The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

This is the final version. Available on open access from eLife Sciences Publications via the DOI in this recordData availability: All data generated or analysed during this study are included in the manuscript, supporting files and source files; Supporting Data files and Source Data have been provided to all figures.The Wnt/β-catenin signalling pathway regulates multiple cellular processes during development and many diseases, including cell proliferation, migration, and differentiation. Despite their hydrophobic nature, Wnt proteins exert their function over long distances to induce paracrine signalling. Recent studies have identified several factors involved in Wnt secretion; however, our understanding of how Wnt ligands are transported between cells to interact with their cognate receptors is still debated. Here, we demonstrate that gastric cancer cells utilise cytonemes to transport Wnt3 intercellularly to promote proliferation and cell survival. Furthermore, we identify the membrane-bound scaffolding protein Flotillin-2 (Flot2), frequently overexpressed in gastric cancer, as a modulator of these cytonemes. Together with the Wnt co-receptor and cytoneme initiator Ror2, Flot2 determines the number and length of Wnt3 cytonemes in gastric cancer. Finally, we show that Flotillins are also necessary for Wnt8a cytonemes during zebrafish embryogenesis, suggesting a conserved mechanism for Flotillin-mediated Wnt transport on cytonemes in development and disease.Medical Research Council (MRC)Biotechnology and Biological Sciences Research Council (BBSRC

Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; ApcCKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC

Apc Mutation Enhances PyMT-Induced Mammary Tumorigenesis

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, ApcMin/+ mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the ApcMin/+ mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the ApcMin/+ mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling

Mutations in Apc and p53 synergize to promote mammary neoplasia

Mutations of Apc and p53 have both been implicated in human and murine mammary neoplasia. To investigate potential interactions between Apc and. p53, we conditionally inactivated Ape in both the presence and the absence oil functional p53. Ape deficiency on its own leads to the development of metaplasia but not neoplasia. We. show here that these areas of metaplasia are characterized by elevated levels of both p53 and p21. In the additional absence of p53, there is rapid progression to neoplasia, with 44.4% of lymphoma-free mice developing a mammary tumor with earliest observed onset at pregnancy. To investigate the mechanism by which p53 deficiency accelerates neoplasia, we used the Rosa26R reporter strain as a marker of Cre-mediated recombination and show a role for p53 in the loss of Apc-deficient cells. This role seems limited to pregnancy and subsequent time points. We therefore show clear synergy between these two mutations in mammary gland neoplasia and present data to suggest that at least one mechanism for this acceleration is the p53-dependent loss of Apc-deficient cells

Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo

Oncogenic mutations in the K-ras gene occur in approximate to 50% of human colorectal cancers. However, the precise role that K-ras oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K-ras(V12) allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K-ras(V12) does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf/MEK/ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K-ras(V12) expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic K-ras(V12) allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf/MEK/ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K-ras oncogene. However, activation of K-ras(V12) after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K-ras oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion

Conditional disruption of Axin1 leads to development of liver tumors in mice.

BACKGROUND & AIMS: Mutations in components of the Wnt signaling pathway, including β-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. METHODS: Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of β-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. RESULTS: Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G(2)/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of β-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of β-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. CONCLUSIONS: Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of β-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC

Conditional disruption of Axin1 leads to development of liver tumors in mice.

BACKGROUND & AIMS: Mutations in components of the Wnt signaling pathway, including β-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. METHODS: Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of β-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. RESULTS: Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G(2)/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of β-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of β-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. CONCLUSIONS: Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of β-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC