Over-expression of PGRMC1 enhances triple-negative breast cancer growth

Sierra Gallaway

doi:10.7273/000003763

Triple-negative breast cancer (TNBC) tumors, which lack expression of the classical progesterone and estrogen receptors, as well as the HER2 receptor, have the highest breast cancer mortality rate due to its resistance to conventional therapies and high rates of recurrence and metastasis. The expression of progesterone receptor membrane component 1 (PGRMC1) is commonly elevated in several women’s cancers, particularly TNBC. While such correlative gene expression studies have been informative, they do not clearly demonstrate that changes in the expression of PGRMC1 contribute to the development and/or progression of these cancers. As such, the objective of this study was to evaluate the role of PGRMC1 in the development and progression of TNBC tumors using a mouse xenograft tumor model. Based on previous PGRMC1 studies, the hypothesis is that PGRMC1 facilitates the progression of TNBC tumor growth, producing larger tumors. Here, MDA-MB-231 (MDA) TNBC cells were transfected with empty vector or PGRMC1-3XFlag lentiviral vector constructs to generate cell lines in which PGRMC1 protein is expressed at endogenous levels (PGRMC1-intact) or is over-expressed (PGRMC1-3XFLAG). A previously generated MDA cell line deficient in PGRMC1 by constitutive siRNA (i.e., PGRMC1-deplete) was also included in these studies. Western blotting and immunocytochemistry were used to validate endogenous PGRMC1 or PGRMC1-3XFLAG expression in these cells lines. As expected, the PGRMC1-3XFLAG cells expressed both the endogenous and Flag tagged PGRMC1 proteins. Intraperitoneal TNBC tumors were developed in immunocompromised nude mice by inoculating each mouse with 1 X 107 PGRMC1-intact, PGRMC1-deplete, or PGRMC1-3XFLAG cells (n=10 each). Tumors were harvested, weighed, and evaluated for mitosis and vascular development prior to 10-weeks post-inoculation. PGRMC1- intact and PGRMC1-3XFLAG tumors grew faster and larger than PGRMC1-deplete tumors (p<0.05). Interestingly, we observed more tissue necrosis in PGRMC1-intact tumors than in PGRMC1-3XFLAG tumors, suggesting that PGRMC1-3XFLAG tumors had a growth advantage. To test this, immunohistochemistry was used to assess mitosis and vascular development. As determined by the marker phospho-histone H3, mitosis was significantly elevated in PGRMC1-3XFLAG tumors compared with PGRMC1-intact tumors despite not being different in overall mass (p<0.05). Likewise, vascular development was also higher in PGRMC1-3XFLAG tumors based on immunostaining for the endothelial cell marker CD31 (p=0.001). In summary, this study shows that elevated expression of PGRMC1 in TNBC cells increases xenograft breast tumor growth through increased mitosis and angiogenesis. A key to understanding PGRMC1 mechanism of action will be to begin identifying PGRMC1-interacting proteins. Given the presence of a ligand binding domain, PGRMC1 may useful for pharmacological or gene therapy targeting as a mechanism to treat TNBC.

Over-expression of PGRMC1 enhances triple-negative breast cancer growth

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