SPTBN1 inhibits inflammatory responses and hepatocarcinogenesis via the stabilization of SOCS1 and downregulation of p65 in hepatocellular carcinoma

Theranostics. 2021 Feb 20;11(9):4232-4250. doi: 10.7150/thno.49819. eCollection 2021.

Abstract

Background: Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein for transforming growth factor beta (TGF-β) signaling, is recognized as a tumor suppressor in the development of hepatocellular carcinoma (HCC); however, the underlying molecular mechanisms of this tumor suppression remain obscure. Methods: The effects on expression of pro-inflammatory cytokines upon the inhibition or impairment of SPTBN1 in HCC cell lines and liver tissues of Sptbn1+/- and wild-type (WT) mice were assessed by analyses of quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), enzyme linked immunosorbent assay (ELISA), Western blotting and gene array databases from HCC patients. We investigated the detailed molecular mechanisms underlying the inflammatory responses by immunoprecipitation-Western blotting, luciferase reporter assay, chromatin immunoprecipitation quantitative real time PCR (ChIP-qPCR), immunohistochemistry (IHC) and electrophoretic mobility shift assay (EMSA). The proportion of myeloid-derived suppressor cells in liver, spleen, bone marrow and peripheral blood samples from WT and Sptbn1+/- mice were measured by fluorescence-activated cell sorting (FACS) analysis. Further, the hepatocacinogenesis and its correlation with inflammatory microenvironment by loss of SPTBN1/SOCS1 and induction of p65 were analyzed by treating WT and Sptbn1+/- mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Results: Loss of SPTBN1 in HCC cells upregulated the expression of pro-inflammatory cytokines including interleukin-1α (IL-1α), IL-1β, and IL-6, and enhanced NF-κB transcriptional activation. Mechanistic analyses revealed that knockdown of SPTBN1 by siRNA downregulated the expression of suppressor of cytokine signaling 1 (SOCS1), an E3 ligase of p65, and subsequently upregulated p65 accumulation in the nucleus of HCC cells. Restoration of SOCS1 abrogated this SPTBN1 loss-associated elevation of p65 in HCC cells. In human HCC tissues, SPTBN1 gene expression was inversely correlated with gene expression of IL-1α, IL-1β and IL-6. Furthermore, a decrease in the levels of SPTBN1 gene, as well as an increase in the gene expression of IL-1β or IL-6 predicted shorter relapse free survival in HCC patients, and that HCC patients with low expression of SPTBN1 or SOCS1 protein is associated with poor survival. Heterozygous loss of SPTBN1 (Sptbn1+/- ) in mice markedly upregulated hepatic expression of IL-1α, IL-1β and IL-6, and elevated the proportion of myeloid-derived suppressor cells (MDSCs) and CD4+CD25+Foxp3+ regulatory T cells (Foxp3+Treg) cells in the liver, promoting hepatocarcinogenesis of mouse fed by DDC. Conclusions: Our findings provided evidence that loss of SPTBN1 in HCC cells increases p65 protein stability via the inhibition of SOCS1 and enhances NF-κB activation, stimulating the release of inflammatory cytokines, which are critical molecular mechanisms for the loss of SPTBN1-induced liver cancer formation. Reduced SPTBN1 and SOCS1 predict poor outcome in HCC patients.

Keywords: NF-κB; SOCS1; SPTBN1; pro-inflammatory cytokines; protein stabilization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carcinogenesis / metabolism*
  • Carcinoma, Hepatocellular / metabolism*
  • Cell Line, Tumor
  • Down-Regulation / physiology*
  • Gene Expression Regulation, Neoplastic / physiology
  • Humans
  • Inflammation / metabolism*
  • Liver / metabolism
  • Liver Neoplasms / metabolism*
  • Mice
  • NF-kappa B / metabolism
  • Signal Transduction / physiology
  • Spectrin / metabolism*
  • Suppressor of Cytokine Signaling 1 Protein / metabolism*
  • T-Lymphocytes, Regulatory / metabolism
  • Up-Regulation / physiology
  • eIF-2 Kinase / metabolism*

Substances

  • NF-kappa B
  • SOCS1 protein, human
  • SPTBN1 protein, human
  • Suppressor of Cytokine Signaling 1 Protein
  • Spectrin
  • eIF-2 Kinase