Inhibition of RhoGEF/RhoA alleviates regorafenib resistance and cancer stemness via Hippo signaling pathway in hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) are vulnerable to drug resistance. Although drug resistance has been taken much attention to HCC therapy, little is known of regorafenib and regorafenib resistance (RR). This study aimed to determine the drug resistance pattern and the role of RhoA in RR. Two regorafenib-resistant cell lines were constructed based on Huh7 and Hep3B cell lines. In vitro and in vivo assays were conducted to study RhoA expression, the activity of Hippo signaling pathway and cancer stem cell (CSC) traits. The data showed that RhoA was highly expressed, Hippo signaling was hypoactivated and CSC traits were more prominent in RR cells. Inhibiting RhoA could reverse RR, and the alliance of RhoA inhibition and regorafenib synergistically attenuated CSC phenotype. Furthermore, inhibiting LARG/RhoA increased Kibra/NF2 complex formation, prevented YAP from shuttling into the nucleus and repressed CD44 mRNA expression. Clinically, the high expression of RhoA correlated with poor prognosis. LARG, RhoA, YAP1 and CD44 show positive correlation with each other. Thus, inhibition of RhoGEF/RhoA has the potential to reverse RR and repress CSC phenotype in HCC.

The role of daurisoline treatment in hepatocellular carcinoma: Inhibiting vasculogenic mimicry formation and enhancing sensitivity to sorafenib.

BACKGROUND: Vasculogenic mimicry (VM) is a newly described tumor vascular phenomenon that is independent of traditional angiogenesis and provides an adequate blood supply for tumor growth. VM has been consistently observed in different cancer types. Hence, inhibition of VM may be considered a new anticancer therapeutic strategy. PURPOSE: This study aimed to elucidate the potential anticancer effect of daurisoline (DS) on hepatocellular carcinoma (HCC) and the potential molecular mechanism by which DS inhibits VM. We also verified whether combination treatment with sorafenib and DS constitutes a novel therapeutic approach to prevent HCC progression. METHODS: The effects of DS on proliferation were evaluated by Cell Counting Kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. 4',6-Diamidino-2-phenylindole (DAPI) staining and flow cytometric analysis were employed to investigate its effects on apoptosis. Western blot analysis, Matrigel tube formation assays, pulldown assays and immunofluorescence staining were applied to validate the potential mechanism by which DS inhibits VM. Mouse xenograft models were used to evaluate anticancer activities. RESULTS: DS inhibited HCC cell proliferation, induced HCC cell apoptosis and repressed VM formation by inactivating RhoA/ROCK2-mediated AKT and ERK-p38 MAPK signaling. Additionally, DS dramatically sensitized HCC cell lines to sorafenib, a curative anticancer drug for patients with advanced HCC. CONCLUSIONS: Our study provides insights into the molecular mechanisms underlying DS-induced inhibition of VM, which may facilitate the development of a novel clinical anti-HCC drug. Moreover, our findings suggest that the combination of DS and sorafenib constitutes a potential therapeutic strategy for HCC.

Targeting cancer stem cells for reversing therapy resistance: mechanism, signaling, and prospective agents.

Cancer stem cells (CSCs) show a self-renewal capacity and differentiation potential that contribute to tumor progression and therapy resistance. However, the underlying processes are still unclear. Elucidation of the key hallmarks and resistance mechanisms of CSCs may help improve patient outcomes and reduce relapse by altering therapeutic regimens. Here, we reviewed the identification of CSCs, the intrinsic and extrinsic mechanisms of therapy resistance in CSCs, the signaling pathways of CSCs that mediate treatment failure, and potential CSC-targeting agents in various tumors from the clinical perspective. Targeting the mechanisms and pathways described here might contribute to further drug discovery and therapy.

Hypoxic induction of vasculogenic mimicry in hepatocellular carcinoma: role of HIF-1 α, RhoA/ROCK and Rac1/PAK signaling.

