Colon Cancer Malignancy Research Articles

Cinobufotalin regulates the USP36/c-Myc axis to suppress malignant phenotypes of colon cancer cells in vitro and in vivo.

Ubiquitin-specific protease 36 (USP36) has been reported to exhibit oncogenic effects in various malignancies, but the function of USP36 in colon cancer progression remains indefinite. Herein, we aimed to determine the role and mechanism of USP36 in malignant phenotypes of colon cancer cells and explore the potential drug targeting USP36. Bioinformatics analyses indicated that USP36 is highly expressed and significantly related to tumor stages in colon cancer. Besides, USP36 was further up-regulated in oxaliplatin (Oxa)-resistant colon cancer cells. Colony formation, Edu staining, Transwell, wound healing, sphere formation, and CCK-8 assays were conducted and showed that the proliferation, Oxa-resistance, migration, stemness, and invasion of HCT116 cells were promoted after overexpressing USP36, while suppressed by USP36 knockdown. Mechanically, USP36 enhances c-Myc protein stabilization in HCT116 cells via deubiquitination. AutoDock tool and ubiquitin-AMC hydrolysis assay identified cinobufotalin (CBF), an anti-tumor drug, maybe a USP36 inhibitor by inhibiting its deubiquitination activity. CBF significantly prohibited proliferation, migration, invasion, and stemness of HCT116 cells and reversed Oxa-resistance, whereas enforced expression of USP36 blocked these effects. Moreover, in vivo analyses confirmed the oncogenic role of USP36 and the therapeutic potential of CBF in the malignancy of colon cancer. In conclusion, CBF may be a promising therapeutic agent for colon cancer due to its regulation of the USP36/c-Myc axis.

LncRNA ELFN1-AS1 enhances the progression of colon cancer by targeting miR-4270 to upregulate AURKB.

The oncogenic role of lncRNA ELFN1-AS1 has been described in different cancers, including colon cancer (CC). However, how ELFN1-AS1 regulates CC malignancy remains unclear. In this study, ELFN1-AS1, AURKB, and miR-4270 expression levels in CC cells and tissues were determined using RT-qPCR and western blotting. CCK-8 and wound healing assays were also performed to analyze alterations in CC cell proliferation and migration. The expression of apoptosis-related proteins (Bax and Bcl-2) was determined via western blot analysis. RNA immunoprecipitation (RIP) assays coupled with luciferase reporter assays were employed to verify the relationship between miR-4270, ELFN1-AS1, and AURKB. An in vivo assay was performed using xenograft tumors in mice to detect the change of tumor growth. It was found that AURKB and ELFN1-AS1 expression was upregulated, whereas miR-4270 was downregulated in CC cells and tissues. ELFN1-AS1 silencing exhibited anti-proliferative, anti-migratory, and pro-apoptotic effects in CC cells. The tumor-suppressive effect of ELFN1-AS1 silencing was verified using in vivo assays. MiR-4270 was predicted to be a target of ELFN1-AS1 and AURKB as a target of miR-4270. Their interactions were further elucidated using luciferase reporter and RNA RIP assays. More importantly, treatment with a miR-4270 inhibitor not only rescued the tumor-suppressing effect of ELFN1-AS1 silencing but also abrogated the tumor suppressor functions of AURKB silencing in CC cells. Taken together, the ELFN1-AS1/miR-4270/AURKB axis facilitates CC tumorigenesis; therefore, targeting this axis might be a promising intervention in preventing CC progression.

A novel mitochondria-related gene signature for controlling colon cancer cell mitochondrial respiration and proliferation.

Colon cancer cells rely on mitochondrial respiration as major source of energy for supporting their proliferation and invasion, thus promoting colon cancer malignancy and progression. In this study, we comprehensively investigated the prognostic significance of mitochondria-related genes in colon cancer and identified the hub genes that control colon cancer cell mitochondrial respiration and proliferation. We first systematically evaluated the prognostic significance of differentially expressed mitochondria-related genes in colon cancer specimens. Furthermore, a protein-protein interaction network was constructed to explore the hub genes. Eventually, five hub genes were identified, namely, POLG, FASTK, MRPS5, AARS2, and VARS2. Functional analyses showed that all these five hub genes are essential for maintaining mitochondrial respiration and proliferation of colon cancer cells. Mechanistic studies revealed the roles of these five hub genes in modulating mitochondrial DNA expression, that in turn influence mitochondrial respiration. In summary, our study demonstrated that POLG, FASTK, MRPS5, AARS2, and VARS2 may potentially serve as prognostic biomarkers and therapeutic targets for colon cancer.

