Tumor Suppressor In Colorectal Carcinoma Research Articles

LINC00294/miR-620/MKRN2 axis provides biomarkers and negatively regulates malignant progression in colorectal carcinoma.

Colorectal carcinoma (CRC) ranks the third most frequent malignancy worldwide. Makorin RING zinc finger-2 (MKRN2) has been identified as a tumor suppressor in CRC, and the bioinformatics prediction indicated that some non-coding RNAs (ncRNAs) that directly or indirectly regulate MKRN2 might play critical roles in CRC progression. This study aimed to analyze the regulatory effect of LINC00294 on CRC progression, and to explore the underlying mechanisms by assessing miR-620 and MKRN2. The potential prognostic value of the ncRNAs and MKRN2 was also investigated. The expression of LINC00294, MKRN2, miR-620 was examined by qRT-PCR. Cell counting kit-8 assay was used to assess the proliferation of CRC cells. Transwell assay was used to evaluate the migration, invasion of CRC cells. Kaplan-Meier method and log-rank test were used to perform comparative analysis of overall survival in CRC patients. Lower expression of LINC00294 was observed in both CRC tissues and cell lines. In CRC cells, LINC00294 overexpression inhibited cell proliferation, migration and invasion, but these effects were directly reversed by the overexpression of miR-620, which was demonstrated as a target of LINC00294. Additionally, MKRN2 was found to be a target gene of miR-620, and might mediate the regulatory function of LINC00294 in CRC progression. In CRC patients, low LINC00294, MKRN2 and high miR-620 expression was associated poor overall survival of CRC. LINC00294/miR-620/MKRN2 axis had the potential to provide prognostic biomarkers for CRC patients, and negatively regulated the malignant progression of CRC cells, including proliferation, migration and invasion.

MiRNA-212 acts as a tumor-suppressor in colorectal carcinoma through targeting SOX4.

Colorectal cancer is a common gastrointestinal cancer, with mortality ranking the third all over the world. MicroRNA-212 (miR-212), located on chromosome 17p13.3, was lowly expressed in a variety of tumors. The purpose of our study was to explore the effects of miR-212 on colorectal cancer (CRC) cells. Quantitative Real Time-Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blot were employed to evaluate the levels of mRNAs and proteins. The transwell assay was performed to calculate the abilities of migration and invasion. The Luciferase reporter assay was utilized to verify miR-212 targeting to the 3'-UTR of SOX4 mRNA. Statistical analysis was applied to analyze the experimental data. MiR-212 was lowly expressed in CRC tissues and cell lines, while the expression of SRY-box 4 (SOX4) was overexpressed. Exogenous increasing of miR-212 or knockdown of SOX4 inhibited the migration, invasion and epithelial-mesenchymal transition (EMT) in CRC cells. Moreover, the expression of miR-212 had a negative connection with SOX4 expression, and miR-212 mediated the expression of SOX4 by directly binding to the 3'-UTR of SOX4 mRNA. In addition, low expression of miR-212 or overexpression of SOX4 was associated with poor prognosis of colorectal cancer patients. MiR-212 inhibited the migration, invasion and EMT by direct targeting to the 3'-UTR of SOX4 mRNA in colorectal cancer. The newly identified miR-212/SOX4 axis provides novel insight for colorectal treatment.

TREM2 Acts as a Tumor Suppressor in Colorectal Carcinoma through Wnt1/β-catenin and Erk Signaling.

