Functional Characterization and Therapeutic Implication of Cd47 in Sorafenib Resistance in Hepatocellular Carcinoma

This dissertation, "Functional Characterization and Therapeutic Implication of CD47 in Sorafenib Resistance in Hepatocellular Carcinoma" by Jessica, Lo, 盧姵岐, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage where surgery cannot provide a cure. Sorafenib is currently a new standard treatment for patients with advanced HCC. However, the survival benefit is modest which may be attributed to drug resistance. Recently, increasing evidence demonstrated the existence of tumor-initiating cells (T-ICs) as the culprit for treatment resistance. To improve HCC outcome, it is of utmost importance to understand the mechanism behind sorafenib resistance. To examine whether sorafenib resistance is due to presence of liver T-ICs, sorafenib-resistant HCC cells were developed successfully both in vitro and in vivo through continuous exposure to sorafenib. Using these models, we found that sorafenib-resistant clones demonstrated enhanced T-IC properties including tumorigenicity, self-renewal and invasiveness. In addition, several T-IC markers were also found to be upregulated, of which CD47 was found to be most significant as evidenced by quantitative polymerase chain reaction analysis, followed by confirmation at protein level with flow cytometry. This result, together with bioinformatics analysis showing two putative binding sites of NF-κB on the CD47 promoter, suggests transcriptional upregulation of CD47 in sorafenib HCC cells. Consistent to this hypothesis, NF-κB was found to be activated in sorafenib-resistant HCC cells. Further implication of the relationship of NF-κB and CD47 was found in clinical HCC samples showing a positive correlation between NF-κB and CD47. To investigate involvement of NF-κB in the upregulation of CD47, the NF-κB stimulator TNF-α and the inhibitor IMD-0354 were used to observe changes in CD47 expression upon alteration of NF-κB levels. Upon stimulation of NF-κB, CD47 expression was found to be increased, while suppression of NF-κB lead to decreased CD47 expression. Confirmation of NF-κB binding to the CD47 promoter was accomplished by ChIP promoter binding assay. Upon stimulation and inhibition of NF-κB, the site occupancy detected increased and decreased, respectively, these findings suggest for the first time that NF-κB-mediated CD47 upregulation promotes sorafenib resistance in HCC. Functional characterization of CD47 in sorafenib resistance of HCC cells was accomplished with lentiviral-based stable knockdown approach, and showed increased sensitization to sorafenib upon knockdown of CD47. Furthermore, blockade of CD47 using anti-CD47 antibody also showed a similar result offering a therapeutic option of combining sorafenib with anti-CD47 antibody. In the evaluation of combination therapy with anti-CD47 antibody in vivo, a patient-derived xenograft mouse model was employed (PDTX#5). The examination of the combined effect of sorafenib (100mg/kg orally) and anti-CD47 antibody (500μg/mouse intraperitoneally) were compared to single agent alone through daily administration for 31 days. Treatment with sorafenib or an anti-CD47 antibody alone resulted in a 2-3-fold suppression in tumor growth compared with the IgG control group. The combined therapy of sorafenib and an anti-CD47 antibody produced an astonishing 8-fold suppression. Further observation by TUNEL assay revealed enhanced suppression of tumor growth in the co-treated group is due to apoptosis of tumor cells. In conclusion, we have delineated the novel mechanism of NF-κB-mediated CD47 upregul