The protein 4.1R downregulates VEGFA in M2 macrophages to inhibit colon cancer metastasis

M2 macrophages are crucial components of the tumour microenvironment and have been shown to be closely related to tumour progression. Co-culture with 4.1R−/− M2 macrophages enhances the malignancy of colon cancer (CC), but the mechanism remains unclear. Here, we report that protein 4.1R knockout reduced the phagocytosis of M2 macrophages (M-CSF/IL-4-treated bone marrow cells) and promoted MC38 colon cancer cell proliferation, migration, invasion, tumour formation and epithelial-mesenchymal transition (EMT), which are regulated by M2 macrophages. Further mechanistic dissection revealed that the 4.1R knockout upregulated vascular endothelial growth factor A (VEGFA) secreted by M2 macrophages and promoted colon cancer progression by activating the PI3K/AKT signalling pathway. In summary, our present study identified that 4.1R downregulates VEGFA secretion in M2 macrophages and delays the malignant potential of colon cancer by inhibiting the PI3K/AKT signalling pathway.

MiR-509-3-5p inhibits colon cancer malignancy by suppressing GTSE1

BackgroundThe overarching goal of this research was to identify the effect of miR-509-3-5p on colon cancer (CC) and its interaction with potential target gene GTSE1 in CC. MethodsThe miR-509-3-5p expression was ascertained after performing qRT-PCR analyses, and the ability of GTSE1 to influence this microRNA was detected after carrying out RNA pull-down assay. CCK-8 assay kit was first employed to determine the proliferation of the cells. To examine the migration and invasion level of HCT116 and SW480 cells, cell wound healing and transwell assay were later performed. After constructing luciferase reporter plasmids, luciferase reporter assay was used to confirm the impacts of miR-509-3-5p on GTSE1 in HCT116 and SW480 cells. ResultsWe found that miR-509-3-5p expression reduced in CC, and its overexpression inhibited the proliferation, migration and invasion of CC cells. We later discovered that miR-509-3-5p could target GTSE1 that was then proved to be an oncogene in CC. ConclusionOur study uncovered that miR-509-3-5p regulated CC malignancy by suppressing target gene GTSE1.

Synchronous primary malignancy of colon cancer and mantle cell lymphoma: A case report

Abstract Multiple primary malignancies in a single patient are relatively rare; however, the frequency of this has increased significantly in recent decades. Here, we retrospectively reported an unusual case of a 70-year-old man who was admitted to the hospital with mantle cell lymphoma (MCL) and colon cancer and aimed to explore measures to reduce missed diagnosis. Based on the data, the investigation of the related literatures, colonoscopy examination, and abdominal computed tomography (CT) scan were conducted for the detection of colon cancer. Following this, a precise diagnosis of MCL was confirmed by immunohistochemistry and bone marrow biopsy, which were performed to analyze the clinical characteristics and essentials for the diagnosis and differential of the disease. The results of colonoscopy showed that the patient had colon cancer, while the abdominal CT scan demonstrated colon cancer accompanied by multiple lymphadenopathy throughout the entire body. Besides, the results of immunohistochemistry confirmed that the patient suffered from MCL. The bone marrow biopsy revealed the active hematopoietic tissue hyperplasia but no tumor issue involvement in the bone marrow. In conclusion, our study combined the analysis and summary of the diagnosis and treatment of the colon cancer with MCL to provide clinical guidance for the rare multiple primary malignancy.

MicroRNA-10a-5p overexpression suppresses malignancy of colon cancer by regulating human liver cancer fibroblasts.

