Study Of Experiment And Clinicopathology On Pathogenesis Of Breast Cancer Stem Cell

Breast cancer is the deadliest form of cancer affecting women worldwide. Although effective therapies have been developed for breast cancer, at present the prognosis of most of patients is poor. A great deal of interest has focused on mutation or aberrant regulation in stem cells as a key factor in carcinogenesis. In 2003, Al-Hajj et al. reported that only 200 phenotypically distinct CD44~+CD24~-/lowLin- tumor-initiating cells were capable of tumorigeness in breast,otherwise 20 000 cells without this phenotypical mark can’t develop breast cancer in immunodeficient nonobese diabetic(NOD)/severe combined immunodeficient(SCID) mice. Likewisely, The enhanced ability of CD44~+ prostate cancer cells to form tumors was confirmed compared with CD24~- cells and showed the stem cell properties of reproductive activity, clonality, tumorigeness and metastasis. The hedgehog(Hh) signalling pathway plays a crucial role in vertebrate embryogenesis by controlling cell fate, tissue homeostasis, regeneration and stem cell maintenance. Inappropriate activation of Hh is implicated in the development of tumor. Nevertheless, the role of this pathway in breast carcinogenesis is far from understood, leaving room for conjecture. There are several indications that, active Hh correlates with therapy resistance, cancer stem cells maintenance and co-operation with other signalling pathways. The better understanding of Hh in breast carcinogenesis may provide a new treatment target. The designs of this study are, first, CD44~+CD24~-cells and non-CD44~+CD24~-cells were selected from breast cancer MCF-7 cells, and Real-time RT-PCR was employed to detect the expression of Hedgehog signalling molecules SHH, PTCH1, SMO and GLI1 mRNAs in these cells, to understand the role of Hh in breast carcinogenesis. Then, inoculation was proceeded by injecting CD44~+CD24~-cells and non-CD44~+CD24~-cells in NOD/SCID mice breast to observe tumor growth. Next, The shRNA Smoothened probe was designed to interfere and reduce the Smoothened gene expression in MCF-7 cell to exploe the effect of Hh on cell proliferation, cell cycle and the expression of cyclinD1 and cyclinE. Last, immunohistochemical method was employed to analyse the expressions of Hh molecules in triple negative breast cancer, simultaneously,expression analysis of Hh molecules in breast cancer, mammary hyperplasia and normal breast tissue was completed to explore the effect of Hedgehog signaling pathway in mammary duct malignant transform.Part I Expression and Mechanism of Hedgehog Signalling Molecule in Cancer Stem Cells Selected from Breast Cancer MCF-7 Cell Objective: To explore the effect of Hedgehog signal pathway in the progress of breast cancer.Methods: CD44~+CD24~- cells and non- CD44~+CD24~- cells were selected by magnetic activated cell sorting system (MACS). Real-time RT-PCR was employed to detect the expression of Hedgehog signal molecules SHH、PTCH1、SMO and GLI1 mRNA in CD44~+CD24~- cells and non-CD44~+CD24~- cells, respectively.Results: CD44~+CD24~- cells accounted for 8.25% of breast cancer cells. The expression levels of SHH、PTCH1、SMO and GLI1 mRNA in CD44~+CD24~- cells were higher than those in non-CD44~+CD24~- cells (p<0.05).Conclusions: Hedgehog signalling pathway is activated in breast cancer CD44~+CD24~- cells and may play a role in the maintenance of CD44~+CD24~- cells morphous and function.Part II To Construct NOD/SCID Mice Breast Cancer Model of Cancer Stem Cell and Study of Breast Cancer PathogenesisObjective: To explore the role of cancer stem cell in breast carcinogenesis. Methods: CD44~+CD24~- cells and non-CD44~+CD24~- cells selected were injected in NOD/SCID mice breast to observe tumor growth.Results: The rate of tumor formation was 100% (3/3) in CD44~+CD24~- cell group contrast to 50% (2/4) in non-CD44~+CD24~- cell group. One mouse had lung metastasis, one mouse had lymph node and lung metastasis, and one mouse had liver and lymph node metastasis in CD44~+CD24~- mice, while no organ metastasis was displayed in non-CD44~+CD24~- mice. Conclusions: The inoculation was succeeded by injecting CD44~+CD24~- cells to induce tumorigeness in NOD/SCID mice. The stem~-like cells importantly plays a role in breast carcinogenesis and may induce a high tumor metastasis rate .art III RNAi on Smoothened Effect on Carcinogenesis of Breast Cancer Objective: To explore the effect of Smoothened gene on biological behaviour of breast cancer cell.Methods: The shRNA Smoothened probe was designed to interfere and reduce the Smoothened gene expression in MCF-7 cell.The RT~-PCR and western blot were used to detected them in mRNA and protein of Smoothened in breast cancer MCF-7 cells, which were transfected for 48 hours by the mediation of LipofectamineTM 2000.Flow cytometry used for cell cycle , CCK~-8 kit employed for cell proliferation and real~-time RT~-PCR introducted for expressions of cyclinD1 and cyclinE mRNA.Results: It was verified by partial nucleotide sequencing that the constructed eukaryotic vectors expressing shRNA of Smoothened were correct. In the SMO shRNA~-1 group, SMO shRNA~-2 group, SMO shRNA~-3 group, SMO shRNA~-4 group, blank group, negative control group and MOCK group of MCF-7 cells, the expression of SMO mRNA relative to GAPDH was 0.77±0.07, 0.98±0.08, 0.71±0.05, 0.57±0.05, 0.96±0.08, 1.01±0.08, 1.00±0.11, respectively. Contrast to MOCK group, the SMO mRNA expression in SMO shRNA~-1 group, SMO shRNA~-3 group, SMO shRNA~-4 group declined(p=0.015, 0.002, 0.000), moreover the SMO mRNA expression in SMO shRNA~-4 group obviously lower than other 3 groups(p=0.007, 0.000, 0.046). The inhibitor rate of SMO shRNA~-4 was 87%, meanwhile the expression of SMO protein decreased. MCF-7 cell proliferation, cyclinD1 mRNA and cyclinE mRNA significantly inhibited under the condition of transfected by SMO shRNA~-4.Conclusions: SMO shRNA visibly inhibited the proliferation of breast cancer cell, Hedgehog signaling could induce cell proliferation by actived cyclinD1 and cyclinE. Part IV Hedgehog Molecules Expression in Triple Negative Human Breast Cancer and Effect on Breast Cancer PathogenesisObjective:To explore the effect of Hedgehog signaling pathway on triple negative breast cancer and breast cancer pathogenesis.Methods:Breast cancer, mammary hyperplasia and normal breast tissue were immunohistochemically analyzed for SHH, PTCH1, SMO and GLI1 expressions. Results:Expressions of SMO and GLI1 were increased in triple negative breast cancer compared with non triple negative breast cancer. In triple negative breast cancer, the level of GLI1 expression was increased in lymph node positive cases, SHH and SMO expressions were increased in high histologic grade, expressions of SMO and GLI1 were correlated with superior tumor stage and GLI1 expression manifested inverse association with ER. Expressions of SHH, SMO and GLI1 were significantly increased in breast cancer and mammary hyperplasia. PTCH1 expression was significantly decreased in breast cancer compared with mammary hyperplasia and normal breast tissue.Conclusions: The present study for the first time provided the clinical evidence in support of important roles of Hh signalling in triple negative breast cancer. Hh signalling participates in the breast ductal changes and malignant transformation. Measures to inhibit Hh activity may improve the prognosis of triple negative breast cancer patients.