Pyruvate Carboxylase is Essential for Breast Cancer Metastasis in Vivo

Abstract

Anaplerosis, or the replenishment of TCA cycle intermediates utilized for biosynthetic pathways, has recently emerged as an integral part of breast cancer cell metabolism. In particular, the enzyme pyruvate carboxylase (PC), responsible for facilitating pyruvate flux through oxaloacetate into the TCA cycle, has gained recognition as an essential component of breast tumor growth and metastatic capability. Therefore, the objective of the present studies was to investigate the role of PC in breast cancer utilizing the MCF10 model of breast cancer progression in vitro as well as in vivo utilizing metastatic murine 4T1 cells in immunocompetent Balb/C mice. PC mRNA expression was not significantly different between MCF10A untransformed and Harvey‐ras oncogene transfected cells (MCF10A‐ras), representing initiation. However metastatic MCF10CA1a cells, representing the latest stage in the model, had a 5.14(±0.21) fold higher PC mRNA abundance relative to both the MCF10A and MCF10A‐ras cells. In addition, PC overexpression achieved through transient transfection of a pCMV6‐PC plasmid increased cell migratory capability of both MCF10A‐ras (183%±46 relative to Neo control) and MCF10CA1a cells (191%±31 relative to Neo control), as measured by a Boyden Chamber migration assay. Interestingly, the same overexpression of PC in untransformed MCF10A cells resulted in their acquisition of a migratory phenotype in vitro (252%±34 relative to Neo vehicle). To test the role of PC in vivo, murine metastatic 4T1 breast epithelial cells expressing either PC silencing shRNA or a scramble control were engrafted onto the mammary fat pad of 6 week old Balb/C mice. Five weeks after engraftment, primary tumor and pulmonary metastasis were analyzed. Surprisingly, there was no difference in primary tumor size or volume between shPC and scramble control 4T1 breast epithelial cells. PC silencing, however, had a drastic impact on metastatic capability of cells with nearly complete inhibition of pulmonary metastasis (pulmonary mets per mouse of shPC#25=6±2 and shPC#28=8±4 scramble control=31±10). Together, these results suggest that PC plays an essential role in breast cancer progression, particularly in metastasis of cells from the primary tumor to distant secondary organs.Support or Funding InformationThis work was supported by a Project Development Team within the ICTSI NIH/NCRR (Grant Number UL1TR001108) and the National Institutes of Health, National Cancer Institute(R25CA128770) Cancer Prevention Internship Program. Additional support was received from the Indiana Elks Charities and the SIRG grant, both administered through the Purdue University Center for Cancer Research (Purdue University, West Lafayette, IN).