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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.

Colorectal cancer (CRC) is one of most common malignancies worldwide and its incidence is still growing. In spite of recent advances in targeted therapies, their clinical efficacy has been limited, non-curative and unaffordable. A growing body of literature indicates that CRC is a multi-modal disease, where a variety of factors within the tumor microenvironment play a significant role in its pathogenesis. For instance, imbalance in gut microbial profiles and impaired intestinal barrier function contribute to the overall intestinal inflammation and initiation of CRC. Moreover, persistent chronic inflammation favors a tumor microenvironment for the growth of cancer. In addition, autophagy or ‘self-eating’ is a surveillance mechanism involved in the degradation of cellular constituents that are generated under stressful conditions. Cancer stem cells (CSCs), on the other hand, engage in the onset of CRC and are able to endow cancer cells with chemo-resistance. Furthermore, the aberrant epigenetic alterations promote CRC. These evidences highlight the need for multi-targeted approaches that are not only safe and inexpensive but offer a more effective alternative to current generation of targeted drugs. Curcumin, derived from the plant Curcuma longa, represents one such option that has a long history of its use for a variety of chronic disease including cancer, in Indian ayurvedic and traditional Chinese medicine. Scientific evidence over the past few decades have overwhelmingly shown that curcumin exhibits a multitude of anti-cancer activities orchestrated through key signaling pathways associated with cancer. In this article, we will present a current update and perspective on this natural medicine – incorporating the basic cellular mechanisms it effects and the current state of clinical evidence, challenges and promise for its use as a cancer preventative and potential adjunct together with modern therapies for CRC patients.

Long noncoding RNAs (lncRNAs) are abnormally expressed in many malignant tumors and involved in regulating the malignant phenotypes of cancer cells. However, the role of LINC00665 in colorectal cancer (CRC) and its regulatory mechanism remain unclear. In this study, real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of LINC00665, miR-9-5p and activating transcription factor 1 (ATF1) mRNA in CRC tissues. The expression of ATF1 in CRC tissues was also detected by immunohistochemistry and Western blot. CCK-8 and colony formation assays were employed to detect cell proliferation. Cell cycle and apoptosis were detected by flow cytometry analysis. Scratch healing assay and Transwell test were exploited to detect cell migration and invasion. The targeting relationships between LINC00665 and miR-9-5p, and miR-9-5p and ATF1 were validated by dual luciferase reporter assay. We found that LINC00665 was significantly overexpressed in CRC tissues, and it was also negatively correlated with the expression of miR-9-5p and positively associated with the expression of ATF1. Besides, LINC00665 promoted the proliferation, migration and invasion of CRC cells, and inhibited cell apoptosis by sponging miR-9-5p. ATF1 was proved to be the downstream target of miR-9-5p and was indirectly regulated by LINC00665. Collectively, it is concluded that LINC00665 contributes to the progression of CRC by regulating miR-9-5p/ATF1 axis.

