Breast cancer-specific mutations in CK1ε inhibit Wnt/β-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration

Introduction Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1ε). Because CK1ε is a crucial regulator of the Wnt signaling cascades, we determined how these CK1ε mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines. Methods We performed in silico modeling of various mutations and analyzed the kinase activity of the CK1ε mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1ε affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1ε in these processes. Results In silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1ε, is involved in positive regulation of the CK1ε activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1ε failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1ε mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1ε mutants acted as loss-of-function in the Wnt/β-catenin pathway, and that CK1ε mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7. Conclusions In summary, these data suggest that the mutations of CK1ε found in breast cancer can suppress Wnt/β-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1ε, which are correlated with decreased phosphorylation activities of mutated forms of CK1ε both in vitro and in vivo, interfere with positive autophosphorylation at Thr 4

Intranasal Administration of poly(I:C) and LPS in BALB/c Mice Induces Airway Hyperresponsiveness and Inflammation via Different Pathways

BACKGROUND: Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role. MATERIALS AND METHODS: To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days. RESULTS: When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation. CONCLUSION: In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another

Wnt signalling and cancer stem cells

[Abstract] Intracellular signalling mediated by secreted Wnt proteins is essential for the establishment of cell fates and proper tissue patterning during embryo development and for the regulation of tissue homeostasis and stem cell function in adult tissues. Aberrant activation of Wnt signalling pathways has been directly linked to the genesis of different tumours. Here, the components and molecular mechanisms implicated in the transduction of Wnt signal, along with important results supporting a central role for this signalling pathway in stem cell function regulation and carcinogenesis will be briefly reviewed.Ministerio de Ciencia e Innovación; SAF2008-0060

A t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide functions as a potent antagonist of Wnt5a-dependent melanoma cell invasion

The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5a-mediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63. Both express Frizzled-5, which has been implicated as the receptor for Wnt5a in melanoma cells. However, only the HTB63 cell line expresses and secretes Wnt5a. In these cells, the cytokine, TGF beta 1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGF beta 1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGF beta 1 was an unsuitable strategy to pursue. We therefore attempted to target Wnt5a signaling directly. Exogenous Wnt5a stimulation of A2058 cells increased adhesion, migration and invasion, all crucial components of tumor metastasis, and the Wnt5a-derived N-butyloxycarbonyl hexapeptide (Met-Asp-Gly-Cys-Glu-Leu; 0.766 kDa) termed Box5, abolished these responses. Box5 also inhibited the basal migration and invasion of Wnt5a-expressing HTB63 melanoma cells. Box5 antagonized the effects of Wnt5a on melanoma cell migration and invasion by directly inhibiting Wnt5a-induced protein kinase C and Ca2+ signaling, the latter of which we directly demonstrate to be essential for cell invasion. The Box5 peptide directly inhibits Wnt5a signaling, representing an approach to anti-metastatic therapy for otherwise rapidly progressive melanoma, and for other Wnt5a-stimulated invasive cancers

The prognostic significance of Wnt-5a expression in primary breast cancer is extended to premenopausal women.

Wnt-5a protein expression in primary tumors from unselected breast cancer patients has revealed a tumor suppressive function of the protein. However, in vitro experiments on human breast cancer cells have reported contradictory results, indicating both a tumor suppressive and promoting functions of Wnt-5a. This could be due to various functions of Wnt-5a in different subgroups of patients. The unselected cohorts analyzed to date for Wnt-5a protein expression contained few premenopausal patients. The aim of the present investigation was to evaluate the prognostic significance of Wnt-5a protein expression in a cohort of premenopausal women with comprehensive data on biomarkers, molecular subtypes and long-term outcome. In a randomized trial of adjuvant tamoxifen versus no adjuvant treatment, 564 premenopausal primary breast cancer patients were included. The median follow-up time was 14 years. A tumor tissue array was constructed and 361 samples were evaluated for Wnt-5a reactivity by immunohistochemistry. The primary end-point was recurrence-free survival. Wnt-5a protein expression was reduced or lost in 146/361 of tumors and correlated to younger age, estrogen receptor (ER) negativity and triple-negative phenotype. Wnt-5a was a prognostic factor in the whole cohort (p = 0.003). In patients with ER-positive tumors, Wnt-5a was an independent positive prognostic marker (HR 0.51 95% CI: 0.33-0.78 p = 0.002) and HER2 a negative prognostic marker (HR 2.84 95% CI: 1.51-5.31, p = 0.001) in a Cox multivariate analysis adjusted for standard prognostic markers and tamoxifen treatment. In the ER-negative subset, Wnt-5a added no prognostic information. In a subgroup analysis, Wnt-5a was significantly associated with better prognosis in patients with Luminal A tumors (p = 0.04). Conclusively, our results suggest that loss of Wnt-5a is a valuable prognostic marker in premenopausal breast cancer patients in particular in patients with ER-positive tumors and out-performed conventional prognostic factors in this subset of patients

Wnt5a Exhibits Layer-Specific Expression in Adult Skin, Is Upregulated in Psoriasis, and Synergizes with Type 1 Interferon

Wnt5a is a member of the wingless-type patterning regulators important in pre-natal development. The expression and distribution of Wnt5a and its receptors frizzled (fzd) 3 and fzd 5 in adult human skin have not been comprehensively studied to date.We here show that Wnt5a, fzd3, fzd5, as well as fzd6 are restricted to specific layers in normal epidermis, analogous to their zonal distribution in hair follicles, suggesting a role in adult skin differentiation. In line, Wnt5a and fzd5 are both overexpressed and re-distributed in the epidermis of psoriasis which involves disturbed keratinocyte differentiation. Functionally, Wnt5a lowers the concentration of IFN required to induce target genes, and increases the magnitude of IFN target gene induction, suggesting a molecular mechanism underlying IFN hypersensitivity in psoriasis. Finally, we identify nedd8 and the amyloid precursor APP, previously shown to be upregulated in psoriasis, as targets of synergistic IFNalpha/Wnt5a induction.The present data (i) suggest that Wnt5a regulates epidermal differentiation even in adult skin and (ii) identify synergistic induction of type 1 IFN target genes as a novel mode of Wnt5a action. Targeting Wnt5a in the skin may reduce IFN hypersensitivity and be of therapeutical value

WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells

Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44