Dual action of apolipoprotein E-interacting HCCR-1 oncoprotein and its implication for breast cancer and obesity

Abstract

Obese women have an increased risk for post-menopausal breast cancer. The physiological mechanism by which obesity contributes to breast tumourigenesis is not understood. We previously showed that HCCR-1 oncogene contributes to breast tumourigenesis as a negative regulator of p53 and detection of HCCR-1 serological level was useful for the diagnosis of breast cancer(.) In this study, we found that the HCCR-1 level is elevated in breast cancer tissues and cell lines compared to normal breast tissues. We identified apolipoprotein E (ApoE) interacting with HCCR-1. Our data show that HCCR-1 inhibits anti-proliferative effect of ApoE, which was mediated by diminishing ApoE secretion of breast cancer cells. Finally, HCCR-1 induced the severe obesity in transgenic mice. Those obese mice showed severe hyperlipidaemia. In conclusion, our results suggest that HCCR-1 might play a role in the breast tumourigenesis while the overexpression of HCCR-1 induces the obesity probably by inhibiting the cholesterol-lowering effect of ApoE. Therefore, HCCR-1 seems to provide the molecular link between the obesity and the breast cancer risk.

Figure 1

HCCR-1 and ApoE expressions in human tissues. (A, B) Northern analyses of HCCR-1 (A) and ApoE (B) in human breast tissues and cell lines. Comparison of HCCR-1 mRNA expressions in breast cancer cell lines (BT-474, MCF-7 and MDA-MB-231), and fresh primary breast cancer tissues (C) and their corresponding normal counterparts (N). Human β-actin cDNA was used as a control probe (bottom panel). (C, D) mRNA expression levels of ApoE in various normal human tissues and cancer cell lines. Northern-blots consisting of 8 human cancer cell lines (C) and 12 normal human tissues (D) were probed with radioactively labelled ApoE cDNA (top panel).

Figure 2

Interaction of HCCR-1 with ApoE and their co-localization in the mitochondria. (A) Co-immunoprecipitation experiment of HCCR-1 and ApoE showing the direct interaction between HCCR-1 and ApoE. Cell lysate from MCF-7 cells expressing either HCCR-1-V5 or ApoE-Myc, or both was immunoprecipitated with mAb specific for Myc and HCCR-1 was detected by Western blotting using V5 mAb (left-hand panel). Likewise, ApoE interacting with HCCR-1 was detected by Western blotting using Myc mAb after co-immunoprecipitation with V5 mAb (right-hand panel). (B) Subcellular fractionation of HCCR-1 and ApoE in HEK293 cells. Cytosolic (C), nuclear (N) and mitochondrial (M) fractions were prepared from HEK293 cells transfected with HCCR-1, ApoE or both and analysed by SDS-PAGE and immunoblotting using antibodies, V5 and c-Myc for HCCR1 (top panel) and ApoE (bottom panel), respectively. The Voltage Dependant Anion Channel 1 (VDAC1) was used as a mitochondrial marker. (C) Fluorescence microscopy. Cells were transiently transfected with pEGFP-HCCR-1 (left-hand panel) and pEGFP-ApoE (right-hand panel) using LipofectAMINE 2000 (Invitrogen). Cells were incubated with 25 nM MitoTracker (Molecular Probes). Cells were then mounted using ProLong Gold Antifade Reagents. Fluorescent images were analysed by a Bio-Rad MRC-1024MP laser scanning confocal microscope (Bio-Rad).

Figure 3

HCCR-1 negatively regulates ApoE function in breast cancer. (A, B) Anti-proliferative effect of ApoE in breast cancer. (A) MCF-7cells were transfected with HCCR-1, ApoE or both and vector as a negative control. The data are the number of viable cells grown for 10 days in culture and represent the mean ± S.D. of triplicate determinations. (B) Transfection with ApoE cDNA induces apoptosis in breast cancer cells. Electrophoretic analysis of DNAs from MCF-7 cells transfected with vector alone, HCCR-1, ApoE or both. Cells were cultured for 1, 3, 5 and 7 days, respectively, and their genomic DNAs were analysed on 2% agarose gel stained with ethidium bromide. (C, D) ApoE secretion by MCF-7 cells. ApoE was measured in MCF-7 cells by ELISA, as described in ‘Materials and Methods.’ Results are represented as mean ± S.D. of triplicate determinations. The ApoE concentration was determined by sandwich ELISA using a commercially available kit (MBL ApoE4/Pan-ApoE ELISA kit, MBL Co.).

Figure 4

HCCR-1 interacting with ApoE induced the severe obesity in T/G. (A, B) Phenotypic analyses of HCCR-1 T/G. (A) The obesity of T/G. T/G were generated using standard pronuclear microinjection. For microinjection, the fragment of transgene, CMV-HCCR-1-bGH, was microinjected into the pronuclei of one cell-stage fertilized embryos from C57BL/6N (Charles River, Japan). Obese T/G and non-T/G are shown together with their body weights represented as the mean ± S.D. of 7-times determinations. (B) Haematoxylin-eosin staining of omentum, liver, pancreas and heart taken from the male (top panel) and female TG (second panel) together with the control male non-TG (bottom panel). Original magnification × 200. (C) Expression levels of ApoE and HCCR-1 in various organs of obese T/G and control mice. To detect the ApoE and HCCR-1 expressions, mAbs (A1.4) raised against ApoE of human origin (Santa Cruz biotechnology) and HCCR-1 polyclonal Ab were used in the immunoblot consisting of multiple tissues taken from T/G and non-T/G such as brain, lung, liver, kidney, intestine and omentum. (D) Serum profiles of cholesterol, triglyceride, ApoE, leptin and insulin in obese and control mice. Results are expressed as mean ± S.D. of triplicate determinations.