Β-catenin In Colorectal Cancer Research Articles

The RNA helicase DDX21 activates YAP to promote tumorigenesis and is transcriptionally upregulated by β-catenin in colorectal cancer

The RNA helicase DDX21 is vital for ribosome biogenesis and is upregulated in CRC, but the mechanism by which DDX21 is dysregulated and by which DDX21 promotes tumorigenesis in CRC remains poorly understood. Here, we showed that DDX21 is a direct transcriptional target gene of β-catenin and mediates the protumorigenic function of β-catenin in CRC. DDX21 expression is correlated with the expression and activity of β-catenin, and high DDX21 expression is associated with a poor prognosis in CRC patients. Loss of DDX21 leads to cytoplasmic translocation and decreased transcriptional activity of YAP and suppresses the proliferation and migration of CRC cells, which can be partially rescued by YAP reactivation. Importantly, by using translation elongation inhibitors and DNA intercalators, we showed that ribosomal stress upregulates DDX21 expression and induces the downregulation of LATS and the activation of YAP, probably through the ZAKα-MKK4/7-JNK axis. Overall, our study revealed the transcriptional activation mechanism of DDX21 in CRC and the activation of YAP in the ribosomal stress response, indicating the potential of combination therapy involving the induction of ribosomal stress and YAP inhibition.

The DEAD-box RNA helicase DDX5 (p68) and β-catenin: The crucial regulators of FOXM1 gene expression in arbitrating colorectal cancer

Forkhead box M1 (FOXM1), a vital member of the Forkhead box family of transcription factors, helps in mediating oncogenesis. However, limited knowledge exists regarding the mechanistic insights into the FOXM1 gene regulation. DDX5 (p68), an archetypal member of the DEAD-box family of RNA helicases, shows multifaceted action in cancer progression by arbitrating RNA metabolism and transcriptionally coactivating transcription factors. Here, we report a novel mechanism of alliance between DDX5 (p68) and the Wnt/β-catenin pathway in regulating FOXM1 gene expression and driving colon carcinogenesis. Initial bioinformatic analyses highlighted elevated expression levels of FOXM1 and DDX5 (p68) in colorectal cancer datasets. Immunohistochemical assays confirmed that FOXM1 showed a positive correlation with DDX5 (p68) and β-catenin in both normal and colon carcinoma patient samples. Overexpression of DDX5 (p68) and β-catenin increased the protein and mRNA expression profiles of FOXM1, and the converse correlation occurred during downregulation. Mechanistically, overexpression and knockdown of DDX5 (p68) and β-catenin elevated and diminished FOXM1 promoter activity respectively. Additionally, Chromatin immunoprecipitation assay demonstrated the occupancy of DDX5 (p68) and β-catenin at the TCF4/LEF binding element (TBE) sites on the FOXM1 promoter. Thiostrepton delineated the effect of FOXM1 inhibition on cell proliferation and migration. Colony formation assay, migration assay, and cell cycle data reveal the importance of the DDX5 (p68)/β-catenin/FOXM1 axis in oncogenesis. Collectively, our study mechanistically highlights the regulation of FOXM1 gene expression by DDX5 (p68) and β-catenin in colorectal cancer.

Wnt signaling modulator DKK4 inhibits colorectal cancer metastasis through an AKT/Wnt/β-catenin negative feedback pathway

Aberrant activation of the Wnt/β-catenin signaling pathway is implicated in most malignant cancers, especially in the initiation and progression of colorectal cancer (CRC). DKK4 is a classical inhibitory molecule of the Wnt/β-catenin pathway, but its role in CRC is ambiguous, and the molecular mechanism remains unclear. Here, we determined DKK4 expression was significantly upregulated in 23 CRC cell lines and 229 CRC tissues when analyzed by quantitative PCR and immunohistochemistry, respectively. Our analysis of tissue samples indicated the survival time of CRC patients with high DKK4 expression was longer than that of patients with medium-low DKK4 expression. We examined the effects of DKK4 on cell proliferation and metastasis by cell counting kit-8 assays, transwell assays, and subcutaneous and metastatic mouse tumor models, and we discovered that DKK4 silencing promoted the metastasis of CRC cells both invitro and invivo. Our RNA-seq analysis revealed that AKT2, FZD6, and JUN, which play important roles in AKT and Wnt signaling, were significantly increased after DKK4 knockdown. DKK4 represses Wnt/β-catenin signaling by repressing FZD6 and AKT2/s552 β-catenin in CRC. Further experiments revealed recombinant Wnt3a and LiCl could induce DKK4 expression. Moreover, our bioinformatics analysis and luciferase reporter assays identified posttranscriptional regulators of DKK4 in CRC cells. In summary, DKK4 is elevated in CRC and inhibits cell metastasis by a novel negative feedback mechanism of the Wnt3a/DKK4/AKT/s552 β-catenin regulatory axis to restrict overactivation of Wnt activity in CRC. Therefore, DKK4 restoration may be applied as a potential CRC therapeutic strategy.

KCTD9 inhibits the Wnt/β-catenin pathway by decreasing the level of β-catenin in colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer mortality worldwide. However, the molecular mechanisms underlying CRC progression remain to be further defined to improve patient outcomes. In this study, we found that KCTD9, a member of the potassium channel tetramerization domain-containing (KCTD) gene family, was commonly downregulated in CRC tissues and that KCTD9 expression was negatively correlated with the clinical CRC stage. Survival analysis showed that patients whose tumors expressed low KCTD9 levels had poorer outcomes. Functional analyses revealed that KCTD9 overexpression inhibited CRC cell proliferation and metastasis, whereas KCTD9 knockdown promoted CRC cell proliferation and metastasis in both in vitro and in vivo models. Manipulating KCTD9 levels in CRC cells via overexpression or knockdown showed KCTD9 expression positively influenced the degradation of β-catenin levels leading to inhibition of Wnt signaling and reductions in Wnt pathway target gene expression. Mechanistically, we found KCTD9 associated with ZNT9 (Zinc Transporter 9), a coactivator of β-catenin-mediated gene transcription. The overexpression of KCTD9 or knockdown of ZNT9 in CRC cells increased the polyubiquitination and proteasomal degradation of β-catenin. In turn, the KCTD9-ZNT9 interaction disrupted interactions between β-catenin and ZNT9, thereby leading to decreased β-catenin target gene expression and the inhibition of Wnt signaling. In conclusion, our findings propose that KCTD9 functions as a tumor suppressor that inhibits CRC cell proliferation and metastasis by inactivating the Wnt/β-catenin pathway. Moreover, its frequent downregulation in CRC suggests KCTD9 as a potential prognostic and therapeutic target in CRC.

PGM3 regulates beta-catenin activity to promote colorectal cancer cell progression.

The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis. However, the regulation mechanisms of HBP and its role in colorectal cancer (CRC) progression remain unexplained. This study analyzed the expression level of phosphoglucomutase 3 (PGM3), a key enzyme in HBP, and identified its function in CRC cell lines. Analysis of publicly available CRC microarray data determined that PGM3 is upregulated in CRC tumor tissues. Furthermore, functional experiments emphasized the significant roles of PGM3 in facilitating CRC cell proliferation and migration. Mechanistically, we demonstrated that the activity of β-catenin in CRC was maintained by PGM3-mediated O-GlcNAcylation. PGM3 knockdown or inhibition of O-GlcNAc transferase decreased β-catenin activity and the expression levels of its downstream targets. Collectively, our findings indicate that PGM3 exhibits tumor-promoting roles by elevating O-GlcNAcylation level and maintaining β-catenin activity, and might serve as a prognostic biomarker and treatment target in CRC.

KCTD9 Inhibits the Wnt/β-Catenin Pathway by Decreasing the Level of β-Catenin in Colorectal Cancer

KCTD9 Inhibits the Wnt/β-Catenin Pathway by Decreasing the Level of β-Catenin in Colorectal Cancer

The involvement of miR-150/β-catenin axis in colorectal cancer progression

Colorectal cancer remains as a serious global cause of morbidity and mortality. The current therapies for colorectal cancer treatment are still unsatisfactory and thus, identification of novel targets is an urgent requisite. Recent evidence has reported miRNAs are closely correlated with colorectal cancer development. miR-150 has been identified in tumor progression in various cancers. Nevertheless, its roles in colorectal cancer remain poorly known. In our study, a decreased miR-150 expression in colorectal cancer tissues and cells was observed. Meanwhile, we reported a negative correlation between miR-150 and β-catenin in colorectal cancer. β-catenin can participate in the physiological mechanism of many types of cancers. Then, miR-150 was overexpression in SW480 and HT-29 cells and it was show that miR-150 repressed SW480 and HT-29 cell viability, proliferation and colony formation capacity. Moreover, colorectal cancer cell progression was triggered by the inhibition of miR-150 via negatively regulating β-catenin. Subsequently, the direct binding correlation between miR-150 and β-catenin was demonstrated. β-catenin, c-myc and CyclinD1 level was significantly restrained by the up-regulation of miR-150 in SW480 and HT-29 cells. Finally, it was proved that miR-150 depressed colorectal cancer growth through modulating β-catenin in vivo. Overall, it was implied that miR-150 could inhibit colorectal cancer progression and serve as a tumor suppressor via inactivating β-catenin pathway.