The crosstalk between tumor and stroma plays a critical role in cancer metastasis. However, the function of miR-10a-5p on liver fibroblasts in the metastatic microenvironment of colon cancer (CC) and the effect of activated fibroblasts on CC cells are still unclear. In our study, miR-10a-5p overexpression inhibited the proliferation, migration, and IL-6/IL-8 level of LX-2 cells and human liver cancer fibroblasts (HLCFs). Moreover, miR-10a-5p had lower expression in HLCFs than in human liver normal fibroblasts (HLNFs). The conditioned medium (CM) from LX-2 cells with miR-10a-5p overexpression or HLNFs could inhibit the invasion, migration, and stemness of CC SW480 cells, whereas HLCFs CM could promote these malignant phenotypes of SW480 cells. The present study illustrates the effect of miR-10a-5p on the liver fibroblasts and the altered liver fibroblasts in the microenvironment on CC cells induced by miR-10a-5p, which may aid the understanding of the mechanisms underlying CC liver metastasis.

LINC01123 facilitates proliferation, invasion and chemoresistance of colon cancer cells.

Colon cancer is one of the major causes of cancer-related deaths worldwide. Long non-coding RNA (lncRNA) LINC01123 has been suggested to act as an oncogene in non-small cell lung cancer and a prognostic signature in head and neck squamous cell carcinoma. However, its role in colon cancer remains obscure. From TCGA database, LINC01123 was observed to be up-regulated in colon adenocarcinoma (COAD). Subsequently, the up-regulated LINC01123 was also detected in colon cancer cells. Functionally, LINC01123 could enhance cell proliferation, migration, invasion and angiogenesis. Moreover, the chemoresistance of colon cancer cells was verified to be promoted by LINC01123. Afterward, LINC01123 was found to bind with Ago2 and miR-34c-5p. Besides, miR-34c-5p was confirmed to inhibit the cellular process and chemoresistance of colon cancer cells. Then, VEGFA was disclosed to coexist with LINC01123 and miR-34c-5p in RNA-induced silencing complex. And TCGA database suggested that its expression was correlated with different stages of COAD. Moreover, it was uncovered that VEGFA could bind with miR-34c-5p and its expression positively correlated with LINC01123 expression. Finally, LINC01123 was proofed to regulate colon cancer progression and cells chemoresistance via VEGFA. In conclusion, LINC01123/miR-34c-5p/VEGFA axis promotes colon cancer malignancy and cells chemoresistance.

Carbohydrate response element binding protein (ChREBP) correlates with colon cancer progression and contributes to cell proliferation

Cancers are characterized by reprogrammed glucose metabolisms to fuel cell growth and proliferation. Carbohydrate response element binding protein (ChREBP) is a glucose-mediated transcription factor that strongly regulates glycolytic and lipogenic pathways. It has been shown to associate with metabolic diseases, such as obesity, diabetes and non-alcoholic fatty liver diseases. However, how it associates with cancers has not been well understood. In this study, ChREBP expression was assessed by immunohistochemistry in colon tissue arrays containing normal colon tissue and cancer tissue at different clinical stages. Tissue mRNA levels of ChREBP were also measured in a cohort of colon cancer patients. We found that ChREBP mRNA and protein expression were significantly increased in colon cancer tissue compared to healthy colon (p < 0.001), and their expression was positively correlated to colon malignancy (for mRNA, p = 0.002; for protein p < 0.001). Expression of lipogenic genes (ELOVL6 and SCD1) in colon cancer was also positively associated with colon malignancy (for both genes, p < 0.001). In vitro, ChREBP knockdown with siRNA transfection inhibited cell proliferation and induced cell cycle arrest without changes in apoptosis in colon cancer cell lines (HT29, DLD1 and SW480). Glycolytic and lipogenic pathways were inhibited but the p53 pathway was activated after ChREBP knockdown. Taken together, ChREBP expression is associated with colon malignancy and it might contribute to cell proliferation via promoting anabolic pathways and inhibiting p53. In addition, ChREBP might represent a novel clinical useful biomarker to evaluate the malignancy of colon cancer.

Nuclear-localized costimulatory molecule 4-1BBL promotes colon cancer cell proliferation and migration by regulating nuclear Gsk3β, and is linked to the poor outcomes associated with colon cancer

ABSTRACT Anti-tumor immune response and the prognosis of tumor are the results of competition between stimulatory and inhibitory checkpoints. Except for upregulating inhibitory checkpoints, lowering some immune accelerating molecules to convert an immunostimulatory microenvironment into an immunodormant one through “decelerating the accelerator” might be another effective immune escape pattern. 4-1BBL is a classical transmembrane costimulatory molecule involving in antitumor immune responses. In contrast, we demonstrated that 4-1BBL is predominantly localized in the nuclei of cancer cells in colon cancer specimens and is positively correlated with tumor size, lymph node metastasis, and a lower survival ratio. Furthermore, the nuclear localization of 4-1BBL was also ascertained in vitro. 4-1BBL knockout (KO) arrests the proliferation and impaired the migration and invasion ability of colon cancer cells in vitro and retarded tumor growth in vivo. 4-1BBL KO increased the accumulation of Gsk3β in the nuclei of colon cancer cells and consequently decreased the expression of Wnt pathway target genes and thus alter tumor biological behavior. We hypothesized that unlike membrane-expressed 4-1BBL, which stimulates the 4-1BB signaling of antitumor cytotoxic T cells, the nuclear-localized 4-1BBL could facilitate the malignant behavior of colon cancer cells by circumventing antitumor signaling and driving some key oncotropic signal pathway in the nucleus. Nuclear-localized 4-1BBL might be an indicator of colon cancer malignancy and serve as a promising target of immunotherapy.

Livin promotes colon cancer progression by regulation of H2A.XY39ph via JMJD6

Livin is an important member of the human inhibitor of apoptosis proteins (IAPs) family. IAPs are proteins with antiapoptotic abilities, and their functions are different from the Bcl-2 (B-cell lymphoma-2) family proteins. However, the precise role of Livin in colon cancer progression remains unclear. The purpose of this study is to assess the effect of overexpression Livin in colon cancer cells and to examine its molecular mechanism. We demonstrated that Livin induced a colon cancer phenotype, including proliferation and migration, by regulating H2A.XY39ph (histone family 2A variant (H2AX) phosphorylated on the 39th serine site). We elucidated that Livin degraded Jumonji-C domain-containing 6 protein (JMJD6), which was mediated by the proteasome murine double minute 2 (MDM2), thereby regulating H2A.XY39ph. Above all, the overexpression of JMJD6 recovered H2A.XY39ph in colon cancer cells with a high level of Livin, thus inhibiting colon cancer malignancy progression. These results reveal a previously unrecognized role for Livin in regulating the tumor-initiating capacity in colon cancer and provide a novel treatment strategy in cancer via the interruption of H2A.XY39ph function and the interaction between H2A.XY39ph and JMJD6.

Abstract 1905: New targeted therapeutics for gastric cancer

Abstract Gastric cancer is a health disparity with 2.3 times higher incidence rates in Hispanics than in Non-Hispanic Whites (NHWs). Very few studies have been conducted to characterize this health disparity in Hispanics; however, studies from East Asian patients, where gastric cancer is also a health disparity, have revealed that p21-activated kinases (PAKs 1,2 and 4) are biomarkers for gastric cancer. Even though inhibition of PAKs impedes gastric cancer progression, current PAK inhibitors have failed to successfully complete clinical trials. Thus, we hypothesize that the inhibition of upstream effectors of PAK, the Rho GTPases Rac and Cdc42 is a rational treatment for gastric cancers with high PAK expression. The purpose of this study is to correlate Rac/Cdc42 and PAK expression and activation with Hispanic gastric cancer malignancy, and to test the efficacy of our recently developed Rac/Cdc42 inhibitor MBQ-167 (Humphries-Bickley, et al., Mol Cancer Therap. 2017, 16:805-818). In a related Aim, to identify new targets and biomarkers for Hispanic gastric cancer, gastric cancer tissues and normal gastric mucosa from biopsies and/or surgery were collected from Puerto Rican patients for expression profiling with customized qRT-PCR arrays for mRNA and Agilent arrays for non-coding RNAs (ncRNAs), and western blotting. Western blotting of gastric cancer cell lines revealed that NCI-N87 metastatic gastric cancer cells express more PAK isoforms and phospho (active)-PAK compared to the AGS non-metastatic gastric cancer cells; accordingly, MBQ-167 reduces the viability of NCI-N87 cells without affecting AGS cells. Therefore, to test the efficacy of Rac/Cdc42 inhibition in gastric cancer, immunocompromised mice with subcutaneous tumors from GFP-NCI-N87 cells were treated 3X week with vehicle or 1mg/kg BW of MBQ-167. Tumor growth calculated by image J analysis of fluorescent digital images. A drastic reduction in NCI-N87 tumor growth was observed in the MBQ-167 treated mice with a 61% inhibition in tumor size compared to the controls, and a tumor growth delay of &amp;gt;90% after the second doubling time. Gene expression profiling revealed that 13 genes were overexpressed (&amp;gt;1.5 fold change) in Puerto Rican gastric cancer tissue in comparison to the normal gastric mucosa, including Rac1, PAK1, STAT3, KDR (VEGFR2), CXCR4, IGF1, VEGFA, and IL-6, all of which are associated with Rac/Cdc42/Pak signaling. Differential profiling using a custom-made Agilent array for ncRNAs demonstrated upregulation of a number of pro-cancer microRNAs (miRNAs) such as miR-106b and downregulation of tumor suppressors such as let-7a and miR-145, as well as upregulation of the long ncRNA CCAT2, which we have shown to be important for colon cancer malignancy. Taken together, our data show that Hispanic gastric cancers have known and novel biomarkers, and MBQ-167 is a viable targeted therapeutic option for gastric cancers with elevated Rac/Cdc42/PAK signaling. Citation Format: Jean F. Ruiz Calderon, Marcia Cruz-Correa, George A. Calin, Linette Castillo-Pichardo, Suranganie Dharmawardhane. New targeted therapeutics for gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1905.

Abstract A44: Establishment of patient-derived Hispanic gastric cancer xenografts to test targeted therapeutics

Abstract Gastric cancer is a health disparity with 2.3 times higher incidence rates in Hispanics than in non-Hispanic Whites (NHWs). Very few studies have been conducted to characterize this health disparity in Hispanics; however, studies from East Asian patients, in whom gastric cancer is also a health disparity, have revealed that p21-activated kinases (PAKs 1,2 and 4), which are known to regulate cancer metastasis, are biomarkers for gastric cancer. Even though inhibition of PAKs impedes gastric cancer progression, current PAK inhibitors have failed to successfully complete clinical trials. Thus, we hypothesize that the inhibition of upstream effectors of PAK, the Rho GTPases Rac and Cdc42, is a rational treatment for gastric cancers with high PAK expression. The purpose of this study is to identify the Rac/Cdc42/PAK pathway as novel biomarkers for Hispanic gastric cancer and to test targeted therapeutics in patient-derived gastric cancer xenografts (PDGCXs). To achieve this aim, gastric cancer tissues and normal gastric mucosa from biopsies and/or surgery are being collected for expression profiling with customized qRT-PCR arrays for mRNA and Agilent arrays for noncoding RNAs (ncRNAs), and Western blotting. Using immunodeficient mice, xenografts have been established from the NCI-N87 gastric cancer cell line (from ATCC) and a tumor from a 58-year-old Hispanic (Puerto Rican) female (San Juan Oncology Hospital). As demonstrated by Western blotting, compared to the AGS nonmetastatic gastric cancer cell line, the NCI-N87 metastatic gastric cancer cells express more PAK isoforms and phospho (active)-PAK. We have also shown that our recently developed Rac/Cdc42 inhibitor MBQ-167 reduces the viability of the NCI-N87 cell line (Humphries-Bickley et al., Mol Cancer Therap 2017;16:805-18). Therefore, we are currently testing the effect of MBQ-167 in SCID mice with NCI-N87 xenografts. Gene expression profiling of the gastric cancer tissue from the Hispanic patient revealed that 13 genes were overexpressed (&amp;gt;1.5-fold change) in gastric cancer tissue in comparison to the normal gastric mucosa, including Rac1, PAK1, STAT3, KDR (VEGFR2), CXCR4, IGF1, VEGFA, and IL-6, all of which are associated with Rac/Cdc42/Pak signaling. Differential profiling using a custom-made Agilent array for ncRNAs demonstrated upregulation of a number of procancer microRNAs (miRNAs) such as miR-106b and downregulation of tumor suppressors such as let-7a and miR-145, as well as upregulation of the long ncRNA CCAT2, which we have shown to be important for colon cancer malignancy. Overall, our data show that a number of unique and established biomarkers are present in Hispanic gastric cancer, including elevated Rac/Cdc42/PAK signaling. Therefore, treatment with drugs targeting the Rac/Cdc42/PAK pathway represents a novel therapeutic option for such gastric cancers. In conclusion, modeling gastric cancer in PDGCXs is expected to enable the testing and design of targeted and individualized treatments for Puerto Rican patients. Citation Format: Jean F. Ruiz-Calderon, Marcia Cruz-Correa, George A. Calin, Suranganie Dharmawardhane. Establishment of patient-derived Hispanic gastric cancer xenografts to test targeted therapeutics [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A44.

Novel STAT binding elements mediate IL-6 regulation of MMP-1 and MMP-3

Dynamic remodelling of the extracellular matrix (ECM) is a key feature of cancer progression. Enzymes that modify the ECM, such as matrix metalloproteinases (MMPs), have long been recognised as important targets of anticancer therapy. Inflammatory cytokines are known to play a key role in regulating protease expression in cancer. Here we describe the identification of gamma-activated site (GAS)-like, signal transducer and activator of transcription (STAT) binding elements (SBEs) within the proximal promoters of the MMP-1 and MMP-3 genes, which in association with AP-1 components (c-Fos or Jun), bind STAT-1 in a homodimer like complex (HDLC). We further demonstrate that MMP expression and binding of this complex to SBEs can either be enhanced by interleukin (IL)-6, or reduced by interferon gamma (IFN-γ), and that IL-6 regulation of MMPs is not STAT-3 dependent. Collectively, this data adds to existing understanding of the mechanism underlying cytokine regulation of MMP expression via STAT-1, and increases our understanding of the links between inflammation and malignancy in colon cancer.

Loss of claudin-3 expression induces IL6/gp130/Stat3 signaling to promote colon cancer malignancy by hyperactivating Wnt/β-catenin signaling.

The Hyperactivated Wnt/β-catenin signaling acts as a switch to induce EMT and promote colorectal cancer. However, due to its essential role in gut homeostasis, therapeutic targeting of this pathway has proven challenging. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways and key mucosal barrier components in regulating colon tumorigenesis and cancer progression remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue specific role of claudin-3, a tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Loss of claudin-3 also predicted poor patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated role of the epigenetic regulation. Claudin-3−/− mice revealed dedifferentiated and leaky colonic epithelium, and developed invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3β independent manner, differentiated colon cancer in claudin-3−/− mice versus WT-mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune components. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/β-catenin activation, which is further exacerbated by Stat-3-activation and help promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy.

MiR-223 promotes colon cancer by directly targeting p120 catenin

microRNA (miRNA) dysregulation is frequently observed in colon cancer. Previous studies found that miR-223 is upregulated in colon cancer and functions as an oncogene. Conversely, p120 is often downregulated or even absent in colon cancer, and is a likely tumor suppressor. The present study showed that increased miR-223 and decreased p120 levels are associated with colon cancer malignancy, and p120 expression is negatively correlated with miR-223 expression. A dual luciferase reporter assay showed that miR-223 directly targets p120. miR-223 upregulation in a colon cancer cell line upregulated c-Myc, cyclinD1, MMP7, and vimentin expression, downregulated E-cadherin, increased nuclear expression of β-catenin, and enhanced RhoA activation. We suggest miR-223 may promote colon cancer cell invasion and metastasis by downregulating p120, thereby reducing intercellular adhesion, promoting RhoA activity, and activating β-catenin signaling. Thus miR-223 functions as an oncogene in colon cancer and may be a potential diagnostic and therapeutic target for anti-colon cancer treatment.

