CRBP suppresses breast cancer cell survival and anchorage-independent growth.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Retinol conversion to retinoic acid is impaired in breast cancer cell lines relative to normal cells.

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.

Methylation of conserved CpG sites neighboring the beta retinoic acid response element may mediate retinoic acid receptor beta gene silencing in MCF-7 breast cancer cells.

We investigated the mechanism of retinoic acid receptor (RAR) beta2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous beta2 promoter (-1470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARbeta2. This was true even in the context of replicated chromatin, suggesting a cis-acting rather than a trans-acting defect. Cytosine methylation, a cis-acting DNA modification, has been implicated in RARbeta2 silencing in cancer cells. Upon bisulfite genomic sequencing, we found that 3 CpG sites in the beta2 RARE region were variably methylated in MCF-7 cells but were not methylated in MTSV1.7 cells or in 2 MDA-MB-231 subclones that differed in RARbeta2 expression (high in clone A2, low in clone A4). However, the 5'-UTR region was hypermethylated in clone A4 relative to clone A2 cells. Following 5-azacytidine treatment, RA and trichostatin A markedly induced RARbeta2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A beta2 RARE reporter construct in which the methylation-susceptible cytosines in the sense strand were replaced by thymine displayed marked loss of activity in a replicated chromatin-dependent manner. We conclude that cytosine methylation contributes to RARbeta2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important. Oncogene (2000) 19, 4066 - 4070.

Cellular retinol-binding protein expression and breast cancer.

BACKGROUND: The biologic activity of vitamin A depends, in part, on its metabolism to active nuclear receptor ligands, chiefly retinoic acid. The cellular retinol-binding protein (CRBP) binds vitamin A with high affinity and is postulated to regulate its uptake and metabolism. In this report, we analyze the expression of CRBP in normal and malignant breast tissues. METHODS: We evaluated CRBP expression by in situ hybridization in six reduction mammoplasty specimens and 49 human breast carcinoma specimens by use of digoxigenin-labeled RNA probes and in nine cultured mammoplasty specimens by northern or western blot analysis. Statistical significance was evaluated with the chi(2) test or Fisher's exact test if the sample sizes were small. All P values are from two-sided tests. RESULTS: CRBP was expressed in all 15 mammoplasty specimens (normal breast tissue) and in 33 of 35 available specimens of normal tissue adjacent to carcinoma. In contrast, 12 (24%) of 49 carcinoma lesions were uniformly negative for CRBP (P =.023 for comparison with adjacent normal breast tissue). The loss of CRBP expression was as frequent in ductal carcinoma in situ (six [27%] of 22) as in invasive lesions (six [22%] of 27), suggesting that it is a relatively early event in carcinogenesis and not associated with patient age, tumor grade, and expression of steroid receptors or c-Myc. Preliminary experiments did not find an association between CRBP and retinoic acid receptor beta loss, but most (four of five) CRBP-negative tumors were also retinoic acid receptor beta negative. CONCLUSION: CRBP is underexpressed in 24% (95% confidence interval = 12.5%-36.5%) of human breast carcinomas, implying a link between cellular vitamin A homeostasis and breast cancer. We hypothesize that the loss of CRBP restricts the effects of endogenous vitamin A on breast epithelial cells.