Ewoud N. Speksnijder

Developmental stage-specific contribution of LGR5 + cells to basal and luminal epithelial lineages in the postnatal mammary gland

The leucine‐rich repeat‐containing heterotrimeric guanine nucleotide‐binding protein‐coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mammary epithelial stem cells, we performed in situ lineage‐tracing studies and mammary gland reconstitutions with LGR5‐expressing mammary epithelial cells. Interestingly, the LGR5 progeny population in mammary epithelium switches from the luminal to the myoepithelial compartment during the first 12 days of postnatal development, likely reflecting local changes in Wnt signalling. Together, our findings point to a stage‐specific contribution of LGR5‐expressing cells to luminal and basal epithelial lineages during postnatal mammary gland development. Copyright

Mice Expressing a Mammary Gland–Specific R270H Mutation in the p53 Tumor Suppressor Gene Mimic Human Breast Cancer Development

The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as approximately 30% of sporadic tumors acquire p53 mutations and Li-Fraumeni syndrome patients carrying germ line p53 mutations frequently develop breast tumors at early age. In the present study, conditional expression of a targeted mutation is used to analyze the role of the human R273H tumor-associated hotspot mutation in p53 in mammary gland tumorigenesis. Heterozygous p53(R270H/+)WAPCre mice (with mammary gland-specific expression of the p53.R270H mutation, equivalent to human R273H, at physiologic levels) develop mammary tumors at high frequency, indicating that the R270H mutation predisposes for mammary gland tumor development and acts in a dominant-negative manner in early stages of tumorigenesis. Spontaneous tumor development in these mice is further accelerated by 7,12-dimethylbenz(a)anthracene (DMBA) treatment at young age. The majority of spontaneous and DMBA-induced carcinomas and sarcomas from p53(R270H/+)WAPCre mice is estrogen receptor alpha positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53(R270H/+)WAPCre mice provide a well-suited model system to study the role of p53 in breast tumorigenesis and the responsiveness of mammary gland tumors to chemotherapeutics.

Dominant-Negative but not Gain-of-Function Effects of a p53.R270H Mutation in Mouse Epithelium Tissue after DNA Damage

p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage.

Development of metastatic HER2(+) breast cancer is independent of the adaptive immune system.

The tumour‐modulating effects of the endogenous adaptive immune system are rather paradoxical. Whereas some clinical and experimental observations offer compelling evidence for the existence of immunosurveillance, other studies have revealed promoting effects of the adaptive immune system on primary cancer development and metastatic disease. We examined the functional significance of the adaptive immune system as a regulator of spontaneous HER2+ breast tumourigenesis and pulmonary metastasis formation, using the MMTV–NeuT mouse model in which mammary carcinogenesis is induced by transgenic expression of the activated HER2/neu oncogene. Although T and B lymphocytes infiltrate human and experimental HER2+ breast tumours, genetic elimination of the adaptive immune system does not affect development of premalignant hyperplasias or primary breast cancers. In addition, we demonstrate that pulmonary metastasis formation in MMTV–NeuT mice is not dependent on the adaptive immune system. Thus, our findings reveal that spontaneous HER2‐driven mammary tumourigenesis and metastasis formation are neither suppressed, nor altered by immunosurveillance mechanisms, nor promoted by the adaptive immune system. Copyright

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1F/F; Trp53F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright

Human TP53 polymorphism (rs1042522) modelled in mouse does not affect glucose metabolism and body composition

Variation in TP53 has been associated with cancer. The pro-allele of a TP53 polymorphism in codon 72 (rs1042522) has been associated with longevity. Recently, we showed that the same allele might be involved in preservation of glucose metabolism, body composition and blood pressure during ageing. Here, we assessed glucose tolerance and body composition in mice carrying the human polymorphism. Our data do not support the previous findings in humans, suggesting that this polymorphism does not play a major role in development of glucose metabolism and body composition during ageing. Alternatively, the mouse model may not be suitable to validate these rs1042522-associated traits up to the age tested.