TREM2 (triggering receptor expressed on myeloid cells) is involved in the development of malignancies. However, the function of TREM2 in colorectal cancer has not been clearly elucidated. Here, we investigated TREM2 function for the first time in colorectal epithelial cancer cells and demonstrated that TREM2 is a novel tumor suppressor in colorectal carcinoma. Blockade of TREM2 significantly promoted the proliferation of HT29 colorectal carcinoma cells by regulating cell cycle-related factors, such as p53 phosphorylation and p21 and cyclin D1 protein levels. HT29 cell migration was also increased by TREM2 inhibition via MMP9 (matrix metalloproteinase 9) expression upregulation. Furthermore, we found that the tumor suppressor effects of TREM2 were associated with Wnt/β-catenin and extracellular signal-regulated kinase (ERK) signaling. Importantly, the effect of TREM2 in the suppression of tumor development was demonstrated by in vivo and in vitro assays, as well as in human colon cancer patient tissue arrays. Overall, our results identify TREM2 as a potential prognostic biomarker and therapeutic target for colorectal cancer.

SATB2-AS1 Suppresses Colorectal Carcinoma Aggressiveness by Inhibiting SATB2-Dependent Snail Transcription and Epithelial-Mesenchymal Transition.

Accumulating evidence suggests that long noncoding RNA (lncRNA) plays important regulatory roles in cancer biology. However, the involvement of lncRNA in colorectal carcinoma progression remains largely unknown, especially in colorectal carcinoma metastasis. In this study, we investigated the changes in lncRNA expression in colorectal carcinoma and identified a new lncRNA, the antisense transcript of SATB2 (SATB2-AS1), as a key regulator of colorectal carcinoma progression. SATB2-AS1 was frequently downregulated in colorectal carcinoma cells and tissues, and patients whose tumors expressed SATB2-AS1 at low levels had a shorter overall survival and poorer prognosis. Downregulation of SATB2-AS1 significantly promoted cell proliferation, migration, and invasion in vitro and in vivo, demonstrating that it acts as a tumor suppressor in colorectal carcinoma. SATB2-AS1 suppressed colorectal carcinoma progression by serving as a scaffold to recruit p300, whose acetylation of H3K27 and H3K9 at the SATB2 promoter upregulated expression of SATB2, a suppressor of colorectal carcinoma growth and metastasis. SATB2 subsequently recruited HDAC1 to the Snail promoter, repressing Snail transcription and inhibiting epithelial-to-mesenchymal transition. Taken together, these data reveal SATB2-AS1 as a novel regulator of the SATB2-Snail axis whose loss facilitates progression of colorectal carcinoma. SIGNIFICANCE: These data show that the lncRNA SATB2-AS1 mediates epigenetic regulation of SATB2 and Snail expression to suppress colorectal cancer progression.See related commentary by Li, p. 3536.

MicroRNA-202-5p functions as a tumor suppressor in colorectal carcinoma by directly targeting SMARCC1

Recently, microRNAs (miRNAs) have been emerged as critical regulators for human diseases and as prognostic markers in several tumors, including colorectal carcinoma (CRC). Herein, we identified a tumor-suppressive miRNA, miR-202-5p, which may suppress CRC tumorigenesis. SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily c member 1 (SMARCC1) is a susceptibility gene in CRC. However, the role of SMARCC1 in CRC tumorigenesis has not been elucidated. In our present study, we demonstrated that miR-202-5p was a tumor-suppressive miRNA in CRC progression. We found that expression of miR-202-5p was obviously decreased in CRC tissues. Down-regulation of miR-202-5p was associated with postoperative survival. Overexpression of miR-202-5p inhibited the growth and metastasis of CRC cells. The SMARCC1 was a direct target of miR-202-5p and promoted the growth and metastasis of CRC cells. Further study showed that SMARCC1 could reverse the inhibitory effect of miR-202-5p on growth and metastasis of CRC cells. In conclusion, our data highlight the key role of miR-202-5p in the progression of CRC. Thus, miR-202-5p may be a potential prognostic marker and of treatment relevance for CRC progression intervention.

