Redundant expression of canonical Wnt ligands in human breast cancer cell lines

Human breast cancer displays nuclear accumulation of β-catenin and induction of cyclin D1 expression, which suggests that canonical Wnt/β-catenin signaling is activated. In other cancers, the activation of canonical wnt/β-catenin signaling is associated with APC, CTNNB1 or AXIN1 mutations. However, these mutations are rare or absent in breast cancer. In search of alternative mechanisms, we performed comprehensive expression analysis of Wnt signaling molecules, including 19 Wnt ligands, ten Frizzled receptors, two co-receptors and four Lef/TCF transcription factors in immortalized normal human mammary epithelial cells (HMEC) and six breast cancer cell lines. HMEC expressed all Frizzled receptors except FZD9 and FZD10. They also expressed LRP5 and LRP6 co-receptors, as well as four Lef/TCF transcription factors. HMEC cells also expressed many Wnt ligands, including WNT1, WNT2B, WNT3, WNT5A, WNT5B, WNT7B, WNT9A, WNT10B and WNT16 . Redundant expression of Wnt ligands, Frizzled receptors, co-receptors and Lef/TCF transcription factors was maintained in breast cancer cell lines with some exceptions. The most important changes in cancer cell lines concerned Wnt ligand expression. We noticed that most breast cancer cell lines overexpressed WNT3A, WNT4, WNT6, WNT8B, WNT9A and WNT10B. In contrast, the expression of WNT5A, WNT5B and WNT16 was usually down-regulated. It is noteworthy that all six Wnt ligands that are overexpressed in malignant cell lines are known to signal through the canonical Wnt/β-catenin signaling pathway, whereas down-regulated WNT5A and WNT5B ligands signal via the non-canonical pathway. The expression of both canonical Wnt ligands and most Frizzled receptors in breast cancer cell lines suggests that canonical Wnt/β-catenin signaling is activated in these cell lines by an autocrine/paracrine mechanism. In support of this prediction, we observed nuclear β-catenin accumulation and cyclin D1 induction in breast cancer cell lines, but not in HMEC. These results imply that ligand-dependent canonical Wnt/β-catenin signaling is active in human breast cancer

PORCN moonlights in a wnt-independent pathway that regulates cancer cell proliferation

10.1371/journal.pone.0034532PLoS ONE74

The Internally Truncated LRP5 Receptor Presents a Therapeutic Target in Breast Cancer

BACKGROUND: Breast cancer is a common malignant disease, which may be caused by a number of genes deregulated by genomic or epigenomic events. Deregulated WNT/beta-catenin signaling with accumulation of beta-catenin is common in breast tumors, but mutations in WNT signaling pathway components have been rare. An aberrantly spliced internally truncated LRP5 receptor (LRP5Delta666-809, LRP5Delta) was shown recently to be resistant to DKK1 inhibition, and was required for beta-catenin accumulation in hyperparathyroid tumors and parathyroid tumor growth. METHODOLOGY/PRINCIPAL FINDINGS: Here we show, by reverse transcription PCR and Western blot analysis, that LRP5Delta is frequently expressed in breast tumors of different cancer stage (58-100%), including carcinoma in situ and metastatic carcinoma. LRP5Delta was required in MCF7 breast cancer cells for the non-phosphorylated active beta-catenin level, transcription activity of beta-catenin, cell growth in vitro, and breast tumor growth in a xenograft SCID mouse model. WNT3 ligand, but not WNT1 and WNT3A augmented the endogenous beta-catenin activity of MCF7 cells in a DKK1-insensitive manner. Furthermore, an anti-LRP5 antibody attenuated beta-catenin activity, inhibited cell growth, and induced apoptosis in LRP5Delta-positive MCF7 and T-47D breast cancer cells, but not in control cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the LRP5Delta receptor is strongly implicated in mammary gland tumorigenesis and that its aberrant expression present an early event during disease progression. LRP5 antibody therapy may have a significant role in the treatment of breast cancer

The Wnt Receptor, Lrp5, Is Expressed by Mouse Mammary Stem Cells and Is Required to Maintain the Basal Lineage

Background: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development. Methodology/Principal Findings: Here, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%). Stem cell activity can be enriched by.200 fold (over 80 % of activity), based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42 % basal/total epithelial cells to 22%) and Lrp52/2 mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16 Ink4a and TA-p63. Conclusions/Significance: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component o

Key signaling nodes in mammary gland development and cancer: β-catenin

β-Catenin plays important roles in mammary development and tumorigenesis through its functions in cell adhesion, signal transduction and regulation of cell-context-specific gene expression. Studies in mice have highlighted the critical role of β-catenin signaling for stem cell biology at multiple stages of mammary development. Deregulated β-catenin signaling disturbs stem and progenitor cell dynamics and induces mammary tumors in mice. Recent data showing deregulated β-catenin signaling in metaplastic and basal-type tumors suggest a similar link to reactivated developmental pathways and human breast cancer. The present review will discuss β-catenin as a central transducer of numerous signaling pathways and its role in mammary development and breast cancer

Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE