More than 90% of breast cancers arise from epithelial cells or from progenitors with stem cell properties. Cell polarity is essential for stem cell asymmetric divisions, and failures in polarization can result in tumorigenesis. The Par polarity proteins are key regulators of asymmetric cell division. Par proteins also control polarization of differentiated epithelial cells. Yet the molecular mechanisms underlying polarity in mammary morphogenesis and the role of Par proteins in breast cancer remain largely unknown. We have developed methods that facilitate the study of mammary morphogenesis and tumorigenesis. To manipulate gene expression in mammary glands, stem cells are transduced with lentivirus, and mammary glands are regenerated in host mice from these cells. Lentiviral transduction enables rapid analysis of molecular mechanism through a knock-down/knock-in approach. Using these methods, an essential role for the Par3 polarity protein in mammary morphogenesis was discovered. In addition, silencing of Par3 potentiated tumor growth and metastasis. The goals are as follows: 1. How does Par3 regulate stem/progenitor self-renewal and differentiation during mammary gland morphogenesis? In the absence of Par3, atypical protein kinase C (aPKC) is mislocalized from the luminal apical surface. A knock-down/knock-in strategy will be used to ask if fate determination can be rescued by apically-tethered aPKC and by Par3 mutants. A novel miRNA sensor will be used to identify stem cells and to ask if Par3 is required for asymmetric division. Differential functions of Par3 in luminal and myoepithelial cells will be tested using new methods for cell-type specific RNAi silencing. 2. How does Par3 function to suppress tumor growth and metastasis? Preliminary data suggest that Par3 might limit mammary cell proliferation by suppressing Rac activity in the NICD cells, while separately controlling cell lineage determination and metastasis through aPKC. Lentivirus will be used to manipulate gene expression in primary cells, which are tested using the in vivo transplant model, tail vein injections, and in vitro mammosphere cultures. 3. Can we identify multiple additional metastasis suppressors/inducers by in vivo screening?. Mammary stem cells from ErbB2 mice will be infected with pooled lentiviruses that express a library of cDNAs, then implanted into cleared fat pads of wild type host mice. The viruses also express a red fluorescent protein. RFP-positive lung metastases will be isolated and screened by PCR to identify the cDNA associated with each metastatic colony. Positives will be prioritized based on expression patterns in human tumors for mechanistic studies. Together, these studies will provide insights into polarity protein signaling during stem/progenitor cell self-renewal and lineage determination, and identify new players in mammary tumor metastasis.

Public Health Relevance

Breast cancer is one of the leading causes of death among women. Traditional approaches to studying the molecular basis for breast cancer, using genetically-engineered mice, are expensive and time-consuming;and studies in cell culture do not recapitulate the complex processes involved in building an organ or in tumor progression. New approaches are needed to address these issues. We have developed methods that facilitate the rapid analysis of gene function in mammary gland development and cancer. We will use these methods to identify new cancer genes, and to understand how breast cancers develop and metastasize.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA132898-04
Application #
8446157
Study Section
Molecular Oncogenesis Study Section (MONC)
Program Officer
Snyderwine, Elizabeth G
Project Start
2010-09-01
Project End
2015-02-28
Budget Start
2013-03-18
Budget End
2014-02-28
Support Year
4
Fiscal Year
2013
Total Cost
$338,737
Indirect Cost
$121,193
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Archibald, A; Mihai, C; Macara, I G et al. (2015) Oncogenic suppression of apoptosis uncovers a Rac1/JNK proliferation pathway activated by loss of Par3. Oncogene 34:3199-206
Rodriguez-Boulan, Enrique; Macara, Ian G (2014) Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15:225-42
Macara, Ian G; Guyer, Richard; Richardson, Graham et al. (2014) Epithelial homeostasis. Curr Biol 24:R815-25
Macara, Ian G; McCaffrey, Luke (2013) Cell polarity in morphogenesis and metastasis. Philos Trans R Soc Lond B Biol Sci 368:20130012
Pasic, Lejla; Eisinger-Mathason, T S Karin; Velayudhan, Bisi T et al. (2011) Sustained activation of the HER1-ERK1/2-RSK signaling pathway controls myoepithelial cell fate in human mammary tissue. Genes Dev 25:1641-53