Reduced RCE1 expression predicts poor prognosis of colorectal carcinoma

BackgroundAs an end-proteolytic enzyme that cleaves the last three residues of proteins with a terminal CAAX, Ras-converting enzyme 1 (RCE1) has an essential role in multiple signaling pathways and take part in the process of differentiation, proliferation and carcinogenesis. The aim of the study is to investigate expression pattern of RCE1 and its prognosis in colorectal carcinoma (CRC).MethodsThe expression of RCE1 and phospho-MAPK family members was confirmed by immunohistochemical staining of CRC tissues. miR-RCE1 lentiviral vectors were transduced into HCT116 and SW489 cells. Reverse transcription PCR (RT-PCR) and western blot were conducted to measure the transfection efficiency. Transwell assays were used to detect the invasiveness of CRC cells.ResultsIn the present study, we assessed RCE1 expression in 244 CRC specimens and matching adjacent, non-tumorous tissues by immunohistochemistry (IHC). Compared with the matched adjacent non-tumor tissue samples, the RCE1 reduced in the tumor tissue samples (p < 0.001). RCE1 expression was significantly decreased in 106 of 244 (43.4%) CRC cases. In univariate and multivariate analyses, Decreasing expression of RCE1 independently predicts poor prognosis for patients in both overall survival and disease-free survival. Further study indicated that RCE1 influenced tumor invasion through the p38 pathway. Knockdown of RCE1 reduced phosphorylation and significantly increased the invasive capacity of CRC cells.ConclusionTaken together, the outcomes of this study indicate that RCE1 acts as a tumor suppressor in CRC, as its reduced expression may increase CRC cell invasion and independently predict an unsatisfactory prognosis in CRC patients.

CBX4 Suppresses Metastasis via Recruitment of HDAC3 to the Runx2 Promoter in Colorectal Carcinoma.

Polycomb chromobox (CBX) proteins participate in the polycomb repressive complex (PRC1) that mediates epigenetic gene silencing and endows PRC1 with distinct oncogenic or tumor suppressor functions in a cell-type-dependent manner. In this study, we report that inhibition of cell migration, invasion, and metastasis in colorectal carcinoma requires CBX4-mediated repression of Runx2, a key transcription factor that promotes colorectal carcinoma metastasis. CBX4 inversely correlated with Runx2 expression in colorectal carcinoma tissues, and the combination of high CBX4 expression and low Runx2 expression significantly correlated with overall survival, more so than either CBX4 or Runx2 expression alone. Mechanistically, CBX4 maintained recruited histone deacetylase 3 (HDAC3) to the Runx2 promoter, which maintained a deacetylated histone H3K27 state to suppress Runx2 expression. This function of CBX4 was dependent on its interaction with HDAC3, but not on its SUMO E3 ligase, its chromodomain, or the PRC1 complex. Disrupting the CBX4-HDAC3 interaction abolished Runx2 inhibition as well as the inhibition of cell migration and invasion. Collectively, our data show that CBX4 may act as a tumor suppressor in colorectal carcinoma, and strategies that stabilize the interaction of CBX4 with HDAC3 may benefit the colorectal carcinoma patients with metastases. Cancer Res; 76(24); 7277-89. ©2016 AACR.

Reduced expression of EphA5 is associated with lymph node metastasis, advanced TNM stage, and poor prognosis in colorectal carcinoma.

Colorectal carcinoma (CRC) is the third most common cancer and a major cause of morbidity and mortality throughout the world. The prognosis of patients has improved markedly over the last 15 years because of the introduction of new therapy including molecular target drugs. To comprehensively understand the molecular process of carcinogenesis of colorectal carcinoma is essential for the diagnosis, prognosis and treatment. EphA5 is a member of the Eph family and plays a critical role in carcinogenesis of lung cancer, prostate cancer, and breast cancer. The expression profile and the role of EphA5 in colorectal carcinoma have not been well investigated till now. In this study, a set of colorectal carcinoma specimens was subjected to immunohistochemical assay using an EphA5 specific antibody. The relationship between the expression of EphA5 and clinicopathological parameters was statistically analyzed. EphA5 was positively expressed in all tested normal mucosa specimens (120/120, 100%) and partly in colorectal carcinoma specimens (70/120, 58.3%). The loss of EphA5 protein was associated with depth of wall invasion (P=0.002), poor tumor differentiation (P<0.001), lymph node metastasis (P<0.001), and advanced TNM stage (P<0.001). The survival analysis showed that patients with reduced expression of EphA5 had a poor overall survival (P=0.017). Our data indicate that EphA5 receptor may be a tumor suppressor in colorectal carcinoma and it may be a new therapeutic target for colorectal carcinoma.

