Functional relevance of ARC in mammary gland morphogenesis and breast cancer

Abstract

Cell death by apoptosis is necessary during development and in adult tissue homeostasis. Evasion of apoptosis is critical for tumorigenesis. ARC (Apoptosis Repressor with CARD Domain), an inhibitor of both the death receptor and mitochondrial apoptosis pathways, is induced in human epithelial cancers including breast cancer. To explore the role of ARC in acinar formation and carcinogenesis MCF-10A cells were applied in a three dimensional basement membrane (Matrigel) cell culture model. Exogenous over expression of ARC did not prevent, nor delay apoptosis of acini lumens. However, endogenous ARC mRNA and protein levels in MCF-10A acini increased incrementally after day 5 in culture. The depletion of ARC prevented acinar growth and induced a G1 cell cycle arrest. This data suggests that ARC has a function in normal mammary morphogenesis. To test ARC'S relevance in an in vivo model of breast cancer, ARC4-/- mice were bred with MMTV-PyMT transgenics which have mammary specific activation of oncogenic pathways common to human breast cancer, including the induction of ARC. At 14 weeks of age, PyMTtg/;ARC -/- mice exhibited a 34% decrease in primary tumor burden with no change in tumor onset and multiplicity. The reduced tumor burden can be accounted for by decreased proliferation in PyMTtg/;ARC-/- tumors while baseline apoptosis remained comparable. PyMTtg/;ARC -/- mice also have a 44% reduction in the number of lung metastases compared to PyMTtg/;ARC+/+. In vivo invasion assays revealed that PyMTtg/;ARC -/- tumors have a reduced number of invading cells that are chemo-sensitive in response to tamoxifen. PyMTtg/;ARC-/- mice also displayed a decreased number of circulating tumor cells as revealed by blood burden intravasation assays. Overexpression of ARC in a mouse metastatic cell line caused a significant increase in basement membrane invasion in vitro and lung metastases when injected into mice by tail vein. The knock down of ARC in the LM2 human metastatic cell line resulted in a 50% reduction of orthotopic tumor volume and a substantial decline in invasion compared to control tumors. This data implicates that ARC promotes primary tumor growth and improves the efficiency of metastasis while rendering the invading cell population resistant to chemotherapies.;Cell death by apoptosis is necessary during development and in adult tissue homeostasis. Evasion of apoptosis is critical for tumorigenesis. ARC (Apoptosis Repressor with CARD Domain), an inhibitor of both the death receptor and mitochondrial apoptosis pathways, is induced in human epithelial cancers including breast cancer. To explore the role of ARC in acinar formation and carcinogenesis MCF-10A cells were applied in a three dimensional basement membrane (Matrigel) cell culture model. Exogenous over expression of ARC did not prevent, nor delay apoptosis of acini lumens. However, endogenous ARC mRNA and protein levels in MCF-10A acini increased incrementally after day 5 in culture. The depletion of ARC prevented acinar growth and induced a G1 cell cycle arrest. This data suggests that ARC has a function in normal mammary morphogenesis. To test ARC's relevance in an in vivo model of breast cancer, ARC-/- mice were bred with MMTV-PyMT transgenics which have mammary specific activation of oncogenic pathways common to human breast cancer, including the induction of ARC. At 14 weeks of age, PyMT tg/;ARC-/- mice exhibited a 34% decrease in primary tumor burden with no change in tumor onset and multiplicity. The reduced tumor burden can be accounted for by decreased proliferation in PyMTtg/;ARC -/- tumors while baseline apoptosis remained comparable. PyMT tg/;ARC-/- mice also have a 44% reduction in the number of lung metastases compared to PyMTtg/;ARC+/+. In vivo invasion assays revealed that PyMTtg/;ARC -/- tumors have a reduced number of invading cells that are chemo-sensitive in response to tamoxifen. PyMTtg/;ARC-/- mice also displayed a decreased number of circulating tumor cells as revealed by blood burden intravasation assays. Overexpression of ARC in a mouse metastatic cell line caused a significant increase in basement membrane invasion in vitro and lung metastases when injected into mice by tail vein. The knock down of ARC in the LM2 human metastatic cell line resulted in a 50% reduction of orthotopic tumor volume and a substantial decline in invasion compared to control tumors. This data implicates that ARC promotes primary tumor growth and improves the efficiency of metastasis while rendering the invading cell population resistant to chemotherapies.