Intrinsic β-catenin signaling suppresses CD8+ T-cell infiltration in colorectal cancer

Colorectal cancer is the third most common cancer worldwide and shows resistance to immune checkpoint inhibitors which have been demonstrated to be effective in many other types of cancers. Pre-existing T-cell response in tumor microenvironment often determines the therapeutic benefit of immune checkpoint blockade. Tumor-infiltrating CD8+ T-cells are considered as the major effector immune cells in antitumor immunity. In this study, we aimed to identify the intrinsic oncogenic pathway that contributes to a reduction of CD8+ T-cell infiltration in colorectal cancer. To achieve this, human colon adenocarcinoma samples derived from The Cancer Genome Altas (TCGA) were stratified into low T-cell-inflamed and high T-cell-inflamed groups based on the expression of T-cell signature genes. Gene set enrichment analysis of revealed a close correlation between activation of the Wnt/β-catenin signaling pathway and absence of T-cell infiltration. By immunohistochemical analysis of 155 colorectal cancer tissues, we found that tumors with high β-catenin expression showed a significant reduction of CD8+ T-cell infiltration. Mechanistically, β-catenin can regulate CCL4 expression to recruit CD103+ dendritic cells to enable CD8+ T cell activation. Collectively, our data indicate that oncogenic β-catenin signal may mediate colorectal cancer resistance to immunotherapies, pointing to the combined PD-1-immunotherapy with targeting β-catenin in colorectal cancer.

Abstract 2428: Redox regulation of β-catenin in colorectal cancer

Abstract Colorectal cancer (CRC) is the second most newly diagnosed cancer in the United States. β-catenin is the downstream effector of the Wnt/β-catenin signaling pathway. Loss of function (LOF) mutations on adenomatous polyposis coli (APC), a critical member of the β-catenin degradation complex leads to constitutively elevated β-catenin as an early oncogenic event in 85% of CRCs and is associated with decreased CRC patient survival. Increased level of β-catenin augments the nuclear accumulation of β-catenin, where it acts a co-activator of a number of transcription factors leading to the expression of downstream target genes that promote cancer cell proliferation, survival, and migration. There is no effective therapy for β-catenin as a cause of cancer. In addition to CRC, elevated levels of Wnt signaling have been reported in leukemia, melanoma, and breast cancers highlighting the need for novel therapies that attenuate aberrant β-catenin mediated transcription. Here we report on the redox dependency of the Wnt/β-catenin signaling pathway, identified through a forward genetic screen in Drosophila, and identify thioredoxin reductase-1 (TXNR1) as a potential target for the inhibition of aberrant β-catenin transcriptional activity through in vivo studies with the APCmin/+ mouse model. Drosophila harboring a mutation in their thioredoxin reductase (trxr-1) gene and a partial lethality phenotype were used in an enhancer/suppressor forward genetic screen. Increased lethality was observed when the trxr-1 mutation (trxr-1481) was combined with heterozygous mutations and deletions in wnt2, while wnt2 deletion and mutations alone had no effect on lethality. To test the translational value of these findings, APCmin/+ mice were treated with PX-12, a human TXNR1 inhibitor, daily by gavage from day 40 to time of death. We observed a dose dependent reduction in the number and size of intestinal polyps by PX-12 treatment, and an increased survival in treated mice. Furthermore, mechanistic studies utilizing human CRC cell lines harboring LOF mutations to the β-catenin degradation complex revealed β-Catenin mediated transcription, as measured by TOP Flash luciferase assay, was diminished when TXNR1 was silenced by siRNA. Additionally, we observed decreased β-catenin protein level in CRC cell lines treated with PX-12 or siRNA targeting TXNRD1 and its functional partner thioredoxin (TXN). The addition of the pan caspase inhibitor ZVAD but not the proteasome inhibitor MG132 was able to stabilize β-catenin protein upon treatment with PX-12 suggesting β-catenin destabilization is occurring via a caspase dependent mechanism. These results suggest a conserved mechanism for the redox regulation of β-catenin involving TXNR1; furthermore, inhibition of TXNR1 activity destabilizes β-catenin protein and decreases β-catenin mediated transcription in human CRC cell lines and inhibits intestinal neoplasia development in an experimental mouse model for CRC. Citation Format: Alejandro D. Campos, Brian James, Petrus De Jong, Robert Lemos, Nikolas Marino, Garth Powis. Redox regulation of β-catenin in colorectal 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 2428.