Abstract 1342: Loss of claudin-3 expression induces IL6/gp130/Stat3 signaling to promote colon cancer malignancy

Abstract Genetic and epigenetic aberrations drive the formation of a benign colorectal adenoma and its progression to the full-blown carcinoma. Hyperactivated Wnt/β-catenin signaling acts as a switch to induce EMT and promote colorectal cancer. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways in their capacity to communicate with mucosal barrier to promote colon cancer remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue specific role of claudin-3, tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Notably, in our analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (&amp;lt;250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p&amp;lt;0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p&amp;lt;0.05) of the greater levels of claudin-3 expression with patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated differential epigenetic regulation. In further studies, naïve claudin-3-/- mice revealed dedifferentiated {significant down regulation of P-27 (p&amp;lt;0.05) and vimentin expression versus WT mice} and leaky colonic epithelium. Moreover, claudin-3-/- mice demonstrated increased colon tumor burden and invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3β independent manner, characterized colon cancer in claudin-3-/- mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune organization. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/β-catenin activation in Stat-3-dependent manner to promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy. Citation Format: Rizwan Ahmad, Balawant kumar, Zhimin Chen, Steven(Xi) Chen, Dominik Muller, Subodh M. Lele, Mary Kay Washington, Surinder K. Batra, Punita Dhawan, Amar B. Singh. Loss of claudin-3 expression induces IL6/gp130/Stat3 signaling to promote colon cancer malignancy [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 1342. doi:10.1158/1538-7445.AM2017-1342

T lymphocyte SHP2-deficiency triggers anti-tumor immunity to inhibit colitis-associated cancer in mice.

Nonresolving inflammation is involved in the initiation and progression process of tumorigenesis. Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) is known to inhibit acute inflammation but its role in chronic inflammation-associated cancer remains unclear. The role of SHP2 in T cells in dextran sulfate sodium (DSS)-induced colitis and azoxymethane-DSS-induced colitis-associated carcinogenesis was examined using SHP2CD4−/− conditional knockout mice. SHP2 deficiency in T cells aggravated colitis with increased level of pro-inflammatory cytokines including IFN-γ and IL-17A. In contrast, the SHP2CD4−/− mice developed much fewer and smaller tumors than wild type mice with higher level of IFN-γ and enhanced cytotoxicity of CD8+ T cells in the tumor and peritumoral areas. At the molecular level, STAT1 was hyper-phosphorylated in T cells lacking SHP2, which may account for the increased Th1 differentiation and IFN-γ secretion. IFN-γ neutralization or IFN-γ receptor knockout but not IL-17A neutralization, abrogated the anti-tumor effect of SHP2 knockout with lowered levels of perforin 1, FasL and granzyme B. Finally, the expression of granzyme B was negatively correlated with the malignancy of colon cancer in human patients. In conclusion, these findings suggest a new strategy to treat colitis-associated cancer via targeting SHP2.

