Collagen Ⅰ-DDR1 Signaling Promotes Hepatocellular Carcinoma Cell Stemness Via Hippo Signaling Repression

Objective: Cancer stem cells(CSCs)are a minority population of cancer cells with stemness and multiple differentiation potentials,leading to cancer progression and therapeutic resistance.However,the concrete mechanism of CSCs remains obscure.Collagen Ⅰ-DDR1 signaling can response to ECM which can be a physical barrier that protects CSCs from chemotherapy.We explore the clinical significance,biological function and molecular mechanism of Collagen Ⅰ-DDR1 signaling in promoting cancer cell stemness.Methods: Picro-Sirius red staining was performed to distinguish Collagen types.We established DDR1 knock-out,knock-down and overexpression in different HCC cell lines by CRISPR-Cas9 gene editing and lentivirus infection.sphere-forming assay,single-cell colony formation,CD133 positive stem-like-cell proportion,and chemoresistance was performed to assess cancer cell stemness in vitro.Cancer cells limiting dilution implantation assay was conducted to assess stemness in vivo.Stemness and Hippo signaling associated genes expression was determined by quantitative RT-q PCR.Proteins interaction was investigated by mass spectrometry,co-immunoprecipitation,GST pull-down,immunofluorescence and confocal microscopy imaging.The mechanisms of Collagen Ⅰ-DDR1 regulating Hippo signaling were performed by RNA sequencing,immunoprecipitation and immunoblotting.Results: Collagen Ⅰ was the most enrich Collagen type in the ECM of HCC.In advanced HCC tissues,Collagen Ⅰ was upregulated,which was consistent with the expression of its receptor DDR1,high Collagen Ⅰ levels accompanied by high DDR1 expression are associated with a poor prognosis in patients with HCC.Collagen Ⅰ-induced DDR1 activation enhanced HCC cell stemness in vitro and in vivo.Mechanistically,DDR1 interacts with CD44,which acts as a co-receptor that amplifies Collagen Ⅰ-induced DDR1 signaling,and Collagen Ⅰ-DDR1 signaling antagonized Hippo signaling by facilitating the recruitment of PP2 AA to MST1,leading to exaggerated YAP activation.The combined inhibition of DDR1 and YAP synergistically abrogated HCC cell stemness.Conclusions: Our findings reveal the mechanism of Collagen Ⅰ-DDR1 signaling inhibiting Hippo signaling,highlight the role of CD44/DDR1/YAP axis in promoting cancer cell stemness,and the radiomic model have potential of non-invasive predicting Collagen Ⅰexpression,suggesting that DDR1 and YAP may serve as novel prognostic biomarkers and therapeutic targets in HCC.