Background: Lung cancer is one of most common malignancies worldwide. We have previously identified retinoic acid-induced gene G (Rig-G) as a tumor suppressor in not only acute promyelocytic leukemia, but as well in other solid tumors. However, the clinical significance of Rig-G and the underlying mechanism(s) for its biological function in lung cancer remain largely unexplored. Methods: We first compared the expression of Rig-G between lung cancer (n=138) and normal tissues (n=23), from public-available datasets and our patient cohort. We further analyzed the correlation of Rig-G expression with key clinico-pathological features and survival outcomes in a multi-site clinical cohort of 300 lung cancer patients. Functional studies for Rig-G were performed in cell lines, and an animal model to support clinical findings. Findings: We found that Rig-G was frequently downregulated in lung cancer tissues and celllines, and correlated with poor prognosis in lung cancer patients. Overexpression of Rig-G led to significantly reduced cell growth and suppressed migrationin A549 and NCI-H1944 cells, accompanied by reduced epithelial-mesenchymal transition. Likewise, restoration of Rig-G in Lewis lung carcinoma cells permitted development of fewer cancer metastases vs. controls in an animal model. Gene expression profiling results identified p53 pathway as a key downstream target of Rig-G, and p53 inhibition by pifithrin-α caused abrogation of tumor-suppressive effects of Rig-G in lung cancer. Interpretation: We for the first time have identified Rig-G as a novel and important tumor suppressor, which may serve as a potential therapeutic target for restoring p53 expression in lung cancer patients. Funding Statement: This work was supported by the National Cancer Institute, NIH (CA72851, CA184792, CA202797 and CA187956), the National Natural Science Foundation of China (81272603, 81472179 and 81873975), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Clinical Research and Cultivation Project of Shanghai Tongji Hospital grant [ITJ(ZD)1803]. This work was also supported by the National Natural Science Foundation of China (81672826 and 81874179), the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai (2017YQ044), the Shanghai Pujiang Talent Plan (18PJD047), the Natural Science Foundation of Shanghai (19ZR1448800), the Medical Guidance Science and Technology Support Project of Shanghai (9411964800) and the National Natural Science Foundation Training Program of Tongji Hospital (GJPY1804). Declaration of Interests: None of the authors have any potential conflicts to disclose. Ethics Approval Statement: A written informed consent was obtained from all patients and the study was approved by ethics committee of Shanghai Tongji Hospital. All animal experiments were approved by the Tongji Hospital of Tongji University Ethics Committee on the Use and Care of Animals.

Emerging evidence suggests that hypermethylation of HOXD10 plays an important role in human cancers. However, the biological and clinical impacts of HOXD10 overmethylation and its downstream targets in colorectal cancer remain unknown. We evaluated the methylation level of HOXD10 in paired cancer and normal tissues (n = 42) by using pyrosequencing, followed by validation of the methylation status of HOXD10 from The Cancer Genome Atlas (TCGA) datasets with 302 cancer tissues and 38 normal tissues. The biological function of HOXD10 was characterized in cell lines. We further evaluated the effects of HOXD10 and its targets on chemoresistance in our established resistant cell lines and clinical cohort (n = 66). HOXD10 was found frequently methylated in colorectal cancer, and its hypermethylation correlates with its low expression level, advanced disease, and lymph node metastasis. Functionally, HOXD10 acts as a tumor suppressor gene, in which HOXD10-expressing cells showed suppressed cell proliferation, colony formation ability, and migration and invasion capacity. Mechanistically, DNMT1, DNMT3B, and MeCP2 were recruited in the HOXD10 promoter, and demethylation by 5-Aza-2′-deoxycytidine (5-Aza-CdR) treatment or MeCP2 knockdown can sufficiently induce HOXD10 expression. HOXD10 regulates the expressions of miR-7 and IGFBP3 in a promoter-dependent manner. Restoration of the expression of HOXD10 in 5-fluorouracil (5-FU)-resistant cells significantly upregulates the expressions of miR-7 and IGFBP3 and enhances chemosensitivity to 5-FU. In conclusion, we provide novel evidence that HOXD10 is frequently methylated, silenced, and contributes to the development of colorectal cancers. Restoration of HOXD10 activates the expressions of miR-7 and IGFBP3 and results in an inhibited phenotype biologically, suggesting its potential therapeutic relevance in colorectal cancer (CRC).