Abstract 1175: Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival

Abstract Dysregulation of colonocyte differentiation, imposing a crypt progenitor phenotype, characterize colorectal cancer (CRC) progression. Thus, an improved understanding of the molecular processes that regulate colonocyte differentiation can help identify novel therapeutic targets and biomarkers. In this regard, we found claudin-3, a tight junction integral protein, to be the highest expressed cell-cell adhesion moiety in the normal colonic epithelium and particularly concentrated amongst terminally differentiated colonocytes at the crypt top. We therefore postulated a key role for claudin-3 in maintaining colonocyte differentiation and negative association with colon tumorigenesis. In accordance, claudin-3 expression was low in 10 out of 14 CRC cells lines tested and undetectable in poorly differentiated and highly tumorigenic HCT116 and SW620 cell lines. In further analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (&amp;lt;250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p&amp;lt;0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p&amp;lt;0.001) of the greater levels of claudin-3 expression with patient survival. Genetic manipulation studies using colon cancer cells further supported a causal role of claudin-3 in upholding colonocyte differentiation, by modulating ZEB-1 protein synthesis and Wnt-signaling activation (Topflash promoter activity, p&amp;lt;0.05; claudin-3 siRNA versus control cells), and inhibiting cancer cell mobility and invasive ability. Importantly, homozygous deletion of claudin-3 expression in mice similarly inhibited colonocyte differentiation (down-regulated P-27 expression and up-regulated Vimentin expression), and promoted colon cancer growth and invasion when subjected to the AOM-DSS-induced mouse model of colorectal cancer (p&amp;lt;0.001 versus WT littermates). Pharmacological manipulations further revealed epigenetic regulation as potential mechanism for the inhibition of caludin-3 expression in CRC. Taken together, our data reveal a tissue-specific and inverse causal association of claudin-3 expression with CRC progression and patient survival, and highlights key importance of claudin-3 in maintaining colonocyte differentiation in Wnt-ZEB1-dependent manner. Citation Format: Rizwan Ahmad, Zhimin Chen, Balawant Kumar, Xi Chen, Mary kay Washington, Punita Dhawan, Amar B. Singh. Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival. [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 1175.

Abstract 4139: Role of Akt2 in cell survival and metastasis of colorectal cancer (CRC) cells

Abstract Background: Colorectal Cancer (CRC) is the second highest cause of cancer related deaths in United States. Metastatic dissemination of CRC cells to distant organs (primarily to the liver and lungs) makes the disease more lethal and incurable. Inappropriate cell survival signaling mechanisms contribute to successful colonization of cells at metastatic sites. Akt is a major cell survival-signaling node, which undergoes aberrant activation in various types of cancer. The Akt family has three members: Akt1, Akt2 and Akt3 which share structural homology and have similar mechanism of activation; however, differences in the phenotype of Akt1, Akt2 and Akt3 knock out mice suggest that they have non-redundant functional characteristics. Recently Akt2 has been identified as a significant contributor to colon cancer malignancy in addition to Akt1. Earlier reports have shown that knockdown of Akt2 results in reduced liver metastasis of CRC cells, however there are no studies related to the mechanism responsible for the reduction in metastasis. We observed a significant increase in the expression of Akt2 (microarray analysis) in liver metastatic samples formed by highly metastatic IGF1R-dependent CRC cells in comparison to the primary tumor. This was further confirmed by western blot and immunohistochemistry (IHC) analysis. Additionally, correlative studies using human primary colon carcinoma and corresponding liver metastatic samples showed a significant (p&amp;lt;0.001) increase in the expression of Akt2. These preliminary data led us to hypothesize that Akt2 may be playing a role in regulating cell survival and metastasis of CRC cells. Materials and Methods: IGF1R-dependent CRC cells (GEO and CBS) were used for the study. Two doxycycline inducible shRNAs for Akt2 were introduced by lentiviral transfection. Non-Targeting shRNA was used as control. Results: shRNA-mediated knockdown of Akt2 led to reduction in the expression of XIAP and survivin. We and others have shown that these IAPs (XIAP and survivin) are implicated to play an important role in cancer cell survival and metastasis. Furthermore, loss of Akt2 resulted in loss of pBad (S136), which is an essential cell survival marker. Loss of pBad results in increased cell death by PARP activation. Akt2 knockdown also affects cell survival by increasing p21 and decreasing the expression of cyclin E. PCR array analysis for metastatic genes revealed that a metastatic suppressor gene MTSS1 was 9 fold increased on knockdown of Akt2. It has been shown that MTSS1 inhibits metastasis by affecting cell junction assembly and stability. Conclusion: We have shown that loss of Akt2 promotes cell death and inhibits cell proliferation. This is the first study, which suggests the mechanism of Akt2 mediated cell death by XIAP and survivin downregulation. These results suggest that Akt2 inhibits metastasis by increasing the expression of MTSS1. Citation Format: Ekta Agarwal. Role of Akt2 in cell survival and metastasis of colorectal cancer (CRC) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4139. doi:10.1158/1538-7445.AM2015-4139