SUMOylation of SMAD4 by PIAS1 in Conjunction with Vimentin Upregulation Promotes Migration Potential in Non-Small Cell Lung Cancer

Background: The expression of vimentin as a marker of epithelial-to-mesenchymal transition (EMT) has been speculated to be associated with tissue heterogeneity and metastases of non-small cell lung cancer (NSCLC). Methods: This study utilized in vitro co-immunoprecipitation with small interfering RNAs (siRNAs) against protein inhibitors of STAT system type 1 (PIAS1) or SMAD4 in transforming growth factor-beta (TGF- $\beta{}$ ) signaling pathway in combination with SUMOylation assay. Results: We successfully demonstrated that PIAS1 enhanced SUMOylation of SMAD4 by forming a complex PIAS1-SUMO1-SMAD4 protein complex. This, in accordance with subsequently increased production of vimentin microfilaments, led to enhanced migration ability of non-small cell lung cancer (NSCLC) A549 line, observed from wound healing assay. Conclusions: Our results further supported the positive correlation of SUMOylated SMAD4 mediated by PIAS1 and downstream overexpression of vimentin. In addition, the observation that overexpression of vimentin in this certain cell line was not necessarily linked with accelerated relative wound closure raised concerns that further exploration will be needed to confirm if the causal relationship exists between vimentin expression and the metastases of NSCLC, and if so, to what extent vimentin contributes to it.

Keywords

non-small cell lung cancer

SUMOylation

SMAD4

PIAS1

Vimentin

Funding

2020HMZD02/Key Reasearch Foundation of Hwa Mei Hospital, University of Chinese Academy of Sciences

2022HMZD09/Key Reasearch Foundation of Hwa Mei Hospital, University of Chinese Academy of Sciences

PPXK2018-05/Ningbo Health Branding Subject Fund

LTGY23H010001/Basic Public Welfare Research Program of Zhejiang Province

2022KY1125/Medical Scientific Research Foundation of Zhejiang Province

2023KY280/Medical Scientific Research Foundation of Zhejiang Province

2022L004/Ningbo Clinical Research Center for Respiratory System Disease of Zhejiang Province

Figures

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Fig. 1.

PIAS1 enhances SMAD4 SUMOylation by forming PIAS1-SUMO1-SMAD4 multiprotein complex. (A) Cell lysates transfected with vector control or a plasmid containing cDNA encoding Myc-SUMO1, FLAG-SMAD4, and HA-PIAS1, alone or in combination, were subjected to SMAD4 immunoprecipitation (FLAG IP) followed by SMAD4 or Myc immunoblotting (IB). At the same time, 10% lysates were subjected to SMAD4, PIAS1, SUMO1 IB as the input, and $\beta{}$ -actin as the input control. (B) Relative SUMOylated SMAD4 level with vector control, infused with PIAS1, or with endogenous inhibition of PIAS1. Experiments were performed with quadruplicates in each group, quantitative data analyses were conducted in Prism 9.0, the error bar showed standard error (SD), ANOVA was applied, two-tailed p values were indicated as **p $\leq$ 0.01, ****p $\leq$ 0.0001, and Bonferroni’s correction test was conducted for multiple comparisons.

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Front. Biosci. (Landmark Ed) Print ISSN 2768-6701 Electronic ISSN 2768-6698