The Role of RUNX3 in Colorectal Tumor Progression

Objectives: RUNX3 (runt-related-transcription-factor3) is a putative tumor suppressor in colorectal carcinoma (CRC) and other cancers. Recent evidence also suggests that RUNX3 localization to the cytoplasm may play an oncogenic role. We investigated the role of cytoplasmic and nuclear expression of RUNX3 in CRC tumor progression from adenoma to carcinoma. Methods: We compared expression of RUNX3 in CRC and tubulovillous adenomas (TVAs) in …

Recurrent, truncating SOX9 mutations are associated with SOX9 overexpression, KRAS mutation, and TP53 wild type status in colorectal carcinoma.

PurposeThe extent to which the developmental transcription factor SOX9 functions as an oncogene or tumor suppressor in colorectal carcinoma (CRC) is debatable. We aimed to clarify the effect of SOX9 mutations on SOX9 protein expression and their association with known molecular subtypes and clinical characteristics in advanced CRC.Experimental DesignNext generation sequencing data (MSK-IMPACT) from CRC patients was used to interrogate SOX9, KRAS, NRAS, BRAF, TP53, APC, and PIK3CA. Mutant and wild type (WT) SOX9 cases underwent immunohistochemical (IHC) staining to assess protein expression. SOX9 allele-specific copy number was assessed by Affymetrix Oncoscan array.ResultsSOX9 was mutated in 38 of 353 (10.7%) CRC, of which 82% were frameshift or nonsense. Compared to SOX9 WT, SOX9 mutation was strongly associated with coexistent mutant KRAS (p=0.0001) and WT TP53 (p=0.0004). SOX9 was overexpressed in both SOX9 mutant and WT CRC. Among SOX9 mutants, the highest expression was noted for truncating exon 3 mutants (mean H scores 239±105 versus 147±119, p value=0.02). Further, SOX9 truncating mutants with loss of the WT allele demonstrated protein overexpression indicating the WT protein was not required for protein stabilization.ConclusionsSOX9 is overexpressed in CRC, including those with recurrent distal truncating mutations. The latter has structural similarity to the oncogenic isoform MiniSOX9, which is distally truncated due to aberrant splicing. This information suggests that truncated SOX9 has oncogenic features. SOX9 mutations are highly enriched in KRAS mutant and TP53 wild type CRC; and may provide a therapeutic target in approximately 11% of CRC.

MicroRNA-9 suppresses cell migration and invasion through downregulation of TM4SF1 in colorectal cancer.

Transmembrane-4-L6 family 1 (TM4SF1) is upregulated in colorectal carcinoma (CRC). However, the mechanism leading to inhibition of the TM4SF1 is not known. In the present study, we investigated the regulation of TM4SF1 and function of microRNAs (miRNAs) in CRC invasion and metastasis. We analyzed 60 colon cancers and paired normal specimens for TM4SF1 and miRNA-9 (miR-9) expression using quantitative real-time PCR. A bioinformatics analysis identified a putative miR-9 binding site within the 3'-UTR of TM4SF1. We also found that TM4SF1 was upregulated in CRC tissues and CRC cell lines. The expression of TM4SF1 was positively correlated with clinical advanced stage and lymph node metastasis. Moreover, a luciferase assay revealed that miR-9 directly targeted 3'-UTR-TM4SF1. Overexpression of miR-9 inhibited expression of TM4SF1 mRNA and protein, wound healing, transwell migration and invasion of SW480 cells, whereas, overexpression of anti-miR-9 and siRNA-TM4SF1 inversely regulated the TM4SF1 mRNA and protein level in HCT116 cells. Furthermore, miR-9 suppressed not only TM4SF1 expression but also MMP-2, MMP-9 and VEGF expression. In clinical specimens, miR-9 was generally down-regulated in CRC and inversely correlated with TM4SF1 expression. These results suggest that miR-9 functions as a tumor-suppressor in CRC, and that its suppressive effects mediate invasion and metastasis by inhibition of TM4SF1 expression. Our results also indicate that miR-9 might be a novel target for the treatment of CRC invasion and metastasis.

