The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.

Wnt signalling in stem cells and cancer

The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.

Generation of a functional mammary gland from a single stem cell

4839 The mammary gland can be functionally regenerated in mice by serial transplantation of epithelial fragments, providing evidence for the existence of self-renewing, multipotential mammary stem cells (MaSCs). Recently the concept has emerged that MaSCs play a central role in breast tumorigenesis. However, the identity and purification of MaSC has proved elusive due to the lack of defined markers. Using specific cell surface markers and flow cytometry, we have identified a distinct subpopulation that is enriched for MaSCs, demonstrated by transplantation into cleared mammary fat pads at limiting dilution. Remarkably, a single mammary epithelial cell from this population, carrying the lacZ transgene, was found to generate a complete mammary gland in vivo. These cells contributed to both the luminal and myoepithelial lineages in transplanted virgin mammary glands, and extensive lobuloalveolar units were generated during pregnancy. Serial transplantation of the clonal outgrowths also yielded complete mammary glands, confirming that the cells were capable of self-renewal. These data establish that single cells from the enriched population have multipotential and self-renewing capacity, a hallmark of stem cells. It will be of interest to determine whether the stem cell is a target of transformation mammary tumorigenesis models.

Since the initial discovery of the oncogenic activity of WNT1 in mouse mammary glands, our appreciation for the complex roles for WNT signalling pathways in cancer has increased dramatically. WNTs and their downstream effectors regulate various processes that are important for cancer progression, including tumour initiation, tumour growth, cell senescence, cell death, differentiation and metastasis. Although WNT signalling pathways have been difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can alter WNT signalling in preclinical models, thus setting the stage for clinical trials in humans.

WNT signalling pathways as therapeutic targets in cancer

Wnt signaling is one of the key cascades regulating development and stemness, and has also been tightly associated with cancer. The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer, but aberrant Wnt signaling is observed in many more cancer entities. Here, we review current insights into novel components of Wnt pathways and describe their impact on cancer development. Furthermore, we highlight expanding functions of Wnt signaling for both solid and liquid tumors. We also describe current findings how Wnt signaling affects maintenance of cancer stem cells, metastasis and immune control. Finally, we provide an overview of current strategies to antagonize Wnt signaling in cancer and challenges that are associated with such approaches.

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Wnt signaling in cancer.

Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing β-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.

https://www.pnas.org/content/pnas/100/26/15853.full.pdf

Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells

Background
In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development.

/pdf/changes-in-gene-expression-during-the-development-of-mammary-1o4t4u13xh.pdf

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice.

A missense mutation has been identified in the human phenylalanine hydroxylase [PAH; phenylalanine 4-monooxygenase; L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] gene in a Chinese patient with classic phenylketonuria (PKU). A G-to-C transition at the second base of codon 413 in exon 12 of the gene results in the substitution of Pro413 for Arg413 in the mutant protein. This mutation (R413P) results in negligible enzymatic activity when expressed in heterologous mammalian cells and is compatible with a classic PKU phenotype in the patient. Population genetic studies reveal that this mutation is tightly linked to restriction fragment length polymorphism haplotype 4, which is the predominant haplotype of the PAH locus in the Oriental population. It accounts for 13.8% of northern Chinese and 27% of Japanese PKU alleles, but it is rare in southern Chinese (2.2%) and is absent in the Caucasian population. The data demonstrate unambiguously that the mutation occurred after racial divergence of Orientals and Caucasians and suggest that the allele has spread throughout the Orient by a founder effect. Previous protein polymorphism studies in eastern Asia have led to the hypothesis that "northern Mongoloids" represented a founding population in Asia. Our results are compatible with this hypothesis in that the PKU mutation might have occurred in northern Mongoloids and subsequently spread to the Chinese and Japanese populations.

https://www.pnas.org/content/pnas/88/6/2146.full.pdf

Founder effect of a prevalent phenylketonuria mutation in the Oriental population.

IntroductionDespite advances in early detection and adjuvant targeted therapies, breast cancer is still the second most common cause of cancer mortality among women. Tumor recurrence is one of the major contributors to breast cancer mortality. However, the mechanisms underlying this process are not completely understood. In this study, we investigated the mechanisms of tumor dormancy and recurrence in a preclinical mouse model of breast cancer.MethodsTo elucidate the mechanisms driving tumor recurrence, we employed a transplantable Wnt1/inducible fibroblast growth factor receptor (FGFR) 1 mouse mammary tumor model and utilized an FGFR specific inhibitor, BGJ398, to study the recurrence after treatment. Histological staining was performed to analyze the residual tumor cells and tumor stroma. Reverse phase protein array was performed to compare primary and recurrent tumors to investigate the molecular mechanisms leading to tumor recurrence.ResultsTreatment with BGJ398 resulted in rapid tumor regression, leaving a nonpalpable mass of dormant tumor cells organized into a luminal and basal epithelial layer similar to the normal mammary gland, but surrounded by dense stroma with markedly reduced levels of myeloid-derived tumor suppressor cells (MDSCs) and decreased tumor vasculature. Following cessation of treatment the tumors recurred over a period of 1 to 4 months. The recurrent tumors displayed dense stroma with increased collagen, tenascin-C expression, and MDSC infiltration. Activation of the epidermal growth factor receptor (EGFR) pathway was observed in recurrent tumors, and inhibition of EGFR with lapatinib in combination with BGJ398 resulted in a significant delay in tumor recurrence accompanied by reduced stroma, yet there was no difference observed in initial tumor regression between the groups treated with BGJ398 alone or in combination with lapatinib.ConclusionThese studies have revealed a correlation between tumor recurrence and changes of stromal microenvironment accompanied by altered EGFR signaling.

Additional file 2: Figure S1. of Upregulation of EGFR signaling is correlated with tumor stroma remodeling and tumor recurrence in FGFR1-driven breast cancer

Shixia Huang

Papers

Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice.

Founder effect of a prevalent phenylketonuria mutation in the Oriental population.

Additional file 2: Figure S1. of Upregulation of EGFR signaling is correlated with tumor stroma remodeling and tumor recurrence in FGFR1-driven breast cancer