Transcription Factor Gli1 Is Involved In The Transition Of Breast Cancer Cells From Estrogen Dependent To Independent Stage

Breast cancer is one of the most commonly found solid tumors in women. It has long been established that estrogen sustains the growth of breast cancer cells expressing functional estrogen receptors (ER). Although the majority of breast cancers are ERαpositive and respond to antiestrogen therapy which aims to block estrogen stimulation of breast cancer cells, many of them will inevitably progress to the estrogen-independent stage and resist to antiestrogen therapy. Understanding molecular mechanisms involved in the development of estrogen independence in breast cancer cells is thus crucial for developing more effective therapies for estrogen-independent breast cancers.Hedgehog (Hh)/ Gli signaling controls a variety of developmental processes such as pattern formation, differentiation, proliferation, and organogenesis. Besides its crucial roles in embryogenesis, Hh/Gli signalling has recently attracted considerable interest in the field of cancer research, as a series of publications has implicated aberrant pathway activation in the growth and maintenance of common malignancies such as basal cell carcinoma (BCC), lung, esophageal and biliary cancer, as well as pancreatic and prostate cancer. Binding of Hh-protein to the Patched1 (Ptc1) receptor abolishes the inhibitory effect of Ptc1 on Smoothened (Smo), allowing Smo to transduce the signal towards the nucleus, a process regulated by complex interactions and modifications of a large number of cytoplasmic proteins. The net effect of pathway stimulation is the activation of members of the Gli family of zinc finger transcription factors, which translate the extra-cellular Hh-stimulus into defined transcriptional programs in a context-dependent and cell-type specific manner.Vertebrates have at least 3 distinct Gli genes, Gli1, Gli2 and Gli3. The highly conserved DNA binding domain of Gli proteins comprises 5 zinc finger domains of the C2-H2 class and all Gli proteins were shown to bind to the consensus sequence GACCACCCA. In addition, Gli1 proteins contain a C-terminal transactivation domain, but not an N-terminal repression domain. Gli1 is also a target gene of Hh signaling pathway. Gli1 is mainly regulated at the transcriptional level. In fact, induction of Gli1 mRNA expression by Hh-signalling is a reliable marker for pathway activity. A common property of HH-associated cancers is the elevated expression level of one or more Gli transcription factors. Keratin5-promoter driven expression of Gli1 in the epidermis of transgenic mice leads to development of epidermal tumours with characteristics of BCC. Evidence that Gli1 is required for proliferation of tumour cells in man has come from RNAi-based loss-of-function studies showing that primary prostate carcinoma cells cease to proliferate when treated with Gli1 siRNA. Gli1 also appears to control the metastatic properties of prostate cancer cells, since its overexpression is sufficient to convert low-metastatic prostate carcinoma cells into highly metastasizing lines. Furthermore, in vivo Gli1 is mediating critical downstream processes in Hh-associated brain tumours, despite the fact that Gli1 is dispensable for mouse development.The Hh/Gli pathway has also been established as an important signaling system in normal mammary gland development. High Gli1 levels were note in more than 60% of the breast cancers samples. In addition, treatment with the cyclopamine, a steroidal alkaloid that blocks the Hh pathway, suppresses the expression of Gli1 and growth of the Hh pathway-activated breast carcinoma cells.There is no direct evidence that whether Gli1 itself has any effect on the growth of breast cancer cells and whether it is involved in the transition of breast cancer from estrogen-dependent to–independent states.We examined the endogenous mRNA expression of ERα, ERβ, Gli1 and Patch1 in human breast cancer cells lines by Quantitative real-time PCR (QRT) and found that mRNA levels of Gli1 and Patch1 were obviously higher than that in estrogen-dependent cells. In addition, data indicated there was a negative relationship between the mRNA levels of ER and Gli1/Patch1. On the basis of these results, we did the following work: 1. We established stable Gli1 transfectants in MCF-7 cells and found Gli1 mRNA levels of Gli1-clone1 and Gli1-clone2 were about 12- fold and 6 -fold of the control cells ( pcDNA3.1-MCF-7) respectively.2. Using stable Gli1 transfectants, we found that expression of Gli1 in MCF-7 cells induced estrogen-independent proliferation, indicating that Gli1 may be involved in the development of estrogen independence in these cells.3. Moreover overexpression of Gli1 attenuated response to estrogen-induced growth and antiestrogen-induced inhibition of cell growth in vitro. At the condition of without supplying estrogen pellets, pcDNA3.1 cells did not form tumors, but Gli1-clone1 cells formed very small tumors in nude mice. The Gli1-transfectants (Gli1-clone1) form xenografts more rapidly in nude mice supplied with estrogen and continued growth, compared with pcDNA3.1-MCF-7 tumors, whose growth is inhibited by the estrogen withdrawal either alone or in the presence of tamoxifen.4. Then we found Gli1-reduced response was correlated with down-regulation of expression of ERαand PR, and decreased transactivation of ERα, as confirmed by quantitative real-time PCR (QRT), western blot analysis and luciferase assays. We also observed Gli1-induced activity of NF-κB, which may be involved in Gli1-induced estrogen-independent development of breast cancer as well.5. In addition, we also found that ectopic expression of ERαcan induce the expression and transcriptional activity of Gli1.In summary, here we demonstrated that the expression of Gli1 in estrogen-independent breast cancer cells was much higher than that in estrogen-dependent cells. Overexpression of Gli1 attenuated the response to estrogen-induced growth and antiestrogen-induced inhibition of cell growth, which was associated with down-regulation of expression of ERαand PR, and also decreased transactivation of ERα. Gli1-induced Activation of NF-κB may also be invoved. In addition, we also found that ectopic expression of ERαcan induce the expression and transcriptional activity of Gli1. Our study indicated that Gli1 may be involved in the transition of breast cancer from estrogen dependent to independent stage by substituting estrogen signaling in sustaining the growth and the survival of breast cancer cells.