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

Research Paper

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

Ling Lin1#, Shuyi Chen2#, Hua Wang3, Bin Gao3, Bhaskar Kallakury1, Krithika Bhuvaneshwar4, Katherine Cahn1, Yuriy Gusev4, Xue Wang5, Yunan Wu1, John L. Marshall1, Xiuling Zhi2✉, Aiwu Ruth He1✉

1. Department of Medicine and Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.
2. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
3. Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA.
4. Innovation Center for Biomedical Informatics, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.
5. Department of Pathology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
#These authors contributed equally to this work.

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Citation:
Lin L, Chen S, Wang H, Gao B, Kallakury B, Bhuvaneshwar K, Cahn K, Gusev Y, Wang X, Wu Y, Marshall JL, Zhi X, He AR. SPTBN1 inhibits inflammatory responses and hepatocarcinogenesis via the stabilization of SOCS1 and downregulation of p65 in hepatocellular carcinoma. Theranostics 2021; 11(9):4232-4250. doi:10.7150/thno.49819. Available from https://www.thno.org/v11p4232.htm

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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: SPTBN1, pro-inflammatory cytokines, NF-κB, protein stabilization, SOCS1