GSK-3β inhibitor 6-bromo-indirubin-3'-oxime promotes both adhesive activity and drug resistance in colorectal cancer cells.

Multi-targets inhibitor 6-bromo-indirubin-3'-oxime (BIO) has diverse biological effects on cancer cells. The key component of the β-catenin destruction complex glycogen synthase kinase 3β (GSK-3β), one of the major target for BIO, polyubiquitination and degradation of the main oncoprotein β-catenin in colorectal cancer (CRC). In the present study, we evaluated the effect of BIO on drug resistance and biological properties of CRC cells. Whole-genome transcriptional profiling revealed that differentially expressed genes were mainly centered on well-characterized signaling pathways including stem cell, cell adhesion and cell growth in BIO-treated CRC cells. BIO treatment downregulated migration and invasion abilities of CRC cells, accompanying with MMP-9 downregulated and E-cadherin upregulated CRC cells. BIO treatment decreased apoptosis induced by 5-Fu/DDP in CRC SW480 cells. In addition, BIO treatment reversed the 5-Fu-induced CD133+ cell downregulation trend in CRC SW620 cells. After incubation with BIO, the expression levels of EpCAM, TERT and DCAMKL-1 proteins were upregulated in CRC cells. BIO treatment downregulated the activity of GSK-3β, upregulated and transported β-catenin to the nucleus in CRC cells. Our findings reveal that BIO treatment upregulated stemness, adhesive and chemoresistance of CRC cells. GSK-3β inhibition and WNT/β-catenin activation by BIO, may partly result in the biological behavior alterations in CRC cells.

Expression of regulatory factor R-spondin family in Wnt signaling pathway in colorectal cancer and its clinical significance

To explore the expression of R-spondin family in colorectal cancer tissues and adjacent tissues, and to evaluate its relationship with clinic-pathological stage. Methods: A total of 64 samples of colorectal cancer tissues and adjacent tissues were collected from the patients, who received radical surgery in Xiangya Hospital, Central South University between January 2014 and August 2014. The mRNA and protein expression levels of R-spondin 1-4 and β-catenin in the colorectal cancer tissues and adjacent tissues were detected by qRT-PCR and immunohistochemistry. The relationship between the expression level of R-spondin 1-4 and the clinic-pathological factors were analyzed to explore the correlation between the expression level of R-spondin 1-4 and β-catenin in colorectal cancer. Results: Compared with the adjacent tissues, the mRNA and protein expression levels of R-spondin 1 were elevated in the colorectal cancer tissues (P<0.05). The mRNA and protein expression levels of R-spondin 2-4 were increased in the colorectal cancer tissues than those in the normal tissues (P<0.05), but there was no significant difference between the colorectal cancer tissues and adjacent tissues (P>0.05). The expression level of R-spondin 1 was positively correlated with the nuclear expression of β-catenin in the colorectal cancer tissues (r=0.6307, P<0.05). Conclusion: Compared with the adjacent tissues, the mRNA and protein expression levels of R-spondin 1 are significantly elevated in the colorectal cancer tissues. R-spondin 1 may play a role in promoting carcinogenesis by regulating the activity of β-catenin in the downstream of Wnt signaling pathway.

The lncRNA H19 Promotes Cell Proliferation by Competitively Binding to miR-200a and Derepressing β-Catenin Expression in Colorectal Cancer.

