Nuclear factor kappa B–mediated CD47 up‐regulation promotes sorafenib resistance and its blockade synergizes the effect of sorafenib in hepatocellular carcinoma in mice

Abstract

Sorafenib is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, the survival benefit of this treatment is modest, partly owing to drug resistance. Recent evidence has demonstrated the existence of tumor-initiating cells (T-ICs) as the culprit for treatment resistance. To examine whether sorafenib resistance was a result of the presence of liver T-ICs, we developed sorafenib-resistant HCC cells both in vitro and in vivo through continuous exposure to sorafenib. Using these models, we found that sorafenibresistant clones demonstrated enhanced T-IC properties, including tumorigenicity, selfrenewal, and invasiveness. In addition, several T-IC markers were found to be up-regulated, among which CD47 was found to be most significant. Using chromatin immunoprecipitation assays and expression analyses, CD47 expression was found to be regulated by nuclear factor kappa B (NF-jB) through a specific response element in the promoter of CD47, and the site occupancy and expression were increased and decreased upon stimulation and inhibition of NF-jB, respectively. Consistently, NF-jB was activated in sorafenib-resistant HCC cells, and this finding was confirmed in clinical HCC samples, which showed a positive correlation between NF-jB and CD47 expression. Functional characterization of CD47 in sorafenib-resistant HCC cells was evaluated using a lentivirus-based knockdown approach and showed increased sensitization to sorafenib upon CD47 knockdown. Furthermore, blockade of CD47 using anti-CD47 antibody (Ab) showed a similar effect. Using a patientderived HCC xenograft mouse model, we found that anti-CD47 Ab (500 lg/mouse) in combination with sorafenib (100 mg/kg, orally) exerted synergistic effects on tumor suppression, as compared with sorafenib and anti-CD47 Ab alone. Conclusions: NF-jB-mediated CD47 up-regulation promotes sorafenib resistance, and targeting CD47 in combination with sorafenib is an attractive therapeutic regimen for the treatment of HCC patients. (HEPATOLOGY 2015;62:534-545) L iver cancer (hepatocellular carcinoma; HCC) is a major malignancy worldwide, 1 and the front-line treatment for this disease is liver transplantation and surgical resection. However, most HCCs are inoperable, given that patients typically present at advanced stages, and even after surgical resection, the long-term prognosis of HCC remains unsatisfactory owing to its high recurrence rate. For advanced-staged HCC

Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells

“…Reported previously by us and others (60,61), SIRPα expression in macrophages is decreased following LPS stimulation, suggesting a dynamic nature for the CD47-SIRPα-mediated inhibition especially on infection or activation of TLR. The expression of CD47 on cells can also be changed under different conditions (62)(63)(64). Also reported by us, alteration of clustering structures of SIRPα on macrophages or CD47 on tissue cells affects phagocytosis (65,66).…”

HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells

“…Blocking the interaction of CD47 with its receptor, SIRPα, on macrophages enables phagocytosis and inhibits tumor growth (35,36,(44)(45)(46). However, the molecular mechanisms regulating the expression of CD47 by cancer cells have not been delineated.…”

“…The results presented here represent to our knowledge the first identification of a transcriptional regulator of CD47 expression in breast cancer cells and further studies are required to determine whether HIF-1 cooperates with other transcription factors that are induced by the tumor microenvironment, such as CREB, NF-κB, SMAD2, or STAT3. Increased NF-κB activity was observed in hepatocellular carcinoma cells that developed sorafenib resistance (36), which is of interest because the antiangiogenic effects of sorafenib induce intratumoral hypoxia that causes HIF-1-dependent induction of breast CSCs (19). Immunosuppressive functional interactions of HIF-1 and CREB have been proposed in T cells (47), but may also occur in cancer cells.…”

“…CD47 is preferentially expressed on bladder, liver, and pancreatic CSCs compared with the bulk cancer cells (non-CSCs) (33)(34)(35)(36). To investigate whether CD47 plays a role in breast CSCs, we first analyzed CD47 mRNA levels in SUM159 cells, which were cultured as standard adherent monolayers or as nonadherent spheroids (mammospheres), which are highly enriched for CSCs (37).…”

Inflammation, phagocytosis and cancer: another step in the CD47 act

“…Though the notion that cancer cells frequently up-regulate CD47 compared to their normal counterparts is now widely accepted, the underlying up-and down-stream regulatory mechanisms are not fully elucidated. Previous studies including breast cancer identified that the promoter region for the CD47 gene is regulated by binding of several transcription factors, including MYC and NF-κB (8,9). However, the exact mechanism behind the overexpression of CD47 in cancer cells is insufficiently understood (10).…”