Identification of TIMP3 as a regulator for metastasis in neuroblastoma

• Main Authors: Mati Kargren, 卡爾根馬緹
• Other Authors: Hsinyu Lee
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/3k5qn5

碩士 === 國立臺灣大學 === 生命科學系 === 107 === Neuroblastoma is a tumor disease of the sympathetic nervous system and it is the most common type of extra-cranial solid tumors in children. Despite improvements in available treatment options, prognosis for high-risk neuroblastoma patients remains unfavorable, with metastatic spread being one of the reasons behind it. In our study we tested the ability of JQ1, a Bromodomain and Extra-Terminal (BET) inhibitor, and CL-387,785, an EGFR/ErbB2 dual inhibitor, to block metastatic properties of neuroblastoma cells. BET inhibitors target MYCN, an oncogene whose amplification is associated with high-risk neuroblastoma. Additionally, both EGFR and ErbB2 were shown to be highly expressed in NB cells and tissues, and have been implicated in metastasis of various tumors. Using wound healing and transwell assays, we were able to show that both of the compounds significantly reduce migration and invasion of neuroblastoma cells. Consistently, colony formation assays showed that both compounds are able to inhibit anchorage-independent growth of neuroblastoma cells. Using a human tumor gene microarray, we later identified tissue inhibitor of metalloproteinases 3 (TIMP3) as one of the significantly upregulated genes in neuroblastoma cells treated with JQ1 or CL-387,785. These results were confirmed by quantitative real-time PCR, Western blotting and immunocytochemistry. Furthermore, analysis of neuroblastoma patient data from the web-based R2 platform revealed that patients with tumors expressing high levels of TIMP3 have a significantly increased overall and event-free survival probability. Lastly, quantitative real-time PCR, Western blotting and immunohistochemical analysis of tumor samples from a Taiwanese cohort of neuroblastoma patients showed that TIMP3 is expressed at higher levels in more differentiated neuroblastoma tumors compared to less differentiated ones. In summary, our results show that JQ1 and CL-387,785 are able to significantly inhibit molecular processes involved in metastasis of NB, and that expression of TIMP3 is higher in cells treated by these compounds, as well as tumor samples from NB patients with favorable prognoses. Our findings provide the proof-of-principle evidence that JQ1 and CL-387,785 could be further developed to become the target therapies for high-risk neuroblastoma, and also verify the prognostic merit of TIMP3 in neuroblastoma.