Fig 2: Mutant p53 activates CPT1C expression through the miR-200c-ZEB2 axis. (A) miR-200c-deficiency leads to enhanced expression of CPT1C in mammary epithelial cells. The expression of CPT1C and Actin in control (Ctrl) and miR-200c-KO MCF12A cells (MCF12A-Sg1 and MCF12A-Sg2) were examined by immunoblotting. (B) The box and whisker plot of CPT1C expression in p53 wide-type breast cancer patients (n = 657) from TCGA with high (n = 328) or low miR-200c expression (n = 329). The statistical significance of differential CPT1C expression in miR-200c high expression and low expression groups was determined by Wilcoxon rank-sum test. p: corresponding p-value. (C, D) Restoring the expression of miR-200c compromises the enhanced CPT1C expression caused by mutant p53. The expression of CPT1C in control (MCF12A-Ctrl), p53 mutant cells (MCF12A-p53R249S, MCF12A-p53R273H and MCF12A-p53R280K) and miR-200c-overexpressing p53 mutant cells (MCF12A-p53R249S-miR-200c, MCF12A-p53R273H -miR-200c and MCF12A-p53R280K-miR-200c) were examined by Q-PCR (C) or immunoblotting (D). (E) The box and whisker plot of CPT1C expression in breast cancer patients (n = 290) harboring p53 missense mutations with high (n = 145) or low miR-200c expression (n = 145). Statistic method: Wilcoxon rank-sum test. p: corresponding p-value. (F) The box and whisker plot of CPT1C expression in total breast cancer patients (n = 1074) from TCGA with high (n = 537) or low miR-200c expression (n = 537). Statistic method: Wilcoxon rank-sum test. p: corresponding p-value. (G) The Spearman correlation plot for CPT1C versus miR-200c in total breast cancer patients from the TCGA database. R: correlation coefficient value; P: corresponding p-value. (H–J) Knockdown of ZEB2 interferes with the enhanced expression of CPT1C in miR-200c-KO (H). MCF12A-miR-200c-Sg2), p53 mutant-overexpressing (I), MCF12A-p53 R273H), and BT549 (J) cells. (K) The Pearson’s pairwise correlation plot for CPT1C versus ZEB2 in total breast cancer patients from TCGA. R: correlation coefficient value; P: corresponding p-value. The result is derived from bc-GeneExMiner v4.5 (http://bcgenex.ico.unicancer.fr).

Fig 3: Interference of CPT1C expression attenuates mutant p53-mediated biological effects. CPT1C expression was downregulated by RNA interference in mutant p53-overexpressing cells (MCF12A-p53R273H and MCF12A-p53R280K) and BLBC cell lines (BT549 and MDA-MB-231), and these cell lines were then subjected to the following analysis. (A, B) Downregulating CPT1C expression attenuates migration and invasion ability in mutant p53-overexpressing mammary epithelial cells and BLBCs. The migration (A) and invasion (B) ability of CPT1C-KD cells (siPCK2 #1, #3, or #4) were analyzed by migration/invasion assay done in triplicate. The relative migration/invasion ability is presented as a fold change of the numbers of cells passed through trans-wells between control and CPT1C-KD cells (mean ± SD; n = 3). The two sides of the lines above the bars indicated the comparison of each KD clone to the control, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test. *p <0.05; ***p < 0.001. (C) Downregulating CPT1C expression diminishes the anchorage-independent growth in MDA-MB-231 and mutant p53-overexpressing mammary epithelial cells. The anchorage-independent growth ability of CPT1C-KD cells was examined by soft-agar foci formation assay (mean ± SD; n = 3). The two sides of the lines above the bars indicated the comparison of each KD clone to the control, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test. *p <0.05; **p <0.01; ***p < 0.001. (D) Interference of CPT1C expression suppresses stemness/cancer stemness in mutant p53-overexpressing mammary epithelial cells and BLBCs. The stem cell/cancer stem cell properties of CPT1C KD cells were examined by mammosphere forming assay (mean ± SD; n = 3). The two sides of the lines above the bars indicated the comparison of each KD clone to the control, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test. **p < 0.01; ***p < 0.001.