Background Emerging evidence suggests that PIWI-interacting RNAs (piRNAs) may be important epigenetic regulators of gene expression in human cancers; however, their functional and clinical significance in colorectal cancer (CRC) remains unknown. Methods We performed piRNA expression profiling in paired cancer and normal tissues through small RNA-sequencing. The clinical significance of candidate piRNAs was investigated, and independently validated in 771 CRC patients from three independent cohorts. The biological function of piRNAs was characterized in cell lines, followed by identification and validation of downstream target genes in CRC tissues. Results We identified piR-1245 as a novel and frequently overexpressed noncoding RNA in CRC, and its expression significantly correlated with advanced and metastatic disease. Patients with high piR-1245 expression experienced significantly shorter overall survival, and multivariate analysis identified its expression to serve as an independent prognostic biomarker in CRC. Functionally, piR-1245 acts as an oncogene and promotes tumor progression, and gene expression profiling results identified a panel of downstream target-genes involved in regulating cell survival pathway. Based upon piRNA:mRNA sequence complementarity, we identified a panel of tumor suppressor genes (ATF3, BTG1, DUSP1, FAS,NFKBIA, UPP1, SESN2, TP53INP1 and MDX1) as direct targets of piR-1245, and successfully validated an inverse correlation between their expression and piR-1245 in CRC. Conclusions We for the first time have identified the role for a PIWI-interacting noncoding RNA, piR-1245, as a novel oncogene and a potential prognostic biomarker in colorectal cancer. Electronic supplementary material The online version of this article (10.1186/s12943-018-0767-3) contains supplementary material, which is available to authorized users.

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.

Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients. Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings. Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae (P = 0.0018), and its overexpression was associated with poor patient survival (P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918–28. ©2017 AACR.

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.

Background We evaluated the diagnostic value of serum-CD166 in patients with hepatocellular carcinoma (HCC). Methods Tissue-CD166 was measured using immunohistochemistry. Cell proliferation and migration were evaluated using MTT and Transwell assays, respectively. Serum-CD166 was examined using ELISA and western blotting. Results CD166 was up-regulated in HCC compared to those in normal liver tissues. Cell proliferation was positively correlated and cell migration was negatively correlated with endogenous CD166 expression in HCC cells. CD166 inhibition using specific shRNA decreased cell proliferation but increased cell migration. Serum CD166 concentrations were much higher in HCC than in colon cancer, hepatitis B, hepatitis C, cirrhosis, gastric cancer, breast cancer, lung cancer and healthy individuals. Serum CD166 also decreased dramatically after curative surgery. A positive correlation was found between serum CD166 and AFP (R = 0.7141, p = 0.000). Serum CD166 was also positively correlated with γ-GT, bile acid, ALT, AST, and ALP but was negatively correlated with Alb and pre-Alb. The area under the receiver operating characteristic curve for serum-CD166 was 0.9860, which was better than AFP (AUC-ROC, 0.9354) for the differentiation of HCC patients from healthy individuals, with a cut-off of 261 ng/ml (sensitivity: 100.00%, specificity: 89.41%). Conclusion Serum CD166 is a novel diagnostic tumor marker for HCC.

Renal cell carcinoma (RCC) is a common urological malignancy. It remains unclear, however, whether Yin Yang 1 (YY1) plays a functional role in the development of human RCC. In the present study, we demonstrated that levels of YY1 were significantly increased in primary RCC tissues when compared to these levels in the matched healthy tissues. YY1 knockdown inhibited cell growth, migration and invasion of RCC cells. Additionally, we highlighted a positive feedback-loop pathway resulting in YY1 upregulation. We observed that overexpression of YY1 caused repression of C/EBPα and the inhibition of C/EBPα led to the suppression of miR-34a. Since YY1 is a direct target of miR-34a, the low level of miR-34a increased the expression of YY1, promoting the aggressiveness of RCC cells. Furthermore, this feedforward mechanism was found in RCC tissues. We observed that miR-34a was downregulated in the pools of cancer tissues when compared to that of the normal tissues. The expression of miR-34a displayed an inverse correlation with YY1, but a positive correlation with C/EBPα. In conclusion, our study highlights the importance of a novel regulatory circuitry for YY1 activation in maintaining the aggressive phenotypes of RCC.

