Tuberin and hamartin are aberrantly expressed and linked to clinical outcome in human breast cancer:: The role of promoter methylation of TSC genes

Purpose: The tuberous sclerosis (TSC) genes TSC1 and TSC2 encode the protein products hamartin and tuberin, respectively, and are putative tumour suppressor genes. Germ-line mutation of either TSC gene leads to the development of the heritable disorder TSC. This disorder is characterized by the development of hamartomas in many organs and is associated with the proliferative lung disease, lymphangioleiomyomatosis, the brain tumour giant cell astrocytoma and occasionally with renal cell carcinoma. However, the TSC genes have not been studied in breast cancer. The current study investigated the expression of the TSC gene products and the potential mechanisms of their aberrancy in human breast cancer cells and tissues. Experimental design and results: Using immunohistochemical analysis, both hamartin and tuberin were found to be strongly stained in normal mammary epithelial cells and weakly in stromal cells. In invasive tumour tissues, however, the staining of both proteins were to be markedly reduced (P < 0.01). At message level, although normal and tumour tissues expressed both TSC products, the transcript levels of tuberin was significantly lower in tumour tissues compared with normal tissues (P < 0.05). There was no statistical difference between node negative and node positive tumours with both hamartin and tuberin. Tumours from patients who developed recurrence and died from breast cancer had significantly low levels of tuberin compared with those who remained disease free (P = 0.03 and 0.05, respectively). Likewise, hamartin levels were significantly lower in patients with metastasis, recurrence and mortality, when compared with those remained disease free (P = 0.001, 0.041 and 0.003, respectively). Using methylation specific PCR, the TSC1 promoter was found to be heavily methylated in ZR751, MDA MB 435, and BT549, but not in MCF-7 which expressed highly level of hamartin. TSC1 promoter methylation was also seen in most breast tumours, but only in a limited number of normal tissues. The methylation of TSC2 promoter appears to be less frequent. MDA MB 468, MDA NIB 483, MDA MB 435S and weakly MDA MB 435 were found to have methylated TSC2 promoter. In breast tissues, however, a very small number of samples were found to have methylation of the TSC2 promoter. Conclusion: TSC1 genes are aberrantly expressed in human breast cancer cell lines and breast tumour tissues and their promoters are seen to be methylated in breast tumour tissues. The expression of TSC1 is associated with an unfavourable clinical outcome in patients with breast cancer. (c) 2005 Elsevier Ltd. All rights reserved.