Fig 4: CPT1C regulates mutant p53-mediated biological effects through modulating redox status. (A–D) Total cellular ROS level is elevated by knockdown of CPT1C in MCF12AR273H (A, B) and MDA-MB-231 (C, D) cells and is compromised by N-acetyl cysteine (NAC) treatment. Cells were stained with CellROX™ Green Reagent (A, C) or DCFDA (B, D), then the fluorescence was measured by FACS to determine the total cellular ROS. Similar trends in the representative images were observed in three independent experiments. (E, F) CPT1C deficiency-mediated suppression of cancer stemness and migration ability are rescued by NAC treatment. MCF12AR273H [(E, F), left], MDA-MB-231 [(E, F) middle], and BT549 [(E, F) right] cells treated with NAC (0, 5, or 10 mmol/L) for 1 day and its Ctrl cells were subjected to mammosphere formation assay and transwell migration assay (mean ± SD; n = 3). The two sides of the lines above the bars indicated the comparison of the two groups, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001. (G) The ratio of NADPH/NADP+ is upregulated by mutant p53. The absolute amount of NADPH and NADP+ were measured by CE-TOFMS and CE-QqQMS analysis, and the ratio of NADPH/NADP+ in MCF12A-ctrl and MCF12A-p53R280K were compared and shown in bar graphs (mean ± SD, n = 2).

Fig 5: CPT1C induces EMT and enhances migration, invasion, and stemness in mammary epithelial cells. (A) Ectopic expression of CPT1C induces EMT in mammary epithelial cells. The expressional levels of N-Cadherin, E-Cadherin, and Actin in control (Ctrl) and CPT1C-overexpressing (CPT1C) MCF12A cells were examined by Western Blotting. Relative mRNA expression of N-cadherin and E-cadherin between control cells and CPT1C-overexpressing (CPT1C) MCF12A cells were determined by Q-PCR (mean ± SD, n = 3). (B) CPT1C shows a slight to moderate positive association with EMT in breast cancer patients. Correlation map showing the Pearson’s pairwise correlations among CPT1C, CDH1 (E-cadherin), CDH2 (N-cadherin), VIN (vimentin), and FN1 (fibronectin) in breast cancer patients (N = 4712). Each cell represents a statistical relation between two genes and is colored according to the value of the Pearson correlation coefficient ranging from dark blue (coefficient = -1) to dark red (coefficient = 1). R: correlation coefficient value; P: corresponding p-value. Results are derived from bc-GeneExMiner v4.5 (http://bcgenex.ico.unicancer.fr). (C, D) Ectopic expression of CPT1C enhances cell migration (C) and invasion (D) Migration and invasion ability of CPT1C-overexpressing MCF12A cells were analyzed by migration and invasion assay. The relative migration/invasion ability is presented as a fold change of the numbers of cells passed through trans-wells between control and CPT1C groups (mean ± SD; n=3). The two sides of the lines above the bars indicated the comparison of CPT1C-overexpressing cells to the control cells, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test. **p <0.01; ***p < 0.001. Representative micrographs of migrated (C) or invaded (D) cells were shown on the right. (E) CPT1C overexpression enhances anchorage-independent growth. A soft-agar foci formation assay was performed to determine the anchorage-independent growth ability of CPT1C-overexpressing MCF12A cells. Foci number in triplicate dishes was counted and presented as fold change between control and CPT1C groups (mean ± SD; n = 3). The two sides of the line above the bars indicated the comparison of CPT1C-overexpressing cells to the control cells, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test. ***: p < 0.001. (F) Overexpression of CPT1C enhances stemness. CPT1C-overexpressing MCF12A cells exhibit higher mammosphere forming ability. Control or CPT1C-overexpressing MCF12A cells were grown in suspension culture for 7-8 days to form mammospheres. Mammospheres with a diameter larger than 40 μm were counted and presented as fold change. Results were derived from experiments done in triplicate (mean ± SD; n = 3). The two sides of the line above the bars indicated the comparison of CPT1C-overexpressing cells to the control cells, and the value of fold changes with the asterisks representing significance was marked above the line. Data were analyzed by unpaired t-test. ***p < 0.001. Representative micrographs of mammospheres under a microscope are shown on the right. (G) CPT1C shows a slight to moderate positive association with stemness markers in breast cancer patients. Correlation map showing the Pearson’s pairwise correlations among CPT1C and stemness markers, ALDH1A1, ALDH1A2, ALDH1A3, and CD133 (PROM1) in breast cancer patients. R: correlation coefficient value; P: corresponding p-value. Results are derived from bc-GeneExMiner v4.5 (http://bcgenex.ico.unicancer.fr).