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. The increased incidence rates of CRC places a considerable burden on individuals and society in Asian countries. Although surgical resection remains the standard treatment for CRC patients, accumulating evidences showed advanced patients derived an additional benefit from molecular targeted agents. Therefore, there is a great need to identify novel cellular targets in CRC that can be exploited for therapeutic benefit. Trefoil factor 3 (TFF3), is a member of a family of small secretory proteins characterized by three intrachain disulfide bonds constituting the trefoil motif. TFF3 was initially found as a mediator of mucosal healing and play a critical role in epithelial restitution. Recent studies showed that TFF3 may contribute to tumorigenesis. However, its role in CRC remains unclear. Herein, the protein expression of TFF3 was measured in colorectal adenoma polyps (PLP, n=20), cancer tissues (n=20) and matched adjacent healthy tissues (n=20) by immunohistochemical (IHC) assay. We further evaluated serum and urine level of TFF3 from healthy volunteers (n=40), PLP patients (n=40) and CRC patients (n=40) by using ELISA assay. To investigate the functional or mechanistic role of TFF3 in CRC, we conducted several in vivo and in vitro approaches. The IHC showed that TFF3 was frequently overexpressed in cancer tissues (12/20, P Note: This abstract was not presented at the meeting. Citation Format: Ying Long, Tsuyoshi Ozawa, Hanhua Li, Weijie Pan, Wenhao Weng. Novel evidence for Trefoil Factor 3 as an oncogenic mediator of disease progression, and a potential predictor for neoadjuvant chemotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3161.

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. Conclusion: 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. (Hepatology 2013;53:1011–1020)

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.

Purpose: Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. But the process is still unknown. Here, we analyzed genetic differences between primary tumors associated with liver metastasis and those without liver metastasis and predicted clinical significance. Experimental design: Eight primary tumors associated with liver metastasis and twelve without liver metastasis of CRC were analyzed by whole-exome sequencing. The functional characterisation for the role of metastatic-related genes was investigated through in vitro migration and invasion assays. For clinical relevance, TMAs were used for immunohistochemistry assay. Results: The mutation spectrum was similar to that of previous studies. TP53, APC and KRAS were the three most frequently mutated genes, with no significant difference of mutation frequency between primary tumors from patients with liver metastasis and those from other patients. Notably, four recurrently mutated genes (FBXW7, EDIL3, KCNK9 and PCDHGA7) were identified exclusively in the tumors associated with liver metastasis. Furthermore, in vitro analyses showed that EDIL3 and KCNK9 overexpression resulted in enhanced cell migration and invasion, while CRISPR-mediated deletion of FBXW7 and PCDHGA7 exhibited enhanced migratory and invasive properties as well. For clinical relevance, highly expressed EDIL3 and KCNK9 was significantly associated with liver metastasis, whereas the expression of FBXW7 and PCDHGA7 negatively correlated with it in our cohorts. Pathway enrichment analysis also indicated a role for cell adhesion signaling in liver metastasis of CRC patients. Conclusion: Our study identified candidate genes, which might response to the initiation of metastasis, exclusively mutated in tumors associated with liver metastasis. These findings also provided potential therapeutic targets for personalized treatment. Note: This abstract was not presented at the meeting. Citation Format: Yanlei Ma, Wenhao Weng, Yongzhi Yang, Qing Wei, Sanjun Cai. Whole exome sequencing analyses of colorectal cancers associated with liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2711. doi:10.1158/1538-7445.AM2017-2711

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.

Colorectal cancer (CRC) remains one of the most common types of cancer and leading causes of cancer death worldwide. Although the introduction of cytotoxic drugs such as oxaliplatin, irinotecan and fluorouracil has improved the treatment of advanced CRC, the individual response to chemoradiotherapy varies tremendously from one patient to another. However, recent progress in CRC molecular therapies may provide new insight into the treatment of this disease. Currently, components of the EGFR, VEGF, Wnt and NF-kB pathways are the most important targets for CRC therapy. This review chronicles the development of molecular CRC therapies over the past few decades. We also provide an update on the current progress of research concerning the molecular pathways leading to CRC and discuss the possible implications for CRC therapy.