BACKGROUND: Vasculogenic mimicry (VM), defined as a capability of aggressive tumor Cells to mimic embryonic vasculogenic networks, caused poor prognosis in hepatocellular carcinoma (HCC). Rho kinases (ROCK), p21-activated kinase (PAK), hypoxia or epithelial-mesenchymal transition (EMT) contributed to the VM potential. However, the details underlying these biological behaviors have not been completely elucidated. METHODS: Kaplan-Meier analysis was conducted to predict relationship with hypoxia Inducible factor (HIF-1α), EMT related markers: Vimentin and patient prognosis. CD34/periodic acid-Schiff (PAS) double staining was examined to differentiate VM-positive (VM+) and VM-negative (VM-) samples. Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on RhoA/ROCK, Rac1/PAK and EMT were evaluated by real time-qPCR and western blot. HIF-1α small interfering RNA (siRNA), overexpressed or short hairpin RNA (shRNA) of ROCK and kinase inhibitors were used to explore the effect of HIF-1α, RhoA/ROCK, Rac1/PAK and Vimentin on VM. RESULTS: HIF-1α or Vimentin was upregulated in VM+ HCC tissues, compared to non-cancerous tissues (P < 0.01), and patients with high expression of HIF-1α or Vimentin had worse prognosis (P < 0.001). We showed hypoxia induced RhoA/ROCK and Rac1/PAK signaling transduction, and EMT could be repressed by HIF-1α siRNA. Notably, RhoA/ROCK or Rac1/PAK stabilized HIF-1α in hypoxia, whereas HIF-1α did not significantly altered RhoA/ROCK or Rac1/PAK signaling in hypoxia. Moreover, we found distinct roles of ROCK1, ROCK2 and PAK in regulating Vimentin phosphorylation. CONCLUSIONS: RhoA/ROCK and Rac/PAK signaling played crucial roles in hypoxia-induced VM via Ser72 and Ser56 Vimentin phosphorylation in HCC.

Overlapping and unique roles played by ROCK1 and 2 in the modulation of coding and long noncoding RNA expression.

BACKGROUND: Our previous study described the crucial role of Rho-associated coiled-coil containing-kinases (ROCK) in hepatocellular carcinoma (HCC). However, the potential significance of long noncoding RNA downstream of ROCK is largely unknown. Here, a comprehensive comparative bioinformatics analysis of a microarray of an MHCC-97H cell line overexpressing ROCK1 or ROCK2 was performed. RESULTS: Numerous lncRNAs and mRNAs were deregulated by Rho-associated coiled-coil containing kinases 1 and 2. These results were consistent with the qRT-PCR results. Compared with MHCC-97H-Con, which was transfected with a null vector, the GO analysis revealed differentially expressed mRNAs (DEmRNAs) in MHCC-97H-ROCK1 (ROCK1 was overexpressed) enriched in apoptotic cell clearance, the cyclooxygenase pathway and bone trabecula morphogenesis; the DEmRNAs in MHCC-97H-ROCK2 (ROCK2 was overexpressed) were enriched in VEGF production, chemokine-associated signaling pathways, acute inflammatory response and vasoconstriction. Compared with MHCC-97H-ROCK2, the DEmRNAs in MHCC-97H-ROCK1 were involved in the JAK-STAT cascade, the Akt signaling pathway and the activity of several different peptidases. The pathway analysis of ROCK1 and ROCK2 revealed an overlap in the VEGF signaling pathway, ECM-receptor interaction, and adhesion and differences in the PPAR signaling pathway and mismatch repair. The predicted targets of the differentially expressed lncRNA (DElncRNAs) were enriched in the p53 signaling pathway, Jak-STAT signaling pathway, etc. Several hub DElncRNAs were identified. CONCLUSIONS: ROCK1 and 2 modulate the expression of numerous mRNAs and lncRNAs and may participate in several signaling pathways in HCC. Several hub molecules were identified in the lncRNA-mRNA networks. Our results provide baseline data for ROCK1 and 2 regulation in HCC that might have implications for further research.