A positive feedback loop between Periostin and TGFβ1 induces and maintains the stemness of hepatocellular carcinoma cells via AP-2α activation

Background: Liver cancer stem cells (LCSCs) play key roles in the metastasis, recurrence, and chemotherapeutic resistance of hepatocellular carcinoma (HCC). Our previous research showed that the POSTN gene is closely related to the malignant progression and poor prognosis of HCC. This study aimed to elucidate the role of POSTN in generating LCSCs and maintaining their stemness as well as the underlying mechanisms. Methods: Human HCC tissues and matched adjacent normal tissues were obtained from 110 patients. Immunohistochemistry, western blotting (WB), and RT-PCR were performed to detect the expression of POSTN and stemness factors. The roles of transforming growth factor (TGF)-beta 1 and AP-2 alpha in the POSTN-induced stemness transformation of HCC cells were explored in vitro and in vivo using LCSCs obtained by CD133(+) cell sorting. Results: The high expression of POSTN was correlated with the expression of various stemness factors, particularly CD133, in our HCC patient cohort and in TCGA and ICGC datasets. Knockdown of POSTN expression decreased the abilities of HCC cell lines to form tumours in xenograft mouse models. Knockdown of POSTN expression also suppressed cell viability and clone formation, invasion, and sphere formation abilities in vitro. Knockdown of AP-2 alpha attenuated the generation of CD133(+) LCSCs and their malignant behaviours, indicating that AP-2 alpha was a critical factor that mediated the POSTN-induced stemness transformation and maintenance of HCC cells. The role of AP-2 alpha was verified by using a specific alpha v beta 3 antagonist, cilengitide, in vitro and in vivo. Activation of POSTN could release TGF beta 1 from the extracellular matrix and initiated POSTN/TGF beta 1 positive feedback signalling. Furthermore, we found that the combined use of cilengitide and lenvatinib suppressed the growth of HCC cells with high POSTN expression more effectively than the use of lenvatinib alone in the patient-derived xenograft (PDX) mouse model. Conclusions: The POSTN/TGF beta 1 positive feedback pathway regulates the expression of stemness factors and the malignant progression of HCC cells by regulating the transcriptional activation of AP-2 alpha. This pathway may serve as a new target for targeted gene therapy in HCC.