Nuclear Translocation Of Beta-catenin Research Articles

A reappraisal of transcriptional regulation by NR5A1 and beta-catenin in adrenocortical carcinoma.

Overexpression of the transcription factor NR5A1 and constitutive activation of canonical Wnt signalling leading to nuclear translocation of beta-catenin are hallmarks of malignancy in adrenocortical carcinoma (ACC). Based on the analysis of genomic profiles in H295R ACC cells, Mohan etal. (Cancer Res. 2023; 83: 2123-2141) recently suggested that a major determinant driving proliferation and differentiation in malignant ACC is the interaction of NR5A1 and beta-catenin on chromatin to regulate gene expression. I reanalyzed the same set of data generated by Mohan etal. and other published data of knockdown-validated NR5A1 and beta-catenin target genes. Beta-catenin is mainly found in association to canonical T cell factor/lymphoid enhancer factor (TCF/LEF) motifs in genomic DNA. NR5A1 and beta-catenin regulate distinct target gene sets in ACC cells. Overall, my analysis suggests a model where NR5A1 overexpression and beta-catenin activation principally act independently, rather than functionally interacting, to drive ACC malignancy.

Localization of beta catenin across the domain of odontogenic lesions: A systematic review.

CTNNB1 gene encodes beta catenin, a transcriptional activator of Wnt pathway involved in the pathogenesis of odontogenic lesions. Though located intramembranously, its translocation into cytoplasm and nucleus could trigger cell proliferation, inhibition of apoptosis, invasion and migration of the tumour cell. Five electronic databases including MEDLINE by PubMed, Google scholar, Scopus, Trip, Cochrane library and EMBASE until 1 January 2023 without period restriction were thoroughly searched. Those articles that identified CTNNB1 mutation and beta catenin in odontogenic lesions were included for review. Risk of bias was analysed for each study using QUADAS 2 tool and Review Manager 5.3 was used to output its result. Thirty four published articles were included for data synthesis. A total of 1092 cases of odontogenic lesions were assessed for both CTNNB1 mutation and beta catenin expression. CTNNB1 mutation was observed in ameloblastoma, calcifying odontogenic cyst, calcifying cystic odontogenic tumour and all malignant odontogenic tumours. The beta catenin expression (nuclear and cytoplasmic) was maximum in odontogenic keratocyst and calcifying odontogenic cyst. The expression was variable in ameloblastomas, membranous in odontomas, calcifying cystic odontogenic tumour and nuclear in all malignant tumours. High recurrence of odontogenic keratocyst and aggressiveness of solid ameloblastoma and malignant odontogenic tumours could be associated with the nuclear translocation of beta catenin. Disparity between CTNNB1 mutation and beta catenin expression within odontogenic lesions suggests alternate routes of beta catenin activation. The review results support the unique localisation of beta catenin as a helpful diagnostic factor in the pathogenesis of odontogenic lesions.

Nuclear translocation of beta catenin in patients with Rathke cleft cysts—clinical and imaging characteristics and risk of recurrence

PurposeAlthough Rathke cleft cysts (RCC) are benign lesions of the sellar region, recurrence is frequent after surgical treatment. Nuclear translocation of ß-catenin (NTßC), a key effector of the wnt-signaling pathway that is responsible for cell renewal, has been shown to act as a proto-oncogene and is considered to be a potential risk factor for increased recurrence in RCC. In this study, we analyzed a surgically treated cohort into patients with and without NTßC expression in order to identify clinical and imaging differences and further evaluate the risk of recurrence.MethodsPatients with resection of RCC between 04/2001 and 11/2020 were included. Histological specimens were immunohistochemically stained for ß-catenin. Study endpoints were time to cyst recurrence (TTR) and functional outcome. Functional outcome included ophthalmological and endocrinological data. Furthermore, MRI data were assessed.ResultsSeventy-three patients (median age 42.3 years) with RCC underwent mainly transsphenoidal cyst resection (95.9%), 4.1% via transcranial approach. Immunohistochemical staining for ß-catenin was feasible in 61/73 (83.6%) patients, with nuclear translocation detected in 13/61 cases (21.3%). Patients with and without NTßC were equally likely to present with endocrine dysfunction before surgery (p = 0.49). Postoperative new hypopituitarism occurred in 14/73 (19.2%) patients. Preoperative visual impairment was equal in both groups (p = 0.52). Vision improved in 8/21 (33.3%) patients and visual field deficits in 22/34 (64.7%) after surgery. There was no difference in visual and perimetric outcome between patients with and without NTßC (p = 0.45 and p = 0.23, respectively). On preoperative MRI, cyst volume (9.9 vs. 8.2 cm3; p = 0.4) and evidence of hemorrhage (30.8% vs. 35.4%; p = 0.99) were equal and postoperative cyst volume decreased significantly in both groups (0.7 vs. 0.5 cm3; p < 0.0001 each). Cyst progression occurred in 13/73 (17.8%) patients after 39.3 ± 60.3 months. Cyst drainage with partial removal of the cyst wall resulted in improved recurrence-free survival without increasing the risk of complications compared with cyst fenestration alone. Patients with postoperative diabetes insipidus had an increased risk for recurrence according to multivariate analysis (p = 0.005). NTßC was evident in 4/15 patients (26.7%) and was not associated with a higher risk for recurrence (p = 0.67).ConclusionTransnasal transsphenoidal cyst drainage with partial removal of the cyst wall reduces the risk of recurrence without increasing the risk of complications compared with fenestration of the cyst alone. Patients with postoperative diabetes insipidus seem to have an increased risk for recurrence. In contrast, NTßC was not associated with a higher risk of recurrence and did not provide stratification for clinically distinct patients.

Novel Recognition of HER2 Amplification in Enteroblastic Colonic Adenocarcinoma. Diagnostic Clues and Future Perspectives

Enteroblastic differentiation in sporadic colonic adenocarcinoma was a rare event that lacks to be fully elucidated. It was characterized by an aggressive biological behavior, with early metastatic spread and poor prognosis. Diagnostic clues and molecular features were not completely elucidated so far. Histological features including tubulo-papillary architecture, cleared neoplastic cells, fetal oncoproteins expression were diagnostic requirements. We describe a new case of enteroblastic colonic adenocarcinoma (CAED) in the sigmoid, exhibiting small intestinal phenotype, higher stage at diagnosis and early liver metastases. At molecular level, CAED shared both TP53 gene mutation and beta-catenin nuclear translocation with conventional colonic adenocarcinoma. Altered human epidermal growth factor receptor (HER) 2 signaling has been described in a small subset of conventional colonic adenocarcinoma. At best of our knowledge our report represents the first immunohistochemical recognition of HER2 gene amplification in CAED. Given its aggressive clinical behavior, clinicians should be aware of the possible HER2 amplification, that could point toward further treatment options.

The novel NSAID-derivative, MCI-030, prevents ovarian cancer in the egg-laying hen by increasing tumor cell apoptosis and decreasing beta-catenin and MAPK oncogenic signaling

Objectives: The egg-laying hen is an ideal model to study chemoprevention of epithelial ovarian cancer (EOC) as aging hens have a high rate of spontaneous EOC and similar clinicopathological characteristics of the disease to women. Our previous research revealed that the novel non-COX inhibitory NSAID-derivative, MCI-030, decreased ovarian cancer incidence in the hen. MCI-030 inhibits PDE10A, which was shown to be an effective therapeutic target in colon and lung cancers, reducing beta-catenin and MAPK signaling. Molecular analysis of MCI-030 treated hens was performed to study the mechanism by which PDE10A inhibition can reduce ovarian cancer risk. Methods: A total of 225 White leghorn hens aged 2.5 to 3 years old were treated through their feed with MCI-030 or its parent compound, sulindac, for 6- and 12-months and compared to a control group (Figure 1A). Necropsy was performed at each time point to formally assess for tumors with 60 hens evaluated at 6-months and 85 at 12-months. Logistic regression with Firth correction to estimate effect size for this pilot study was performed. Ovary and oviduct tissues were harvested and formalin-fixed or flash-frozen then paraffin-embedded or homogenized, respectively. Molecular analyses were performed using fluorescent immunohistochemistry, TUNEL assays, and western blotting. Results: A trend of decreased risk of malignancy was observed in the MCI-030 group at 6- and 12-months versus control with odds ratios (OR) of 0.56 and 0.75, respectively (95% CI 0.121-2.633, 0.22-2.58). In contrast, this trend was not observed for sulindac (6-month OR=1.56, 95% CI 0.405-6.06, 12-month OR=0.94, 95% CI 0.29-3.03). Assessment of apoptosis in hen ovaries by TUNEL assay demonstrated that MCI-030 significantly increased apoptosis both overall and in ovarian tumors (Figure 1B). Western blotting revealed that pERK was significantly decreased in ovaries of sulindac and MCI-030 hens (Figure 1C). Immunohistochemistry showed that MCI-030 significantly decreased nuclear translocation of beta-catenin and levels of active beta-catenin in ovarian tumors (Figure 1D). Download : Download high-res image (224KB) Download : Download full-size image Conclusions: MCI-030 appears to be a promising agent for the chemoprevention and treatment of ovarian cancer. Molecular analysis of its efficacy in hens revealed that MCI-030 increases tumor apoptosis and decreases pERK and beta-catenin oncogenic signaling. Further investigation of MCI-030 in additional model systems and in-human trials is warranted.

Most colitis associated carcinomas lack expression of LGR5: a preliminary study with implications for unique pathways of carcinogenesis compared to sporadic colorectal carcinoma

BackgroundLeucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a component of the Wnt receptor complex, is thought to lineage label gastric and intestinal stem cells. LGR5 expression is increased in colorectal carcinoma (CRC) compared to normal tissue. Colitis associated colorectal adenocarcinoma (CAC) often shows distinct morphologic and molecular phenotypes compared to sporadic cases. However, the expression profile of LGR5, and by extension the potential role of an intestinal stem cell phenotype, has not been well described in a series of human CAC.MethodRNA in situ hybridization (ISH) for LGR5 expression on 30 CACs (12 cases with conventional morphology and 18 cases with non-conventional type morphology) from 29 inflammatory bowel disease (IBD) patients was performed and compared the expression profile to a control group of 10 sporadic CRCs. Immunohistochemistry for beta-catenin and SATB2 was performed on the 30 CACs.ResultLGR5 was positive in 30% (9/30) of CAC cases and 90% (9/10) of sporadic CRCs (p = 0.002). A large majority (89%) of LGR5 positive CACs were of the conventional histologic type, and conventional type CAC showed a significantly higher LGR5 score (median 3.0; interquartile range 1.75–3.25) than non-conventional type CAC (median 1.5; interquartile range 1.00–2.00) (p = 0.034). CAC with conventional morphology did have a lower level of LGR5 expression than sporadic CRC. Sporadic CRCs showed a significantly higher LGR5 level score than non-conventional type CACs (p < 0.001). Nuclear translocation of beta-catenin was strongly associated with LGR5 expression (p = 0.003), however no significant association was identified between SATB2 expression and LGR5 expression status in CACs.ConclusionThese findings suggest that the wider spectrum of tumor morphology in CAC may be associated with absence of a LGR5-expressing intestinal stem cell phenotype.

Immunohistochemical analysis of beta-catenin, E-cadherin and p53 in canine gastrointestinal epithelial tumors.

