Attenuation of Cancer-Initiating Cells Stemness Properties by Abrogating S100A4 Calcium Binding Ability in Head and Neck Cancers；Physiological Function of Tid1 during Myogenesis and Energy Homeostasis

• Main Authors: Li-Hao Cheng, 鄭力豪
• Other Authors: Jeng-Fan Lo
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/41054443783970689174

博士 === 國立陽明大學 === 口腔生物研究所 === 105 === Part I Abstract: Epithelial-mesenchymal transition (EMT) is a process that epithelial cells acquire a migratory mesenchymal phenotype or stem cell (SC) properties. Further, up regulation of Vimentin and Nanog is closely related to EMT and stemness properties, respectively. S100A4, a member of calcium-binding proteins, is competent to induce EMT and directly controlled by Wnt/β-catenin signaling pathway. Calcium binding activity of S100A4 has been highly associated with the regulation of downstream targets and development of a metastatic phenotype. In addition, we recently demonstrate that S100A4 plays a crucial role in the maintenance of head and neck cancer-initiating cells (HN-CIC) population. Moreover, inhibition of S100A4 decreased the HN-CICs stemness and self-renewal property, both in vitro and in vivo. However, the regulation of S100A4 and the molecular targets to control HN-CICs stemness capability are remained unclear. In this study, to further investigate the molecular mechanism, I established the head and neck squamous cell carcinoma (HNSCC) cell lines stably expressing mutant S100A4 proteins with defective calcium-binding sites on either N-terminal (NM) or C-terminal (CM), or a deletion of the last 15 amino-acid residues (CD). I found that cells expressing the mutant S100A4 result in decreasing of anchorage independent growth ability when it was compared to the cells harboring wild-type S100A4 in HNSCC. The immunoblot analyses showed that wild-type S100A4 not only increased the protein level of Vimentin and Nanog but also decreased the expression of E-cadherin. Inversely, the mutant S100A4 reduced the protein level of Vimentin and Naong and increased the expression E-cadherin in HNSCCs. To further investigate the connection between S100A4 and stemness determinants, I enriched the HN-CICs from wild-type S100A4 and mutants S100A4 HNSCC cells, respectively, by sphere formation. I showed that the NM, CM and CD harboring sphere cells that were enriched with HN-CICs population exhibited impaired stemness and malignant properties in vitro, as well as reduced tumor growth ability in vivo. Mechanistically, I demonstrated that mutant S100A4 proteins decreased the promoter activity of Nanog, likely through inhibition of p53. Taken together, my results suggest that both the calcium-binding ability and the C-terminal region of S100A4 are important for HN-CICs to sustain its stemness property and malignancy, and that the mechanism could be mediated by repressing p53 and subsequently activating the Nanog expression. Part II Abstract: Tid1 is a mitochondrial cochaperone protein, and its transcript is abundantly expressed in skeletal muscle tissues. However, the physiological function of Tid1 during skeletal myogenesis remains unclear. In vitro induced differentiation assay of mouse myoblast C2C12 cells was applied to examine the physiological role of Tid1 during skeletal myogenesis. In addition, transgenic mice with muscle specific (HSA-Cre) Tid1 deletion were established and examined to determine the physiological function of Tid1 during skeletal muscle development in vivo. Expression of Tid1 protein was up-regulated in the differentiated C2C12 cells, and the HSA-Tid1f/f mice displayed muscular dystrophic phenotype. Additionally, I found that liver weight, white adipose tissue mass and blood glucose were reduced in postnatal HSA-Tid1f/f mice on postnatal day (P) 8 to 10. Further, all of the HSA-Tid1f/f mice became lethal around P8 to 10. The expression of myosine heavy chain (MyHC), the protein served as the muscular development marker, was reduced in HSA-Tid1f/f mice at (P) 5 and P8. The protein levels of ATP sensor (p-AMPK) and mitochondrial biogenesis protein (PGC-1α) were also significantly reduced in HSA-Tid1f/f mice. Moreover, Tid1 deficiency induced apoptotic marker Caspase-3 in muscle tissues of HSA-Tid1f/f mice. Consistent with the in vivo finding, I observed that down-regulation of Tid1 not only reduced the ATP production but also abolished the differentiation ability of C2C12 cells by impairing the mitochondria activity. Together, our results suggest that Tid1 deficiency reduces ATP production and abolishes mitochondria activity, resulting in energy imbalance and promoting apoptosis of muscle cells during myogenesis. Moreover, these data suggest that Tid1 skeletal muscular deficiency induces energy insufficiency of muscle cells and results in dysregulating energy homeostasis. It will be of importance to understand the function of Tid1 during human muscular dystrophy in the future.