor 4E to c-Jun/c-Fos mRNA. In conclusion, we demonstrated that MCAM may be a potential tumor marker and therapeutic target for the diagnosis and treatment of HCC.

Recent studies are indicative for strong carcinogenetic roles of Runt related transcription factor 2 (Runx2) and Yes associated protein (YAP) in several cancer types. However, whether and how the interaction between Runx2 and YAP plays a role in liver tumorigenesis still remain illusive. Here, we identified a close relationship between Runx2 and YAP in liver cancer cells. Runx2 had a positive role on YAP expression and vice versa. We also found that Rux2 and YAP were capable of inhibiting long non-coding RNA (lncRNA), Metallothionein 1D, Pseudogene (MT1DP) expression through direct promoter binding. Overexpression of MT1DP resulted in reduced cell proliferation and colony formation in soft agar, but increased apoptosis in liver cancer cells, whereas knockdown of this lncRNA had the opposite effect, indicating that MT1DP acts as a tumor suppressor. Furthermore, MT1DP was revealed as a negative regulator of Alfa-fetoprotein (AFP), a classic liver cancer tumor marker, through inhibiting protein synthesis of Forkhead box A1 (FoxA1), an important transcription factor in liver development and cancer progression. Furthermore, we found that FoxA1 plays a positive role on YAP and Runx2 expression. Specially, opening the compacted chromatin by FoxA1 around CREB binding site within the YAP promoter facilitates CREB-mediated YAP transcription. Finally, MT1DP-inhibited in vivo liver cancer cell growth could be rescued by a combination of overexpression of FoxA1, Runx2 and YAP. Taken together, the close relationship between Rnux2 and YAP plays a pro-carcinogenetic role in liver cancer cells through inhibiting tumor suppressor lncRNA, MT1DP in a FoxA1 dependent manner.

Yes-associated protein (YAP), the downstream effector of Hippo signaling pathway as well as c-Myc has been linked to hepatocarcinogenesis. However, little is known about whether and how YAP and c-Myc interacts with each other. In this study, we find YAP-c-Myc interaction is critical for liver cancer cell both in vitro and in vivo. Moreover, both c-Myc and YAP proteins are closely correlated in human liver cancer samples. Mechanistically, YAP promotes c-Myc transcriptional output through c-Abl. By contrast, c-Myc enhances protein expression independent of transcription. Taken together, our study uncovers a novel positive auto-regulatory feedback loop underlying the interaction between YAP and c-Myc in liver cancer, suggesting YAP and c-Myc links Hippo/YAP and c-Myc pathways, and thus may be helpful in the development of effective diagnosis and treatment strategies against liver cancer.

Mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1) as well as Yes-associated protein (YAP), the downstream effector of Hippo signaling pathway, is linked to hepatocarcinogenesis. However, little is known about whether and how MEK1 interacts with YAP. In this study, we find that MEK1-YAP interaction is critical for liver cancer cell proliferation and maintenance of transformed phenotypes both in vitro and in vivo. Moreover, MEK1 and YAP proteins are closely correlated in human liver cancer samples. Mechanistically, inhibition of MEK1 by both PD98059 and U0126 as well as RNAi reduces beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC), which acts as a potential endogenous YAP protector.

Ovarian cancer is one of the most common gynecological malignancies. Limited efficacy of cytotoxic chemotherapy is a key obstacle in the treatment of advanced ovarian cancer. This study aimed to investigate whether Mir-375 enhances Rawq01 (a ruthenium derived compound) induced cell death in ovarian cancer. Methods: Three human ovarian cancer cell lines were selected, and independently treated with Rawq01+mir-375 and Rawq01+control. MTT assay and flow cytometry were performed to detect the growth of ovarian cancer cells. Western blot was carried out to determine the expression of apoptotic associated proteins. In addition, ovarian cancer xenografts were established to explore whether mir-375 increased the in vivo chemosensitivity of ovarian cancer cells to RAWQ01. Results: Over-expression of mir-375 sensitized the ovarian cancer cells to RAWQ01. Mir-375 enhanced the in vitro sensitivity of ovarian cancer cells to RAWQ01 by inducing apoptosis. Mir-375 also increased the in vivo chemosensitivity of ovarian cancer cells to RAWQ01. Conclusion: Mir-375 can enhance Rawq01 induced cell death in human ovarian cancer both in vitro and in vivo.

Yes-associated protein (YAP), the downstream effecter of the Hippo-signaling pathway as well as cyclic adenosine monophosphate response element-binding protein (CREB), has been linked to hepatocarcinogenesis. However, little is known about whether and how YAP and CREB interact with each other. In this study, we found that YAP-CREB interaction is critical for liver cancer cell survival and maintenance of transformative phenotypes, both in vitro and in vivo. Moreover, both CREB and YAP proteins are highly expressed in a subset of human liver cancer samples and are closely correlated. Mechanistically, CREB promotes YAP transcriptional output through binding to -608/-439, a novel region from the YAP promoter. By contrast, YAP promotes protein stabilization of CREB through interaction with mitogen-activated protein kinase 14 (MAPK14/p38) and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC). Gain-of-function and loss-of-function studies demonstrated that phosphorylation of CREB by MAPK14/p38 at ser133 ultimately leads to its degradation. Such effects can be enhanced by BTRC through phosphorylation of MAPK14/p38 at Thr180/Tyr182. However, YAP negatively controls phosphorylation of MAPK14/p38 through inhibition of BTRC expression.There is a novel positive autoregulatory feedback loop underlying the interaction between YAP and CREB in liver cancer, suggesting that YAP and CREB form a nexus to integrate the protein kinase A, Hippo/YAP, and MAPK14/p38 pathways in cancer cells and thus may be helpful in the development of effective diagnosis and treatment strategies against liver cancer.

Mouse embryonic fibroblasts (MEFs) differentiate into fully functional chondrocytes in response to bone morphogenetic protein-2 (BMP-2). However, the comprehensive proteomic aspect of BMP-2-induced chondrogenesis remains unknown. We took advantage of quantitative proteomic analysis based on isobaric tag for relative and absolute quantitation (iTRAQ) and on-line 2D nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify proteins differentially expressed during BMP-2-induced chondrogenic differentiation of MEFs. We found 85 downregulated proteins, and ingenuity pathways analysis (IPA) revealed a protein-protein network with chromodomain-helicase-DNA-binding protein 4 (Chd4) in the center. Chromatin immunoprecipitation (ChIP) and nuclease hypersensitivity assays showed that Chd4, interacting with Hdac1/2, cooperates with its related proteins Kap1 and Cbx1 to bind at -207/-148 of the Sox9 promoter. We also provided evidence that let-7a targets the 3'UTR of Chd4 to promote chondrogenesis of MEFs. Together, our findings indicate that BMP-2 induced the upregulation of let-7a, targeting Chd4 and positively controlling the chondrogenic differentiation of MEFs. These findings illustrate epigenetic regulation of the chondrogenic differentiation process and also expand the understanding of the involved intracellular mechanisms.