Wnt/beta-catenin signaling, E-cadherin and p53 reportedly play important roles in the development and/or progression of human gastrointestinal cancer. The present study evaluated the roles of beta-catenin, E-cadherin and p53 in canine gastrointestinal tumors. Endoscopic biopsy or surgically resected samples, a total of 131, including 38 gastric, 13 small intestinal and 80 large intestinal tumors, were obtained from 95 dogs. Those specimens were examined pathologically. Immunohistochemically, nuclear beta-catenin expression was found in 88% (42/48) of polypoid type adenocarcinomas. Most cases of non-polypoid type adenocarcinomas lacked nuclear expression of beta-catenin with the exception of one case (6%, 1/17). Nuclear beta-catenin expression was not observed in signet ring cell carcinomas (0/15), mucinous adenocarcinomas (0/7) and undifferentiated carcinomas (0/4). The findings indicate that nuclear translocation of beta-catenin is closely related to the development of polypoid type adenocarcinomas but not that of non-polypoid type malignant tumors. The immunoreactivity of E-cadherin for tumor cells tended to decline overall in most of cases including benign tumors. Significant immunoreactivity for p53 was not found in 61% of tumors examined (80/131), including malignant tumors (63%, 57/91), while intense p53-immunoreactivity was rarely found in a few cases of malignant tumors (8%, 7/91). We could not conclude clearly significant correlations between histopathological tumor types and immunohistochemical results of E-cadherin or p53. This paper indicates the importance of the nuclear translocation of beta-catenin for the tumorigenesis of canine intestinal polypoid type adenocarcinomas, especially in the colorectum.

Abstract 3595: DSP-0692, a selective SCD inhibitor, enhances the anti-tumor activity of anti-PD1 antibody

Abstract SCD (Stearoyl-CoA Desaturase), a key enzyme for lipid metabolism, is overexpressed in various types of cancer and plays an important role in cancer cell proliferation. We have previously shown that the identification and the functional characterization of DSP-0692, a novel and selective SCD inhibitor. SCD expression was induced in tumor cells cultured in sphere conditions, which were resistant to conventional agents within therapeutic concentration. DSP-0692 showed strong growth inhibition when tumor cells were cultured in sphere conditions, but had little impact on cells cultured under adherent conditions. In addition, DSP-0692 decreased the expression of stemness-related genes in tumor cells as well as in tumor tissues. Treatment with DSP-0692 suppressed tumor growth in multiple models both in single agent or in combination with conventional chemotherapies. DSP-0692 also inhibited WNT/beta-catenin signaling pathway through two different mechanisms (inhibition of Wnt3A secretion and nuclear translocation of beta-catenin). DSP-0692 exhibited lower IC50 value to sphere cells carrying mutations related to WNT/beta-catenin signaling pathway than those without these mutations. Blockade of the coinhibitory checkpoint molecule PD-1 has emerged as an effective treatment for many cancers. However, despite successes in the clinical setting, only a limited subpopulation respond to PD-1 blockade. We hypothesized that the deregulated lipid metabolism of tumor cells and microenvironment function as a barrier to antitumor immunity, and therefore that normalization of lipid metabolism with DSP-0692 might restore response to immunotherapy. Combination of DSP-0692 with anti-PD-1 antibody in the syngeneic CT-26 colon model, a resistant model to anti-PD-1 antibody, augmented tumor regressions relative to each single agent alone. DSP-0692 induced PD-L1 and PD-L2 expression in CT-26 cells, indicating that it is one of a mechanism of restoration of the effect of anti-PD-1 antibody. Our data suggest that remodeling the lipogenic tumor microenvironment has potential to convert patients resistant to immunotherapy into those who may receive clinical benefit of immunotherapy. Citation Format: Eiji Sugaru, Yudai Furuta, Yoshikazu Nagagaki, Satoshi Ikeda, Yuichi Fukunaga, Hiroki Umehara, Tsuguteru Otsubo, Manabu Watanabe, Shingo Tojo, Miki Hashizume, Yasushi Matsuki, Hitoshi Ban. DSP-0692, a selective SCD inhibitor, enhances the anti-tumor activity of anti-PD1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3595.

