Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Abstract

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.

1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase

“…To test our hypothesis we employed a pCMV6 plasmid expressing PC transcript variant 1 (NM_ 000920.2) inserted downstream of the cytomegalovirus promoter (Origene, Rockville, MD). Previous studies have shown the breast cancer cells predominantly express transcript variant 1 [16], with studies in our laboratory confirming the same pattern of expression in the MCF10 cell model (data not shown). Following transfection with the PC containing pCMV6 plasmid, PC mRNA was significantly increased in MCF10CA1a at two and five days compared to the control empty pCMV6-Neo plasmid, with no significant difference between vehicle and 1,25(OH) 2 D treated cells (Figure 6A).…”

“…Since previous studies [16], as well as studies in our laboratory, confirmed that breast cancer cells predominantly express transcript variant 1, the PC transcript variant 1 (NM_ 000920.2) clone was inserted into the pCMV6-Neo vector downstream of the cytomegalovirus (CMV) immediate-early promoter. For transfection, cells were grown in either 24-well or 6-well plates to 60% confluence and transiently transfected using lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA) for 20 hours according to the manufacturer’s instructions.…”

“…The anaplerotic enzyme pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate and therefore replenishes TCA cycle intermediates [15]. PC was shown to be overexpressed in breast cancer cells and its expression correlates with breast cancer aggressiveness in clinical patients [16]. Interestingly, PC also plays a role in regulation of lipid accumulation and FA synthesis.…”

Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

“…Three alternative 5′ untranslated region (5′UTR) mRNA variants are produced from the two promoters, with variant 2 transcribed from the proximal promoter while variants 1 and 3 are transcribed from the distal promoter. Previous studies demonstrated that human breast cancer cells express primarily transcript variant 1 [33]. To confirm the same pattern of expression in MCF10A- ras cells, real time-PCR analysis of cDNA was performed using three forward primers that bind to the 5′-untranslated regions (UTR) of variant 1, 2 or 3, with a common reverse primer that binds to exon 1 within the coding region to specifically amplify the three different 5′UTR variants.…”

“…An interesting finding in the present study is the characterization of PC as a direct transcriptional target of 1,25(OH) 2 D. The distal P2 promoter, where the VDRE was identified, regulates transcription of PC in breast epithelial cells as well as pancreatic beta cells [33,38]. PC further plays an important role in glucose stimulated insulin secretion in the latter cell type [32].…”