H19, a paternally imprinted noncoding RNA, has been found to be overexpressed in various cancers, including colorectal cancer (CRC), and may function as an oncogene. However, the mechanism by which H19 regulates CRC progression remains poorly understood. In this study, we aimed to assess H19 expression levels in CRC tissues, determine the effect of H19 on CRC proliferation, and explore the mechanism by which H19 regulates the proliferation of CRC. We measured H19 expression using qRT-PCR and analysed the effects of H19 on colon cancer cell proliferation via cell growth curve, cell viability assay, and colony formation assays. To elucidate the mechanism underlying these effects, we analysed the interactions between H19 and miRNAs and identified the target gene to which H19 and miRNA competitively bind using a series of molecular biological techniques. H19 expression was upregulated in CRC tissues compared with adjacent noncancerous tissues. H19 overexpression facilitated colon cancer cell proliferation, whereas H19 knockdown inhibited cell proliferation. miR-200a bound to H19 and inhibited its expression, thereby decreasing CRC cell proliferation. β-Catenin was identified as a target gene of miR-200a. H19 regulated β-catenin expression and activity by competitively binding to miR-200a. H19 promotes cell proliferation by competitively binding to miR-200a and derepressing β-catenin in CRC.

Expression of μ-protocadherin is negatively regulated by the activation of the β-catenin signaling pathway in normal and cancer colorectal enterocytes.

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity.

Expression and clinical significance of SALL4 and β-catenin in colorectal cancer.

Previous studies on the expression of Sal-like 4 (SALL4), a zinc finger transcription factor, had conflicting results in colorectal cancer (CRC). The main aim of this study was to investigate the expression of SALL4 and its relationship with β-catenin in CRC. Immunohistochemistry was performed to examine the expression of SALL4 and β-catenin in a cohort of 149 patients with CRC, 12 with atypical hyperplasia, 25 with benign tumor and 38 with normal tissue. Expression patterns of SALL4 and β-catenin and correlation with clinicopathological features were investigated. The relationship between SALL4 and β-catenin was examined by immunofluorescence and co-immunoprecipitation using CRC cell lines, SW480, SW620, HCT116 and HT29. Immunohistochemical analysis revealed significantly lower expression of SALL4 in CRC (46.3 %) than atypical hyperplasia (68.0 %, p < 0.05) and normal tissue (78.9 %, p < 0.01). Well-differentiated CRC seemed to express more SALL4 (47.6 %) than moderately (45.8 %) and poorly-differentiated cancers (18.8 %) (p < 0.05). However, SALL4 expression positively correlated with lymph node metastasis and tumor-node-metastasis (TNM) and Dukes stages (all p < 0.05) suggesting a new mechanism involved in the function of SALL4 in CRC. β-catenin was expressed significantly higher in CRC (69.1 %) than normal tissue (21.1 %, p < 0.01), and positively correlated with CA19-9 level in serum (p < 0.05). SALL4 and β-catenin were positively correlated in CRC (Spearman correlation coefficient R = 0.536, p < 0.01). The group of co-expression of the two molecules showed advanced lymph node metastasis, TNM stage and Dukes stage (all p < 0.05). Double-labeling immunofluorescence and co-immunoprecipitation indicated that SALL4 and β-catenin co-localized in the nucleus and cytoplasm and interacted. Taken together, our results revealed that SALL4 and β-catenin were positively correlated in CRC. In CRC cells, SALL4 and β-catenin co-localized and interacted. The function of SALL4 in promoting lymph node metastasis and advanced clinical stage might partly be due to the interaction with β-catenin.