Tribbles homolog 2 (TRIB2) is critical for both solid and non-solid malignancies. Recently, TRIB2 was identified as a liver cancer-specific Wnt/β-catenin signaling downstream target and is functionally important for liver cancer cell survival and transformation. TRIB2 functions as a protein that interacts with E3 ubiquitin ligases and thereby modulates protein stability of downstream effectors. However, the regulation underlying TRIB2 protein stability per se has not yet been reported. In this study, we found that TRIB2 was up-regulated and exhibited high stability in liver cancer cells compared with other cells. We performed a structure-function analysis of TRIB2 and identified a domain (amino acids 1–5) at the N terminus that interacted with the E3 ubiquitin ligase Smurf1 and was critical for protein stability. Deletion of this domain extended TRIB2 half-life time accompanied with a more significant malignant property compared with wild type TRIB2. Furthermore, Smurf1-mediated ubiquitination required phosphorylation of TRIB2 by p70 S6 kinase (p70S6K) via another domain (amino acids 69–85) that is also essential for correct TRIB2 subcellular localization. Mutation of Ser-83 diminished p70S6K-induced phosphorylation of TRIB2. Moreover, the high stability of TRIB2 may be due to the fact that both p70S6K and Smurf1 were down-regulated and negatively correlated with TRIB2 expression in both liver cancer tissues and established liver cancer cell lines. Taken together, impaired phosphorylation and ubiquitination by p70S6K and Smurf1 increase the protein stability of TRIB2 in liver cancer and thus may be helpful in the development of diagnosis and treatment strategies against this malignant disease.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. Upregulation of metastasis-associated protein 1 (MTA1) has been reported to contribute to the development of esophageal squamous cell carcinoma. Therefore, the objective of our study was to identify the molecular mechanisms of MTA1 underlying the invasion and metastasis of ESCC. We overexpressed MTA1 in ESCC cells to examine the role of MTA1 in the regulation of the cell invasion. In addition, using luciferase reporter assay and electrophoretic mobility shift assays, we evaluated the binding of MTA1 to the promoter of E-cadherin. We found that MTA1 overexpression promotes invasiveness of the human esophageal carcinoma cell line EC-9706. This effect was accompanied by downregulation of the epithelial cell marker E-cadherin and upregulation of vimentin and MMP-9 luciferase reporter assays showed that MTA1 inhibited the promoter activity of E-cadherin and that this was dependent on Snail, Slug and HDAC1. We also found that Snail and Slug bound the E-boxes in the promoter of E-cadherin and recruited MTA1 and HDAC1 to suppress E-cadherin expression, as confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. MTA1 promotes tumor invasion by downregulation of E-cadherin. These results demonstrate a novel role for MTA1 in the regulation of esophageal squamous cell carcinoma invasion and provide insight into the mechanisms involved in this process.

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.