FAM172A is a tumor suppressor in colorectal carcinoma

The present study was designed to elucidate the regulatory role of a novel protein FAM172A in carcinogenesis of colorectal carcinoma (CRC). Investigation of clinical samples using Western blotting showed that expression of FAM172A is significantly lower in cancerous tissues than in adjacent tissues. Furthermore, we constructed in vitro model for continuous overexpression and silencing of FAM172A with a retroviral vector system. FAM172A suppressed the proliferative and invasive potentials of LOVO cells as shown in MTT test, transwell migration assay, wound healing assay, 3D-culture morphologic study, and xenograft experiment. RT-PCR and Western blotting showed that FAM172A overexpression inhibited expressions of Cyclin D1, CDK2, MMP-2, MMP-9, PERK, elF2α, ATF6, XBP1, and GRP78, while FAM172A silencing induced their expressions. FAM172A might regulate ERS through PERK-elF2α, ATF6-XBP1-GRP78 signal pathway. The results implicated that FAM172A functioned as a tumor suppressor in colorectal carcinoma.

Decreased ARID1A expression facilitates cell proliferation and inhibits 5-fluorouracil-induced apoptosis in colorectal carcinoma.

AT-rich interactive domain 1A (ARID1A) is a key member of the SWI/SNF chromatin-modeling complex, and the gene has emerged as a tumor suppressor in various human cancers. In the present study, we investigated the expression pattern of ARID1A in human colorectal carcinoma. We found that ARID1A expression was decreased in colorectal carcinoma compared with normal tissue. Loss of ARID1A significantly correlated with poor differentiation (p = 0.0009). We also explored the involvement of ARID1A in the biological behavior of colorectal cancer cell lines. ARID1A overexpression by plasmid transfection in SW620 cell line inhibited proliferation and facilitated 5-fluorouracil-induced apoptosis. ARID1A depletion by siRNA in SW480 cell line promoted proliferation ability and inhibited 5-fluorouracil-induced apoptosis. Furthermore, we found that ARID1A regulated the activity of Akt signaling pathway. In conclusion, our data suggested that ARID1A serves as an important tumor suppressor in colorectal carcinoma and regulates proliferation and chemoresistance of colorectal cancer cells.

MiR-155 regulates the proliferation and cell cycle of colorectal carcinoma cells by targeting E2F2.

MicroRNAs play important roles in carcinogenesis by negatively regulating the expression of target genes. Here we explore the biological function of miR-155 and the underlying mechanism in colorectal carcinoma. We validate, for the first time, that E2F2 is a direct target of miR-155 using western blot and a luciferase reporter assay and that miR-155 regulates the proliferation and cell cycle of colorectal carcinoma cells by targeting E2F2 using siRNA technology. We also found, for the first, time that E2F2 acts as a tumor suppressor in colorectal carcinoma. Overall, miR-155 plays an important role in colorectal carcinoma tumorigenesis by negative regulation of its targets including E2F2 and may be a potential therapeutic target for colorectal carcinoma treatment.

Peroxisome proliferator-activated receptor γ-mediated induction of microRNA-145 opposes tumor phenotype in colorectal cancer

MicroRNAs (miRNAs) regulate diverse biological processes by inhibiting translation or inducing degradation of target mRNAs. miR-145 is a candidate tumor suppressor in colorectal carcinoma (CRC). Colorectal carcinogenesis involves deregulation of cellular processes controlled by a number of intertwined chief transcription factors, such as PPARγ and SOX9. Since PPAR family members are able to modulate complex miRNAs networks, we hypothesized a role of miRNA-145 in the interaction between PPARγ and SOX9 in colorectal carcinogenesis.To address this issue, we evaluated gene expression in tissue specimens of CRC patients and we took advantage of invitro models represented by CRC derived cell lines (CaCo2, SW480, HCT116, and HT-29), employing PPARγ activation and/or miRNA-145 ectopic overexpression to analyze how their interplay impact the expression of SOX9 and the development of a malignant phenotype. ResultsPPARγ regulates the expression of miR-145 by directly binding to a PPAR response element (PPRE) in its promoter at −1207/−1194bp from the transcription start site. The binding is essential for miR-145 upregulation by PPARγ upon rosiglitazone treatment. Ectopic expression of miR-145, in turn, regulates SOX9 expression through the binding to specific seed motifs. The PPARγ-miR-145-SOX9 axis overarches cell cycle progression, invasiveness and differentiation of CRC derived cell lines.Together, these results suggest that miR-145 is a novel target of PPARγ, acts as a tumor suppressor in CRC cell lines and is a key regulator of intestinal cell differentiation by directly targeting SOX9, a marker of undifferentiated progenitors in the colonic crypts.

Abstract 1521: Role of the guanylate-binding-protein 1 (GBP-1) in immunoediting of colorectal carcinoma.

Abstract The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by IFN-γ in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), we previously reported that the expression of GBP-1 in the cells of the desmoplastic stroma correlates with the presence of an IFN-γ-dominated Th1 microenvironment and with an increased cancer-related 5-year survival. Moreover, GBP-1 expression in the blood vessels of CRC tissue was associated with a reduced angiogenesis. However, the expression and the role of GBP-1 in the tumor cells of CRC remained unknown. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating a reduced inducibility in colorectal tumor cells. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in IBD. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration, invasion and anchorage-independent growth. Using RNA interference, we showed that GBP-1 mediates the anti-proliferative, anti-migratory, anti-invasive and anti-clonogenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether these results suggested that GBP-1 acts directly as a tumor suppressor in CRC by mediating the effects of IFN-γ. In addition, the loss of GBP-1 expression in tumor cells indicated that tumor evasion from the IFN-γ-dominated Th1 immune response might have occured. This was confirmed by showing, first, that the absence of GBP-1 inducibility in CRC cell lines correlates with resistance to the anti-proliferative and pro-apoptotic effects of IFN-γ and, second, that the resistant cell lines exhibit defects in the IFN-γ signaling pathway. Citation Format: Nathalie Britzen-Laurent, Karoline Lipnik, Matthias Ocker, Elisabeth Naschberger, Vera Schellerer, Roland Croner, Michael Vieth, Maximilian Waldner, Pablo Steinberg, Christine Hohenadl, Michael Stürzl. Role of the guanylate-binding-protein 1 (GBP-1) in immunoediting of colorectal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2013-1521

GBP-1 acts as a tumor suppressor in colorectal cancer cells

The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.

Clinico-pathological significance of TNF alpha-induced protein3 (TNFAIP3) in Middle Eastern colorectal carcinoma