E2A attenuates tumor-initiating capacity of colorectal cancer cells via the Wnt/beta-catenin pathway

BackgroundThe E2A gene, which encodes two basic helix–loop–helix transcription factors, E12 and E47, regulates colorectal cancer progression and epithelial-mesenchymal transition. However, whether E2A regulates the tumor-initiating capacity of colorectal cancer is unclear. Thus, we have studied E2A expression in the initiation of colorectal cancer in vivo and in vitro.MethodsImmunohistochemistry and immunoblot were performed to determine protein levels of E2A in colorectal cancer specimens and cells. RNAi was employed to downregulate E2A expression, and the subsequent change in protein level was evaluated by immunoblot. Sphere-forming assay and enumeration of liver metastasis in mouse models were used to identify the tumor formation ability of colorectal cancer cells.ResultsE2A expression in colorectal cancer clinical specimens was inversely associated with patients’ progression-free survival. Functional studies demonstrated that E2A significantly decreased tumor formation in vitro and in vivo. Furthermore, nuclear translocation of beta-catenin and activation of the Wnt/beta-catenin pathway occurred after suppression of E2A in colorectal cancer cells. FoxM1 was identified as a down-stream target by mRNA microarray, implying that FoxM1 plays a main role in determining how E2A regulates the tumor-initiating capacity of colorectal cancer.ConclusionE2A suppresses tumor-initiating capacity by targeting the FoxM1-Wnt/β-catenin pathway.

ROCK inhibition with Fasudil induces beta-catenin nuclear translocation and inhibits cell migration of MDA-MB 231 human breast cancer cells

Tumor aggressiveness is usually associated with metastasis. MDA-MB 231, a triple-negative breast cancer (TNBC), is an aggressive type of breast cancer and associated with early metastasis. The Rho/ROCK pathway is a key regulator of cell motility involving cytoskeleton regulation through stabilization of actin filaments and stress fiber formation. In this study we show that Fasudil, a ROCK inhibitor, inhibited the migration of MDA-MB 231 and A549 cells, without altering the viability of these cells at the concentration of 10 μM, modified tumor cell morphology, with disorganization of stress fibers and promotes activation of the canonical-Wnt/beta-catenin pathway. Therefore, Fasudil present a promising approach to the prevention of breast cancer metastasis through a different mechanism of action from the well-known one.

Beta-catenin represses protein kinase D1 gene expression by non-canonical pathway through MYC/MAX transcription complex in prostate cancer.

Down regulation of Protein Kinase D1 (PrKD1), a novel serine threonine kinase, in prostate, gastric, breast and colon cancers in humans leads to disease progression. While the down regulation of PrKD1 by DNA methylation in gastric cancer and by nuclear beta-catenin in colon cancer has been shown, the regulatory mechanisms in other cancers are unknown. Because we had demonstrated that PrKD1 is the only known kinase to phosphorylate threonine 120 (T120) of beta-catenin in prostate cancer resulting in increased nuclear beta-catenin, we explored the role of beta-catenin in gene regulation of PrKD1. An initial CHIP assay identified potential binding sites for beta-catenin in and downstream of PrKD1 promoter and sequencing confirmed recruitment of beta-catenin to a 166 base pairs sequence upstream of exon 2. Co-transfection studies with PrKD1-promoter-reporter suggested that beta-catenin represses PrKD1 promoter. Efforts to identify transcription factors that mediate the co-repressor effects of beta-catenin identified recruitment of both MYC and its obligate heterodimer MAX to the same binding site as beta-catenin on the PrKD1 promoter site. Moreover, treatment with MYC inhibitor rescued the co-repressor effect of beta-catenin on PrKD1 gene expression. Prostate specific knock out of PrKD1 in transgenic mice demonstrated increased nuclear expression of beta-catenin validating the in vitro studies. Functional studies showed that nuclear translocation of beta-catenin as a consequence of PrKD1 down regulation, increases AR transcriptional activity with attendant downstream effects on androgen responsive genes. In silico human gene expression analysis confirmed the down regulation of PrKD1 in metastatic prostate cancer correlated inversely with the expression of MAX, but not MYC, and positively with MXD1, a competing heterodimer of MAX, suggesting that the dimerization of MAX with either MYC or MXD1 regulates PrKD1 gene expression. The study has identified a novel auto-repressive loop that perpetuates PrKD1 down regulation through beta-catenin/MYC/MAX protein complex.

