Anticancer activity of the cholesterol exporter ABCA1 gene

Abstract

The ABCA1 protein mediates the transfer of cellular cholesterol across the plasma membrane to apolipoprotein A-I. Loss-of-function mutations in the ABCA1 gene induce Tangier disease and familial hypoalphalipoproteinemia, both cardiovascular conditions characterized by abnormally low levels of serum cholesterol, increased cholesterol in macrophages, and subsequent formation of vascular plaque. Increased intracellular cholesterol levels are also frequently found in cancer cells. Here, we demonstrate anticancer activity of ABCA1 efflux function, which is compromised following inhibition of ABCA1 gene expression by oncogenic mutations or cancer-specific ABCA1 loss-of-function mutations. In concert with elevated cholesterol synthesis found in cancer cells, ABCA1 deficiency allows for increased mitochondrial cholesterol, inhibits release of mitochondrial cell death-promoting molecules, and thus facilitates cancer cell survival, suggesting that elevated mitochondrial cholesterol is essential to the cancer phenotype.

Figure 1. Reconstitution of ABCA1 Expression Inhibits Tumor Formation

(A) Western blot showing murine ABCA1 protein expression in YAMC, mp53, Ras and mp53/Ras cells. (B) Western Blot showing stable re-expression of ectopic murine ABCA1 in mp53/Ras cells to levels found in YAMC parental cells. Tubulin served as loading control for western blots. (C) Vector control or ABCA1-reconstituted mp53/Ras cells were subcutaneously injected in the flanks of nude mice and tumor volumes were measured at indicated times after injection. Values represent average of 16 tumors and error bars indicate standard deviation at each time point. *P <0.001, by student’s t-test. (D) Cell death of vector control and ABCA1-reconstituted mp53/Ras cells (ABCA1). Cell death was measured by trypan blue exclusion (left panel), with no indication for DNA fragmentation/apoptosis, as measured by TUNEL staining (right panel). Values represent the average of three independent experiments. Error bars indicate standard deviation. *P <0.01 versus vector control, by student’s t-test. See also Figure S1

Figure 2. ABCA1 Reconstitution and Knockdown of Lanosterol Cyclase (LC) Decrease Mitochondrial Cholesterol and Increase Sensitivity to Calcium-Induced Cytochrome C Release and Matrix Swelling

(A, B) Cholesterol in mitochondria from vector control, ABCA1-reconstituted (ABCA1), and lanosterol cyclase knockdown (LC kd) mp53/Ras cells and following cholesterol restoration (ABCA1+Chol and LC kd+Chol). Cholesterol levels determined by Amplex Red cholesterol assay and normalized to total mitochondrial protein. Indicated values represent average from three independent experiments and (B) two independent LC shRNA constructs. Error bars indicate standard deviation, **P < 0.01 versus vector by student’s t-test. (C, D) Calcium induced release of cytochrome C (Cyt C) from mitochondria of cells indicated in (A, B). Cyt C was measured by western blot, with cytochrome oxidase I (COX1) serving as loading control. Blots shown are representative of three independent experiments. (E, F) Calcium induced swelling of mitochondria from cell lines indicated in (A, B). Mitochondrial swelling was assessed by measuring changes in absorbance at 540nm. Error bars indicate standard deviation, *P < 0.001 versus vector by student’s t-test. See also Figure S2

Figure 3. Suppression of CypD or Ant1 Restores Death Resistance of ABCA1-Reconstituted mp53/Ras Cells in vitro and Rescues Tumor Growth in vivo

(A, B) Cell death of the indicated derivatives of mp53/Ras cells: vector control (Vector/Vector), ABCA1-reconstituted (ABCA1/Vector), ABCA1-reconstituted plus Cyclophilin D knockdown (ABCA1/CypD-sh3 and -sh5) or Adenine Nucleotide Translocator 1 knockdown (ABCA1/Ant1-sh3 and -sh4), Cyclophilin D knockdown (Vector/CypD-sh3 or -sh5), and Adenine Nucleotide Translocator 1 knockdown (Vector/Ant1-sh3 or -sh4). Cell death was measured by trypan blue exclusion. Values represent the average of three independent experiments and error bars indicate standard deviation. *P < 0.01 versus all other cell lines, by student’s t-test. (C, D) Box plots indicating the volumes of tumors four weeks after subcutaneous implantation of the mp53/Ras cell derivatives indicated in (A, B). Each box shows the range from the first to third quartile of the tumor volumes. The line in the box indicates the median tumor volume. The bars represent the largest and smallest tumors. *P < 0.01 versus all other cell lines, by student’s t-test. (E, F) Western Blots showing expression of ABCA1. Tubulin serves as a loading control. (G, H) Expression of CypD, and Ant1 was measured by real time quantitative PCR. Error bars indicate standard deviation. See also Figure S3

Figure 4. Loss of ABCA1 Efflux Function Correlates with Reduced Tumor Suppression

(A, B) Box plots indicating the volumes of tumors five weeks after subcutaneous implantation of the indicated derivatives of human colon cancer cell lines: (A) HT-29 and (B) DLD-1 expressing wild type human ABCA1 (hABCA1 WT) or the indicated ABCA1 mutants. Each box shows the range from the first to third quartile of the tumor volumes. The line in the box indicates the median tumor volume. The bars represent the largest and smallest tumors. *P < 0.01 versus WT hABCA1 expressing cell line, by student’s t-test. (C, D) Cholesterol efflux capacities of hABCA1 mutants expressed in (A) HT-29 and (B) DLD-1 cells relative to hABCA1 wild type. Values indicate percentage of wild type efflux and represent the average of three independent experiments. Error bars indicate standard deviation. *P < 0.01 versus WT hABCA1 expressing cell line, by student’s t-test. Images below each graph are western blots showing representative protein expression of WT and mutant hABCA1 in cell lines used in (A, B, C, and D). See also Figure S4