Background: Colorectal cancer (CRC) remains a prevalent malignancy worldwide with a high mortality rate. Over the last decade, the roles of several types of noncoding RNAs in oncogenesis have been clarified. Emerging evidence indicates that not only microRNAs, but small nucleolar RNAs (snoRNAs) appear to be dysregulated in various malignancies and they play a critical role in modulating cell transformation, tumor progression and metastasis. Functionally, cancer associated snoRNAs are subdivided into four major categories based on their oncogenic functions: 1) an independent function from imprinting gene in cancer initiation and progression 2) Activation of human telomerase 3) Inhibition of ribosomal maturation 4) Act directly as oncogene or tumor suppressor. Despite these exciting evidences, the role of snoRNAs in CRC remains limited and their clinical significance remains largely unexplored in this malignancy. Aims: We aimed to clarify the clinical relevance of snoRNAs in tumors and evaluate their prognostic potential in CRC. Furthermore, we investigated the functional role of snoRNAs in CRC tumorigenesis. Methods: Initially, we screened for the most commonly differentially expressed snoRNAs from publicly available microarray and RNA-sequence databases for other malignancies. Three clinical cohorts (a total 345 CRC tissues) were then used to select/validate the candidate snoRNAs. The prognostic potential of the candidate snoRNAs were evaluated and the association with clinicopathological features were assessed. CRISPR/Cas9 system was used to knockdown the target snoRNA in multiple CRC cell lines to determine the functional role of the target snoRNA in a series of in vitro assays. Results: In the discovery phase, we identified four differentially overexpressed snoRNAs in human cancers. We then confirmed overexpression of three snoRNAs (SNORA21, SNORA34 and SNORD66) in CRC compared to adjacent normal tissues. Of these three snoRNAs, high SNORA21 expression in tumors resulted in poor prognosis (p = 0.0086, Log-rank test). In an independent validation cohort, we confirmed high SNORA21 expression results in poor overall survival (p = 0.0060), especially in stage IV CRC (p = 0.0272). Clinicopathological analysis showed positive association between SNORA21 and vascular invasion and metastasis (p = 0.030 and 0.011, by Kruskal-Wallis test and Mann-Whitney U test, respectively) indicating that SNORA21 could be involved in metastasis. Moreover, multivariate analysis revealed that increased SNORA21 expression was an independent prognostic factor for overall survival. Using CRISPR/Cas9 stable SNORA21 knocked-down cell lines, we demonstrated that SNORA21 suppression resulted in reduced cell proliferation and colony forming capacity. Conclusion: We firstly identified SNORA21 as a novel oncogene which could also be a potential prognostic marker in patients with CRC. Citation Format: Kazuhiro Yoshida, Shusuke Toden, Wenhao Weng, Takeshi Nagasaka, Toshiyoshi Fujiwara, Ajay Goel. Clinical significance of SNORA21, an H/ACA box snoRNA, as a metastasis predicting and prognostic biomarker in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4926.

Background: Vacuolar (v) -ATPases are expressed in certain tissues and cell types, and hypothesized to detoxify intracellular environments by expelling protons across plasma membranes, thereby contributing to acidified extracellular environments of solid tumors. Acidic microenvironments suppress anti-cancer cytotoxic T-cells and activate metalloproteinases, promoting tumor cell survival, motility and invasion. Scarce nutrients, hypoxia, and accelerated metabolism that favor pathways that produce excess intracellular protons by promoting glycolysis and lactic acid fermentation, lead to selection of tumor cells that adapted to and countered toxic/acidic cytoplasm. It is unclear whether colorectal cancers (CRCs) upregulate proton-expelling mechanisms, and whether associated genes are prognostic biomarkers. Aim: We hypothesized that adaptations through upregulation of proton transporting genes is prognostic for CRC. This study aims to identify transcriptional adaptations that CRCs undergo in response to acidic microenvironments and evaluate their prognostic value in multiple cohorts of CRC patients. Methods: Comprehensive microarray data from 222 CRCs obtained from The Cancer Genome Atlas were used to screen for genes upregulated in CRCs vs. normal mucosae, and stage III and IV vs. early staged tumors. Filtered genes involved in metabolite transport were tested for their ability to predict survival using Kaplan-Meier survival analysis with a stringent median cutoff. A second cohort was used to corroborate initial findings. A third cohort of matching polyps and adenomas was used to determine if candidate gene upregulation can be detected in pre-cancerous lesions. Functional analysis was performed using siRNA knockdown to determine if the candidate gene is critical for CRC proliferation. Results: Progressive upregulation of ATP6V1C2 from early to late stage CRCs was detected in two separate multinational patient cohorts, where its high expression was correlated with shorter overall survival. Receiver operating characteristic (ROC) curve analysis confirmed that ATP6V1C2 expression successfully discriminated between cancerous and non-cancer tissues. Fisher's exact test confirmed that high ATP6V1C2 expression correlated with advanced depth of invasion (T stage), and distant and liver metastasis. ATP6V1C2 expression was increased in pre-cancerous polyps. SiRNA knockdown of ATP6V1C2 inhibited cell proliferation in high expressing cell lines (HCT116 and HCT15) but not in low expressing RKO and HT29. Citation Format: Timothy J. Zumwalt, Kunitoshi Shigeyasu, Wenhao Weng, Yoshinaga Okugawa, Jinsei Miyoshi, Ajay Goel. The ATP6V1C2 (a vacuolar-ATPase gene) is a novel early prognosticator for colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 768.