Abstract 5498: Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer

Abstract Protein kinase D1 (PRKD1) is down regulated in gastric, breast and prostate cancer. Several regulatory mechanisms modulate PRKD1 activity in cancers. In colon cancer, it has been shown that PRKD1 is down regulated by nuclear beta-catenin. We explored whether beta-catenin is involved in regulation of PRKD1 expression in prostate cancer as well. A CHIP assay performed using prostate cancer LNCaP cell lysate and pull down with beta -catenin antibody, which demonstrated recruitment of beta -catenin to promoter of PRKD1 gene as well as at downstream regions in the gene suggesting that beta -catenin could be involved in PRKD1 regulation. To identify the exact binding site of beta-catenin to PRKD1 gene, we carried out CHIP sequencing. The beta -catenin protein complex was bound to a 166 bp sequence near exon 2 (chr14:29899631-29899796). Because beta -catenin is a coactivator and not a known transcription factor, we performed a transcription factor promoter array, which showed that the MYC/MAX transcription factor complex may be the mediator of the regulatory effect of beta -catenin on PRKD1 expression. We validated the results by performing CHIP assay using MYC and MAX antibodies which showed recruitment of both MYC and MAX to the same beta -catenin binding site to PRKD1. In order to assess the functional impact of beta -catenin regulation of PRKD1 in prostate cancer, beta-catenin mutated at threonine 120 residue (we have previously shown that PRKD1 is the only known kinase to phosphorylate the site) demonstrated increased nuclear translocation of beta-catenin and increase down regulation of PRKD1 and increased androgen receptor (AR) activity (increased AR activity is well established to be associated with PC progression). Our data has identified a novel auto-regulatory mechanism of PRKD1 expression through beta-catenin phosphorylation. Citation Format: Bita Nickkholgh, Sittadjody Sivanandane. Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer [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 5498. doi:10.1158/1538-7445.AM2017-5498

P120 regulates beta-catenin nuclear translocation through E-cadherin endocytosis in ventilator-induced lung injury.

Mechanical stretch induces epithelial barrier dysfunction by altering the location and degradation of cellular junction proteins. p120-catenin (p120) is a cell-cell junction protein known to protect against ventilator-induced lung injury (VILI) that results from improper ventilation of patients. In this study, we sought to determine the role of p120 in VILI and its relationship with the cellular response to mechanical stretch. Mouse lung epithelial cells (MLE-12) transfected with p120 siRNA, p120 cDNA, or E-cadherin siRNA were subjected to 20% cyclic stretch for 2 or 4 hours. Wild-type male C57BL/6 mice were transfected with p120 siRNA-liposome complex to delete p120 in vivo and then subjected to mechanical ventilation. Cyclic stretch induced p120 degradation and the endocytosis of E-cadherin, which induced β-catenin translocation into the nucleus, a key event in lung injury progress and repair. These findings reveal that by reducing β-catenin nuclear translocation through inhibition of E-cadherin endocytosis, p120 protects against ventilator-induced lung injury.

