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

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

Suppression of PC expression in MDA-MB-231 lowers invasion ability.

<p>MDA-MB-231 cells were transiently transfected with PC or scrambled control siRNAs. At 48 h post transfection, an <i>in vitro</i> invasion assay was performed for 4 h in the presence of 4 mM (A) or 0 mM (C) glutamine. The number of PC siRNA-transfected cells that invaded the transwell coated with Matrigel was counted in 5 different fields and shown as means <u>+</u> standard deviation in comparison with that of the scrambled control which was arbitrarily set as 100% (B, D). The results were obtained from three independent experiments, each done in duplicate. The statistical analysis was conducted using student’s t-test where ** <i>P</i> < 0.01.</p

Suppression of PC expression in MDA-MB-231 cells reduced migration.

<p>Representative images of wound-healing assays. MDA-MB-231 cells were transiently transfected with PC or scrambled control siRNAs. At 48 h post transfection, wound-healing assays were performed as described in the materials and methods. (A, C) Representative images of the PC knockdown (PC siRNA) or scrambled control cells (Control) migrated across the wound areas in the presence of 4 mM (A) or absence of glutamine (C). The wound’s closure (width) of the PC knockdown was measured and shown as the means <u>+</u> standard deviation of that of the scrambled control which was arbitrarily set as 100% (B, D). The results were obtained from two independent experiments, each in triplicate. The statistical analysis was conducted using student’s t-test where *<i>P</i> < 0.05.</p

Migration ability of various breast cancer cell lines and the expression levels of PC in these cell lines.

<p><b>A</b>, Migration ability of SKBR3, MCF-7, MDA-MB-435 and MDA-MB-231. <b>B</b>, Q-PCR analysis of PC mRNA expression in the above cell lines. The expression of PC was normalized with the expression of 18s rRNA gene and shown as the relative gene expression. The relative PC expression in MCF-7 was arbitrarily set as 1. <b>C</b>, Real time PCR analysis of PC mRNA variant 1 and 2 expression in the above cell lines. The expression of PC was normalized with the expression of 18s rRNA gene and shown as the relative gene expression. The expression of the relative PC mRNA variants in MCF-7 was arbitrarily set as 1. <b>D</b>, Western blot analysis of PC protein in the above cell lines. The blot was also probed with anti-actin antibody to serve as loading control. <b>E</b>, The immunoreactive band intensity of PC in D was quantitated and normalized with that of the β-actin and shown as the relative PC expression. The statistical analysis was conducted using student’s t-test where *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Overexpression of PC in MCF-7 cells increases their proliferation, migration and invasion.

<p>Proliferation rate of MCF-7 cells overexpressing PC grown in the medium containing 4 mM (A) or 0 mM glutamine (B). Insets in A and B are the Western blot analysis of MCF-7 cells transfected with empty vector (+empty, white bars) or with overexpressed PC (+PC, black bars) at days 0, 3 and 7. MCF-7 cells overexpressing PC grown in the presence or absence of glutamine were also subjected to migration (C) and invasion (D) assays. The statistical analysis was conducted using student’s t-test where * <i>P</i> < 0.05, ** <i>P</i> < 0.01.</p

Suppression of PC expression in MDA-MB-231 retarded proliferation both in glutamine-nourished and glutamine-depleted conditions.

<p>MDA-MB-231 cells were transiently transfected with PC or scrambled control siRNAs. At 48 h post transfection cells were trypsinized, re-plated and grown in the presence of 0 mM or 4 mM glutamine for 7 days. The proliferation rate of the PC knocked down (PC siRNA) and the control MDA-MB-231 cell lines (Control) grown in the medium containing 4 mM (A) or 0 mM (C) glutamine. The relative expression of PC mRNA in the knocked down MDA-MB-231 cells grown in the presence of 4 mM (B) or 0 mM (D) glutamine throughout the assay. The results are means obtained from two independent experiments, each in triplicate. The statistical analysis was conducted using ANOVA test where *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Immunohistochemistry staining of PC in paraffin-embedded breast tissue sections of patients with various stages of breast cancer.

<p>(A) Normal adjacent area of breast tissue showing weak staining of PC compared to strong staining in the cancerous area (B) of the same tissue. The representative samples showing different expression levels of PC in different stages of breast cancer: (C) stage 1, (D) stage 2, (E) stage 3 and (F) stage 4. Original magnification, 100x.</p

siRNA-mediated suppression of PC expression in MDA-MB-231 cell line.

<p>Real time PCR analysis of PC mRNA expression in MDA-MB-231 cells transfected with scrambled control (Control) or PC siRNA. The PC mRNA level was determined by Q-PCR at 48 h post-transfection (A). Western blot analysis of PC protein in the PC knockdown MDA-MB-231 and the scrambled control (B). The statistical analysis was conducted using student’s t-test ***<i>P</i> ≤ 0.001.</p