Elucidating the mechanisms of transcription factor Runx2 in promoting osteogenic differentiation of mesenchymal stem cells and malignant progression of breast cancer cells

• Main Authors: Jing-jie Weng, 翁靖傑
• Other Authors: Yeu Su
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/21367616226046440338

博士 === 國立陽明大學 === 生物藥學研究所 === 101 === Runx2, also called Cbfa-1 or AML3, plays important roles in physiological bone formation and pathological malignant progression of breast and prostate cancers. A good understanding of the mechanism of Runx2 in promoting osteoblast differentiation and breast cancer progression is critical for the design of anabolic treatments for bone deficiency diseases and the development of a novel treatment for breast cancer. Alkaline phosphatase (ALP) expressed by osteoblasts plays an important role in promoting bone mineralization by hydrolyzing pyrophosphate. However, the mechanism by which the expression of ALP is regulated during osteoblast differentiation has not been thoroughly investigated. Using chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA), I found that Runx2 bound to the putative core sites within ALP intron 1. Furthermore, with a Runx2 mutant lacking the nuclear matrix-targeting sequence (Runx2ΔNMTS) bound in situ to the ALP gene less efficiently than the wild-type protein and another Runx2 mutant deficient in DNA binding (Runx2K120A) accumulated largely in the nuclear matrix which in addition acted as a dominant negative mutant to suppress ALP expression induced by the wild-type Runx2, I thereby proposed that the nuclear matrix-localization of Runx2 influenced its ALP gene recognition. These results showed for the first time that ALP is a direct target gene of Runx2 and illustrated that nuclear matrix-targeting is a prerequisite for the recognition/binding and activation of the ALP by this transcription factor. A variety of Runx2 isoforms are present due to the usage of different promoters or alternative splicing. Among these variants, mRNA of the Runx2 with truncated exon 6 (Runx2E6) had been shown to be upregulated in the tumor tissues of breast cancer patients. Although Runx2 had previously been reported to promote breast cancer malignancy, designing therapeutic strategies for breast cancer by targeting Runx2 is infeasible due to the possible adverse effects on bone integrity resulted from Runx2 inactivation. In this regard, understanding the role of Runx2E6 in the malignant progression of breast cancer and deciphering the mechanism of its upregulation in cancer tissues as well as the involvement of this variant in osteogenic differentiation of mesenchymal stem/progenitor cells should be helpful in designing better strategy for breast cancer treatment. In this study, we showed that Runx2E6 was better than the wild-type Runx2 in upregulating matrix metalloproteinase-9 (MMP-9) and 13 (MMP-13) expression as well as in promoting the migration of human breast cancer cells. In addition, hnRNP H1 was identified as one of the upstream activators of Runx2E6 expression in breast cancer cells. Finally, barely detectable level of transcript of this splicing variant was present in osteoblastic lineage of cells, suggesting an insignificant role of Runx2E6 in regulating osteocyte formation. Together, these data suggested that stronger malignancy-promoting effects on human breast cancer are associated with an alternatively spliced variant of Runx2 which might be a novel target for treating this deadly disease.