Project 1 Pharmacology and physiology of retinoids in vivo. 1. Identification of Cyp2C39 as a novel retinoic acid metabolizing enzyme of mouse liver and its upregulation during malignant progression. Murine CYP2C39, along with other close isoforms of the CYP2C subfamily of P450, was found to be an active metabolizer of arachidonic acid and to be expressed exclusively in the liver. We have recently identified mCYP2C39 as a novel retinoic acid 4-hydroxylase and proposed that its downregulation offers a molecular basis for liver retinoid accumulation and fibrosis in Aryl Hydrocarbon Receptor (AHR)-null mice. There is clear evidence that AHR status controls the retinoid homeostasis and mCYP2C39 seems to be the key molecule that links the carcinogen metabolism to retinoid homeostasis.It is also well established that CYP26A1, the only other retinoic acid 4-hydroxylase known so far, plays a role in controlling the susceptibility of cancer cells to retinoids; cytochrome P450 retinoic acid 4-hydroxylase inhibitors enhance growth inhibition by RA and are being used as potential agents for cancer therapy. Depletion of retinoids has been shown in tumor tissues in both experimental and clinical hepatocellular carcinogenesis. Both the absence of hepatic stellate cells (HSCs), the retinoid storing cells in liver, and a rapid metabolism of retinoic acid have been implied as responsible for such phenotype.We investigated the potential involvement of the new hepatic retinoic acid 4-hydroxylase, mCYP2C39, in liver carcinogenesis. Initially we checked whether changes in its expression occur during malignant progression of hepatocellular carcinoma and, if so, at which stage they start to be evident; furthermore, we were also interested in evaluating whether these changes are associated with altered retinoid status (retinoid levels, in vitro RA metabolism) and expression of lecithin:retinol acyl transferase (LRAT), the enzyme responsible for liver retinoid storages. To this end, we started a collaboration with Dr Snorri Thorgeirsson's laboratory, where it has been established that cooperative growth stimulus generated by liver targeted MT/TGFa and Alb/c-myc transgene expression has a dramatic synergistic effect in liver carcinogenesis in TGFa/c-myc double transgenic mice. Specifically, TGFa/c-myc mice experience a severe liver cell dysplasia preceding and accompanying the early development of preneoplastic and neoplastic lesions. We checked both mCYP2C39 and LRAT expression (mRNA and protein level) in normal and tumor tissues at different stages of liver carcinogenesis (wild type, early dysplasia, late dysplasia and hepatocellular carcinoma, respectively), obtained from both single (c-myc) and double (TGFa/c-myc) transgenic mice. Real time RT-PCR data showed a gradual increase in mCYP2C39 mRNA expression with tumor progression culminating with a 9-fold increase in hepatocellularcarcinoma samples compared to wild-type control tissues, in both c-myc and TGFa/c-myc mice. Interestingly, mRNA and protein expression levels of mCYP26A1, the only other known liver RA 4-hydroxylase, didn't change in tissues at the different stages of carcinogenesis from both the single (c-myc) and double (TGFa/c-myc) transgenenic mice. LRAT mRNA and protein expression showed an opposite expression pattern compared to mCYP2C39; in fact, its expression was found to gradually decrease with tumor progression. Hepatocellular carcinoma samples showed about a 60% reduction of LRAT mRNA and protein expression compared to the wild-type control tissues in both c-myc and TGFa/c-myc mice. These results could explain the depletion of retinoids associated with hepatocarcinogenesis. 2. Influence of retinoids on normal and malignant human breast epithelial cells. Our previous data has shown that RXRa localizes to the nuclear splicing factor compartments in highly malignant, retinoid resistant MDA-MB-231 cells, causing disruption of the retinoid signaling pathway. We have studied whether nucleoplasmic RXRa was able to restore retinoid sensitivity in MDA-MB-231. Adenoviral RXRa overexpression induced cell growth arrest at the G0/G1 phase in accordance with rapid upregulation of p21WAF1/CIP1 and downregulation of pRB. Ligand-mediated p21 activation was regulated by time, adenovirus, and ligand in a dose-dependent manner. Moreover, Northern blot and p21 promoter assay revealed that p21 expression was regulated at the transcriptional level. Our data demonstrated that an RARa dominant-negative construct did not interfere with either the reduction of RXR mediated p21 upregulation or G1/S arrest. Further RAR did not induce p21 promoter activity in response to RAR ligand, but it reduced RXR-mediated p21 promoter activity presumably due to allosteric inhibition by RAR. This RXRa induced-p21 upregulation was not due to ligand dependent heterodimer, suggesting that either RXR homodimer or RXR ligand-dependent heterodimer with an orphan receptor might regulate p21. Furthermore, reporter assay with a series of p21 promoter deletion mutants demonstrated that RXRa directly targets the p21 promoter region and a point mutation of the putative RXRE abolished ligand mediated-p21 promoter activity. Nuclear extract from MB-231 cells infected with Ad-RXRa was able to bind to the putative RXRE of the p21 promoter region, more than the one with Ad-null, and this retarded band was supershifted by addition of an antibody to RXR. Biotinylated putative p21 RXRE was purified by streptavidin agarose, and confirmed that RXRa was able to bind to the RXRE of p21 promoter. Taken together, our study suggests that RXR homodimer is a functional modulator in the control of the cell cycle through p21.
| Andreola, Fausto; Calvisi, Diego F; Elizondo, Guillermo et al. (2004) Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion. Hepatology 39:157-66 |
| Andreola, Fausto; Hayhurst, Graham P; Luo, Gang et al. (2004) Mouse liver CYP2C39 is a novel retinoic acid 4-hydroxylase. Its down-regulation offers a molecular basis for liver retinoid accumulation and fibrosis in aryl hydrocarbon receptor-null mice. J Biol Chem 279:3434-8 |
| Tanaka, T; Dancheck, B L; Trifiletti, L C et al. (2004) Altered localization of retinoid X receptor alpha coincides with loss of retinoid responsiveness in human breast cancer MDA-MB-231 cells. Mol Cell Biol 24:3972-82 |