OBJECTIVES: The development of colorectal cancer (CRC) is a highly complex process which needs coordinated regulation in the gene regulatory network. Forkhead box (FOX) proteins constitute a large family of transcriptional regulators. Accumulating evidence indicates that several FOX family members are critical regulators of tumor progression. However, the role of FOX proteins in CRC remains vastly unknown. miRNAs are single stranded small non-coding RNAs which can regulate a variety of biological processes in various human diseases including cancer. AIMS: Herein, we aimed to identify the expression patterns of FOX family members in CRC, followed by extensive characterization for their functional and mechanistic role in this malignancy. In addition, we sought to identify specific miRNAs that are responsible for dysregulated expression of FOX proteins in CRC. METHODS: The web-based database ONCOMINE was used to identify most highly expressed FOX family members in CRC. A cohort of 178 frozen tissues including 134 primary CRC tissues, and 44 matched normal mucosa tissues were collected to analyze the expression levels of FOX genes. For functional analysis, cell proliferation, migration and invasion assays were performed following siRNA knockdown of specific FOX proteins in CRC cells. The potential FOX-targeting miRNAs were initially identified using in-silico approaches, followed by luciferase reporter assays to validate their binding to downstream target genes. RESULTS: We systematically analyzed the expression profiles of FOX family members across 24 studies in cancer and normal tissues, and identified FOXM1, FOXQ1 and FOXA2 as the most highly expressed FOX genes in CRC. The over-expression of FOXM1 and FOXQ1 in CRC was confirmed in a cohort of CRC and matched adjacent normal tissues. Furthermore, the analysis of 134 CRC samples revealed that high FOXM1 and FOXQ1 expression resulted in worse overall survival. We next identified miR-342 as a potential regulator of both FOXM1 and FOXQ1, and subsequently validated its direct binding to the 3’UTR regions of both genes. Furthermore, the expression of miR-342 in CRC tissues inversely correlated with both FOXM1 and FOXQ1 expression. In-vitro functional analysis revealed that suppression of FOXM1 or overexpression of miR-342 inhibited CRC cell proliferation, while FOXM1 and FOXQ1 knockdown or miR-342 overexpression suppressed migration and invasion capacity of CRC cell lines. The stable overexpression of miR-342 reduced FOXM1 and FOXQ1 expression and significantly inhibited tumor growth in CRC xenografts in nude mice. CONCLUSIONS: FOXM1 and FOXQ1 promote cancer progression and act as potential prognostic factors in colon cancer. The downregulation of miR-342 in tumor tissues may contribute to the CRC development through inhibition of FOXM1 and FOXQ1 expression, suggesting that miR-342 could be used as a therapeutic target in colorectal cancer. Citation Format: Wenhao Weng, Yoshinaga Okugawa, Shusuke Toden, Ajay Goel. FOXM1 and FOXQ1 are novel oncogenes in colorectal cancer and can be therapeutically targeted through miRNA-based therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1125.

Tribbles homolog 2 (TRIB2) is specifically regulated by Wnt signaling in liver cancer cells but not in colon cancer cells. However, whether and how TRIB2 regulates Wnt signaling in liver cancer cells remains unclear. Here, we report that TRIB2 negatively regulates Wnt activity through a reduction in protein stability of TCF4 and β-Catenin. Mechanistically, TRIB2 associated-ubiquitin E3 ligases beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP), COP1 and Smad ubiquitination regulatory factor 1 (Smurf1) reduced TCF4/β-Catenin expression, and these effects could be enhanced by TRIB2. Moreover, deletion of the binding regions of these E3-ligases within the TRIB2 protein decreased ubiquitination of TCF4/β-Catenin and reduced nuclear accumulation of β-TrCP, COP1 and Smurf1, which suggested that TRIB2 regulated-Wnt activity is closely correlated with its associated E3 ligases.