Abstract 331: Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG

Abstract Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibited colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activate PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduced the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibited the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, which resulted in caspase activation and apoptosis. This was determined to be through a PKG mediated pathway through the use of small molecule inhibitors of PKG and PKA. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues in a cDNA array. We also report the increase in PDE10 mRNA in 50% of a small collection of human clinical specimens collected at the Mitchell Cancer Institute (n = 13). In addition, novel PDE10 inhibitor, MCI-030, reduced tumor size and activated PDE10 signaling mechanisms in vivo. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells. Citation Format: Kevin J. Lee, Ashley S. Lindsey, Luciana Madeira da Silva, Alisa Trinh, Bernard Gary, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Wen-Chi Chang, Margie Clapper, Gary A. Piazza. Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG. [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 331.

Mechanical Induction of the Tumorigenic β-Catenin Pathway by Tumour Growth Pressure in vivo

The tumour microenvironment may contribute to tumorigenesis due to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch+Apc+/1638N mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, associated with increased Ret and beta-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by acoustic strain imaging and shear wave elastography. Exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of beta-catenin on Tyr654, which impairs its interaction with the E-cadherin in adherens junctions, and which was followed by beta-catenin nuclear translocation after 15 days. As a consequence, elevated expression of beta-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic beta-catenin pathway, conserved from early embryos beta-catenin dependent mechanical induction of patterning-genes expression, suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.Fernandez-Sanchez, Barbier et al, Nature 2015, Jul 2;523(7558):92-5. http://dx.doi.org/10.1038/nature14329. Epub 2015 May 11.

Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

Curcumin inhibits breast cancer stem cell migration by amplifying the E-cadherin/β-catenin negative feedback loop

IntroductionThe existence of cancer stem cells (CSCs) has been associated with tumor initiation, therapy resistance, tumor relapse, angiogenesis, and metastasis. Curcumin, a plant ployphenol, has several anti-tumor effects and has been shown to target CSCs. Here, we aimed at evaluating (i) the mechanisms underlying the aggravated migration potential of breast CSCs (bCSCs) and (ii) the effects of curcumin in modulating the same.MethodsThe migratory behavior of MCF-7 bCSCs was assessed by using cell adhesion, spreading, transwell migration, and three-dimensional invasion assays. Stem cell characteristics were studied by using flow cytometry. The effects of curcumin on bCSCs were deciphered by cell viability assay, Western blotting, confocal microscopy, and small interfering RNA (siRNA)-mediated gene silencing. Evaluations of samples of patients with breast cancer were performed by using immunohistochemistry and flow cytometry.ResultsHere, we report that bCSCs are endowed with aggravated migration property due to the inherent suppression of the tumor suppressor, E-cadherin, which is restored by curcumin. A search for the underlying mechanism revealed that, in bCSCs, higher nuclear translocation of beta-catenin (i) decreases E-cadherin/beta-catenin complex formation and membrane retention of beta-catenin, (ii) upregulates the expression of its epithelial-mesenchymal transition (EMT)-promoting target genes (including Slug), and thereby (iii) downregulates E-cadherin transcription to subsequently promote EMT and migration of these bCSCs. In contrast, curcumin inhibits beta-catenin nuclear translocation, thus impeding trans-activation of Slug. As a consequence, E-cadherin expression is restored, thereby increasing E-cadherin/beta-catenin complex formation and cytosolic retention of more beta-catenin to finally suppress EMT and migration of bCSCs.ConclusionsCumulatively, our findings disclose that curcumin inhibits bCSC migration by amplifying E-cadherin/beta-catenin negative feedback loop.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt506) contains supplementary material, which is available to authorized users.

Comment on: Functional MUC4 suppress epithelial-mesenchymal transition in lung adenocarcinoma metastasis. Gao L, Liu J, Zhang B, Zhang H, Wang D, Zhang T, Liu Y, Wang C. Tumour Biol. 2013, in press

Gao and collaborators (Tumour Biol, 2013) have investigated the role of mucin 4 (MUC4) in lung cancer and have concluded that a loss of MUC4 results in epithelial mesenchymal transition via beta-catenin nuclear translocation and that MUC4 expression is correlated with a risk of lymph node metastasis in a cohort of 20 lung adenocarcinoma patients. This surprising analysis is contradictory to most of the scientific knowledge and literature regarding MUC4 contribution in epithelial cancers that is very hardly discussed in their manuscript.