A20, also known as TNF-alpha-induced protein3 (TNFAIP3), is a well-known negative regulator of the NF-kB activation pathway (Coornaert et al.2009; Stilo et al.2008) and functions as a tumor suppressor gene in lymphomas (Honma et al.2009; Kato et al.2009). Aberrant NF-kB regulation in colorectal carcinoma (CRC) is associated with poor prognosis and resistance to therapy (Horst et al.2009; Kojima et al.2004; Rakitina et al.2003; Scartozzi et al.2007). The current study deals with an interesting and novel topic about oncogenic role of inactivation of tumor suppressor gene A20 in colorectal cancer, the mechanism of A20 dysfunction, and its potential as a novel prognostic marker and therapeutic target. This is the first report of A20 alterations in colon cancer where we comprehensively investigated the genomic profile and epigenetic changes in A20 gene. A better understanding of NF-kB modulators could lead to development of effective therapeutic strategies to target activated NF-kB (Rakitina et al.2003). Mechanistically, A20 exerts its NF-kB inhibitory role in TNF signaling by the cooperative and dual-functioning actions of the deubiquitinating and ubiquitinating enzyme activities located at the N and C terminus, respectively, of the A20 protein (Coornaert et al.2009). The amino terminal of A20 protein containing the deubiquitinating enzyme targets removal of ubiquitin moieties from receptor-interacting protein (RIP1), an essential mediator of the proximal tumor necrosis factor receptor signaling complex in NF-kB activation (Wertz et al.2004). This is followed by polyubiquitination of RIP by the ubiquitin ligase located at the carboxy terminal of A20 protein, thereby targeting RIP for proteosomal degradation. Recent studies have shown that A20 functions as a tumor suppressor gene in several types of lymphoma wherein not only is A20 inactivated by promoter methylation (Chanudet et al.2009), but it is also subject to gene deletion and mutation. Although A20 has been reported as a novel tumor suppressor gene in NHLs, the role of A20 in colorectal carcinoma is not yet known. In this study, we screened for A20 gene copy number changes and A20 expression in a large tissue microarray cohort of 434 colorectal carcinomas and adenoma samples. We further investigated the incidence of A20 mutations and A20 promoter methylation and the relationship of A20 alterations with CRC progression and phenotype. The incidence of A20 mutations, A20 deletions, A20 promoter hyper-methylation was 2.5%, 15.3%, and 50.8%, respectively. We analyzed the entire coding region of A20 gene in 116 CRC samples and 14 CRC cell lines and found that only three CRC samples and two CRC cell lines (LOVO and HCT-15) were mutated. Most of the A20 mutations found in Hodgkin's and mediastinal lymphomas were nonsense or frameshift mutations that prevented production of full-length A20 protein (Chanudet et al.2009; Honma et al.2009; Schmitz et al.2009). The three A20 mutations found in our study were replacement mutations. Two of these three mutations in our study were localized in the zinc finger (ZF) domains at the C terminus of the protein. The A20 ZF domain functions as an E3 ubiquitin ligase, adding K48-linked polyubiquitin chains to RIP1 and targeting the protein for proteasomal degradation. Most of the mutations seen in the three CRC samples as well as the two cell lines were missense mutations (four out of five). A20 deletions were seen in 15.3% (65/424) of the CRC samples analyzed by FISH. Homozygous deletions, i.e., presence of two centromeric signals but no A20 signals, were seen in four of the 65 (3.1%) deleted cases. Although A20 mutations were not observed in the CRC with A20 deletions, six cases showed co-existence of A20 methylation and A20 deletions. Thus, A20 alterations are widely prevalent in colorectal carcinoma, and A20 promoter methylation leads to reduced expression of A20 protein. A20 promoter hyper-methylation was correlated with loss of protein expression and associated with histology subtype of adenocarcinomas differentiated tumors and CRC located on the left side. A20 protein expression was significantly higher in colorectal adenomas as compared to colorectal carcinomas. A20 expression identifies a subgroup of colorectal cancers which show a poor outcome across all subgroups, in stage III alone, stage II and III subgroup, and also stage III and IV subgroup. Reduced A20 expression was an independent prognostic marker in colorectal carcinoma. Together, our results establish A20 as a key tumor suppressor molecule in colorectal carcinogenesis and identify a subgroup of CRC patients with reduced A20 expression showing a poor overall survival. A20 promoter methylation is the key epigenetic mechanism leading to suppression of A20 expression. Interestingly, complete loss of A20 expression results from multiple hits like A20 deletion and or mutation with methylation in accordance with Knudson's two-hit hypothesis. Our findings indicate that A20 is a tumor suppressor in CRC that can be used as a potential prognostic biomarker.