Hormone suppression silencing GUCY2C is required for colorectal cancer

Colorectal cancer is the fourth most common cancer and the second leading cause of cancer death. Transformation begins with activation of Wnt signaling through mutations in APC (80%) or its degradation target β-catenin (15%), producing a gain-of-function in TCF-dependent nuclear transcription underlying epithelial dysfunction and tumorigenesis. While a role for APC and β-catenin in colorectal cancer is well-established, steps leading from gene mutation to tumorigenesis, and their reversibility, remain incompletely defined. Guanylin is the paracrine hormone in colorectum for the receptor GUCY2C. This hormone is the most commonly lost gene product in colorectal cancer, and its universal suppression and associated silencing of GUCY2C at the earliest step in neoplasia contributes to tumorigenesis in a mechanism that is conserved across species. GUCY2C regulates homeostatic mechanisms organizing the intestinal crypt-surface axis, and its silencing through guanylin suppression drives hyperproliferation, DNA damage, metabolic reprogramming and desmoplasia contributing to tumorigenesis. Beyond this dysregulation of homeostatic mechanisms contributing to transformation, we recently discovered that guanylin suppression is required for tumorigenesis induced by mutant APC-β-catenin, reflecting a role for GUCY2C in β-catenin degradation that blocks tumorigenesis. Thus, inactivation of APC, or activation of β-catenin, induces TCF-dependent elimination of guanylin transcription and translation in human intestinal cells in vitro and in conditional genetic mouse models in vivo. Conversely, activation of GUCY2C induces cGMP protein kinase-dependent elimination of wild type or mutant β-catenin by amplifying proteosomal degradation, even in the context of mutations which inactivate the APC degradation complex. Importantly, enforced genetic expression of guanylin in intestinal epithelial cells completely eliminated tumorigenesis in all mouse models of colorectal cancer examined. These observations reveal a pathophysiologic model in which mutant APC-β-catenin signaling eliminates guanylin expression as an obligatory step in tumorigenesis. In turn, silencing GUCY2C reversibly disrupts epithelial homeostatic processes corrupted in tumorigenesis, and removes an essential block to transformation, creating a circuit which amplifies mutant APC-β-catenin signaling. These studies shift the prevailing paradigm for colorectal tumorigenesis from an irreversible oncogenomic mechanism to a reversible functional mechanism whose reconstitution abrogates those mutational defects. Indeed, they define a novel molecular pathway that is obligatory for tumorigenesis, to which the transformation process is addicted. In that context, they highlight how this addiction creates a unique disease-specific vulnerability that can be leveraged to eliminate tumorigenesis by GUCY2C hormone replacement. We have translated these observations to an NCI-funded clinical program exploring the utility of oral GUCY2C ligands to prevent colorectal cancer.

Abstract 3529: BET bromodomain proteins regulate the canonical WNT signaling and drug resistance in colorectal cancer

Abstract The canonical WNT signaling is aberrantly activated in the vast majority of colorectal cancer (CRC) and results in activation of beta-catenin and its downstream target MYC. These two oncogenic transcription factors play central roles in CRC pathogenesis, but are refractory to direct pharmacological interventions. The recurrent mutations of APC or β-catenin in CRC also limit the utilities of currently available WNT inhibitors that mostly target upstream regulators. The bromodomain and extra-terminal domain (BET) family epigenetic readers recently emerged as a key regulator of MYC expression. In the present study, we demonstrated that BET proteins regulated beta-catenin-mediated MYC expression in CRC. Inhibition of BET proteins repressed activities of both wild type and constitutively active β-catenin mutants. Pharmacological or genetic targeting of BET proteins broadly impaired proliferation and survival in CRC cells, including KRAS or BRAF-mutant lines. Furthermore, BET bromodomain inhibitors synergistically sensitized CRC cells to inhibitors of the MAPK pathway. The ability of BET inhibitors to augment the efficacy of the MAPK pathway inhibitors is potentially secondary to altered balance among pro-survival and pro-apoptotic regulators. Using KRAS- or BRAF-mutant CRC xenograft models, we showed that a combination of BET inhibitor and MEK inhibitor, but not either agent alone, induced significant tumor regression. Our findings suggest that BET-based epigenetic therapy can be utilized as an alternative approach to block the canonical WNT signaling in CRC and potentially other human cancers, in particular those carrying APC or beta-catenin mutations. Our results also identify a novel combination therapy for treating CRCs, including those refractory to currently available therapeutic options. Citation Format: Lihong Wang, Yufang Ma, Ethan Lee, Jialiang Wang. BET bromodomain proteins regulate the canonical WNT signaling and drug resistance in colorectal cancer. [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 3529. doi:10.1158/1538-7445.AM2015-3529

LEF1 and B9L Shield β-Catenin from Inactivation by Axin, Desensitizing Colorectal Cancer Cells to Tankyrase Inhibitors

Hyperactive β-catenin drives colorectal cancer, yet inhibiting its activity remains a formidable challenge. Interest is mounting in tankyrase inhibitors (TNKSi), which destabilize β-catenin through stabilizing Axin. Here, we confirm that TNKSi inhibit Wnt-induced transcription, similarly to carnosate, which reduces the transcriptional activity of β-catenin by blocking its binding to BCL9, and attenuates intestinal tumors in Apc(Min) mice. By contrast, β-catenin's activity is unresponsive to TNKSi in colorectal cancer cells and in cells after prolonged Wnt stimulation. This TNKSi insensitivity is conferred by β-catenin's association with LEF1 and BCL9-2/B9L, which accumulate during Wnt stimulation, thereby providing a feed-forward loop that converts transient into chronic β-catenin signaling. This limits the therapeutic value of TNKSi in colorectal carcinomas, most of which express high LEF1 levels. Our study provides proof-of-concept that the successful inhibition of oncogenic β-catenin in colorectal cancer requires the targeting of its interaction with LEF1 and/or BCL9/B9L, as exemplified by carnosate.

LincRNA-p21 enhances the sensitivity of radiotherapy for human colorectal cancer by targeting the Wnt/β-catenin signaling pathway

Recent studies show that long intergenic noncoding RNA-p21 (lincRNA-p21) is aberrantly expressed in several types of cancer, including colorectal cancer (CRC), one of the most common cancers in the world. Radiotherapy is considered as a standard preoperative treatment approach to reduce local recurrence for local advanced rectal cancer. However, a considerable number of rectal cancers are resistant to radiotherapy. In the present study, we evaluated the role of lincRNA‑p21 in radiotherapy for CRC and detected the possible molecular mechanism. By expression profile analysis, we demonstrated that lincRNA-p21 decreases in CRC cell lines and tissue samples, which contributes to the elevation of β-catenin in CRC. We further showed that lincRNA‑p21 increases following X-ray treatment, and enforced expression of the lincRNA enhances the sensitivity of radiotherapy for CRC by promoting cell apoptosis. Suppression of the β-catenin signaling pathway and elevation of the pro-apoptosis gene Noxa expression may help explain the role of lincRNA-p21 in CRC radiotherapy. The present study not only deepens our understanding of the mechanism of radiotherapy for CRC, but it also provides a potential target for CRC radiotherapy.

Expression of β-Catenin and Cyclooxygenase 2 in Colorectal Carcinoma: An Immunohistochemical Study

The Wnt/β-catenin pathway plays an important role in the genesis of familial adenomatous polyposis, the most common form of inherited colorectal carcinoma (CRC). Also, the inflammatory bowel diseases (IBDs) predispose to cancer development; and cyclooxygenase 2 (COX-2) seems to be pivotal in their pathogenesis. This study aimed to investigate the relationship between the expression of COX-2 protein and β-catenin in colorectal cancer. The study enrolled 45 patients, all of whom underwent surgery and immunohistochemical staining of tissue specimens for COX-2 and β-catenin was done. Correlation between the two modulators and their relationship with clinicopathological features were examined. In 34 cases (75.56%) of the tumor samples; β-catenin immunoreactivity was found in the cytoplasm and/or membrane compartment. On the other hand, COX-2 immunoreactivity was weakly and/or strongly positive in 32 cases (71.11%) and negative in 13 (28.89%). Positivity was detected in the cytoplasm and in the perinuclear area. Increased expression of β-catenin was correlated to Duke stage (P=0.009). Furthermore, nuclear β-catenin localization showed a correlation to the Duke stage (P=0.029) and insignificant correlation with distant metastases (P = 0.336). Positive COX-2 expression showed a significant relation to, liver metastases (P = 0.042), and Duke stage (P = 0.011) and insignificant correlation to lymph node invasion (P=0.25). These data indicate that cytoplasmic/membrane β-catenin over-expressions as well as positive COX-2 expressions are associated with a more aggressive behavior of the disease.

Significance of combined expression of MUC1 and β-catenin in colorectal cancer

Objective To investigate the clinical significance of combined expression of mucin 1 （MUC1） and β-catenin in colorectal cancer. Methods Ninety colorectal cancer specimens were subjected to immunohistochemistry for detection of MUC1 and β-catenin. The relationships of expression patterns of these two biomarkers and clinicopathological parameters and survival were analyzed. Results Loss of polarity distribution of MUC1 was detected in 54. 44% cases and was related to depth of invasion （ P 〈0. 05）, and nuclear translocation of β-catenin was found in 58. 89% cases and correlated with TNM stage （P 〈 0. 05 ）. The combined expression of MUC1 and nuclear β-catenin was related to lymph node metasta sis and TNM stage （ P 〈 0. 01 ） and also, a significantly lower survival rate （ P 〈 0. 05 ）. Conclusion Combined expression of MUC1 and nuclear β-catenin indicated a worse prognosis in colorectal cancer. Key words: Colorectal carcinoma; MUC1 ; β-catenin