Master chaperone gp96 for TLRs and integrins regulates canonical Wnt pathway and gut homeostasis (P3278)

Abstract gp96 is a master protein chaperone for Toll-like receptors and integrins in the endoplasmic reticulum (ER) and plays critical roles in innate immunity (Yang et al., Immunity, 2007 26:215-226). Using a conditional and lineage-specific knockout strategy, we have unexpectedly observed that gp96 is required for maintaining intestinal integrity. Loss of gp96 in adult mice causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear beta-catenin translocation, loss of crypt-villus structure and impaired barrier function. Mechanistic study revealed that the observed intestinal pathology in gp96 KO mice is unlikely due to compromise in the gut defense or dysregulated unfolded protein response. Rather, gp96 was found to be essential for maturation of Wnt co-receptor LRP6 and thus canonical Wnt signaling. Our study places Wnt pathway under the direct regulation of a TLR/integrin-master chaperone, suggesting that innate immunity and organ development converge on gp96.

Abstract 281: Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer.

Abstract Introduction: Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. An important determinant for metastasis is epithelial to mesenchymal transition (EMT) which is known to be correlated to castrate resistant prostate cancer (CRCP). Previous report demonstrated the overexpression of Hsp27 and its phosphorylated form in poor diagnosis prostate cancer. In addition, both epidermal growth factor (EGF) and its receptor (EGFR) are up–regulated in PCa during progression to CRPC. However, the role of EGF and EGFR in PCa progression remains unclear. Our objective is to establish that Hsp27 is a central “node” for several signaling pathways activated by EGF and known to modulate EMT. Methods: To assess the role of Hsp27 in EMT, gain and loss of Hsp27 function has been conducted in LNCaP cells with or without EGF stimulation. EMT markers expression has been evaluated at the protein (WB) and mRNA level (qRT–PCR). Furthermore, invasion, migration assays in addition with MMP–9 activity have been done to evaluate the migratory capacity of those cells. Also, to better understand the molecular mechanism involved in EMT induction, transactivation and Chip assays have been performed. The in vitro results have been confirmed with an in vivo study using an Hsp27 antisense oligonucleotide (OGX-427) in a murine model. Results: We found that Heat shock protein 27 (Hsp27) is up–regulated with CRPC progression and drives EMT in PCa cells by decreasing epithelial cell markers, up–regulating mesenchymal markers and enhancing cell migration and MMP–9 activity. Hsp27 knockdown induces sequestration of beta–catenin in the cytoplasm by increasing its binding to GSK–3beta and abrogating EGF induced beta–catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta–catenin to slug promoter thereby lessening its transcriptional activity. As Slug is known to be a critical key in EMT induction, we confirmed the role of EGF and Hsp27 in EMT induction and our capacity to abrogate the EGF-induced EMT effect by Hsp27 knockdown. Finally, targeting Hsp27 using antisense oligonucleotide OGX-427 in vivo, significantly reduced tumor cell metastasis in a PCa murine model. Conclusion: This study shows that EGF through Hsp27 induces modifications in gene expression and morphology toward a mesenchymal phenotype. The mechanism involving Hsp27, β-catenin, nuclear β-catenin/TCF and EGF signaling cascades described in the present study contribute to the comprehension of mechanisms driving metastasis in aggressive castration-resistant prostate cancer. This study reveals that Hsp27 is a crucial effector of EGF–dependent and independent EMT and suggests that targeting Hsp27 holds promises for a new strategy in metastatic PCa. Citation Format: Thomas D. Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer. [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 281. doi:10.1158/1538-7445.AM2013-281