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S100A8 Transported by SEC23A Inhibits Metastatic Colonization Via

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S100A8 Transported by SEC23A Inhibits Metastatic Colonization Via Sun et al. Cell Death and Disease (2020) 11:650 https://doi.org/10.1038/s41419-020-02835-w Cell Death & Disease ARTICLE Open Access S100A8 transported by SEC23A inhibits metastatic colonization via autocrine activation of autophagy Zhiwei Sun1,2, Bin Zeng1,2,DoudouLiu1,2, Qiting Zhao1,2,JianyuWang1,2 and H. Rosie Xing2,3 Abstract Metastasis is the main cause of failure of cancer treatment. Metastatic colonization is regarded the most rate- limiting step of metastasis and is subjected to regulation by a plethora of biological factors and processes. On one hand, regulation of metastatic colonization by autophagy appears to be stage- and context-dependent, whereas mechanistic characterization remains elusive. On the other hand, interactions between the tumor cells and their microenvironment in metastasis have long been appreciated, whether the secretome of tumor cells can effectively reshape the tumor microenvironment has not been elucidated mechanistically. In the present study, we have identified “SEC23A-S1008-BECLIN1-autophagy axis” in the autophagic regulation of metastatic colonization step, a mechanism that tumor cells can exploit autophagy to exert self-restrain for clonogenic proliferation before the favorable tumor microenvironment is established. Specifically, we employed a paired lung-derived oligometastatic cell line (OL) and the homologous polymetastatic cell line (POL) from human melanoma cell line M14 that differ in colonization efficiency. We show that S100A8 transported by SEC23A inhibits metastatic colonization via autocrine activation of autophagy. Furthermore, we verified the clinical relevance of our experimental findings by bioinformatics analysis of the expression of Sec23a and S100A8 and the clinical- pathological associations. We demonstrate that higher Sec23a and Atg5 expression levels appear to be protective factors and favorable diagnostic (TNM staging) and prognostic (overall survival) markers for skin cutaneous 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; melanoma (SKCM) and colon adenocarcinoma (COAD) patients. And we confirm the bioinformatics analysis results with SKCM biopsy samples. Introduction cells enter a period of dormancy to withstand the restrains Metastasis is the main cause of failure of cancer treat- posted by the foreign microenvironment and immune – ment1 3. It is characterized by discrete multi-steps: surveillance. Second, survived tumor cells activate clo- acquisition of the invasive phenotype, local invasion into nogenic proliferation in order to form micro-metastases. surrounding stroma and hematogenous circulation, sur- Finally, upon establishing favorable tumor micro- vival in the circulation, extravasation and invasion into environment, micro-metastases will develop into clinically distant organs, survival at the secondary site, and colo- detectable macro-metastases. – nization to form micro- and macro-metastases2,4 6. The Interactions between the tumor cells and their micro- last stage, i.e., the colonization of invaded tumor cells, is environment play a vital role in the entire metastatic – regarded most rate-limiting for metastasis. Colonization is cascade, especially in the colonization at the distant site6 8. also a multi-step process2,5,6: first, the extravasated tumor However, mechanistic understanding of the interactions between the tumor cells and their microenvironment at the site of metastasis has been largely focused on how Correspondence: Jianyu Wang ([email protected])or H. Rosie Xing ([email protected]) tumor cells will overcome the restrains of the foreign 1Institute of Life Sciences, Chongqing Medical University, Chongqing, China microenvironment to develop into micro- and macro- 2 Laboratory of Translational Cancer Stem Cell Research, Chongqing Medical metastatic lesions, little is known about whether tumor University, Chongqing, China Full list of author information is available at the end of the article cells exert self-restrains upon extravasation. Edited by G.M. Fimia © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Sun et al. Cell Death and Disease (2020) 11:650 Page 2 of 13 The secretome of tumor cells can effectively reshape autophagy in extravasated tumor cells. Furthermore, the tumor microenvironment via autocrine regulation Sec23a and Atg5 are favorable diagnostic and prognostic of tumor cells or paracrine interactions with the stro- markers for human melanoma and colon cancer. mal cells. SEC23A is an important constituent of coat protein complex II (COPII) that is responsible for the Materials and methods transportation of secreted proteins from rough endo- Cell lines and cell culture – plasmic reticulum to Golgi apparatus9 11. And SEC23A M14 cells were kindly provided by Dr. Robert Hoffman has been reported to participate in chondrogenesis12,13 (University of California San Diego). By isolation of M14 – and suppress tumor metastasis14 17 by regulating cells from the lung of mice exhibited oligometastasis or tumor cell protein secretion, i.e., the secretome. Our polymetastasis, respectively, in vivo, followed by three previous study has characterized alterations in the rounds of in vivo validation, OL and POL cell lines from composition of the SEC23A secretome upon Sec23a human melanoma cancer cell line M14 were estab- silencing in M14 human melanoma cells and identified lished18,42. M14, OL and POL cell lines and its derivative S100A8 on the list of the significantly decreased cell lines were cultured in DMEM (Hyclone) supple- secreted proteins18. S100A8 is a multi-functional pro- mented with 10% fetal bovine serum (FBS) (Gibco) and – tein19 21. It is a calcium-binding protein and poly- 1% penicillin-streptomycin (Hyclone). merizes with S100A9 to form calprotectin for metals iron, manganese and zinc detention via chelation. Lentivirus production Secreted S100A8 may regulate inflammatory response The sequence for the sh-RNAs targeting Sec23a was 5′- within the tumor microenvironment. However, the role GGAAGCTACAAGAATGGTTGT-3′. Sec23a over- of S100A8 in metastasis has not been characterized expression (OE) plasmid pLVX-Puro-mRuby-Sec23a prior to this study. (Plasmid #36158) was provided by Addgene. The lenti- Autophagy is an evolutionarily conserved biological virus particles of shSec23a and Sec23a-OE were prepared process of energy metabolism22,23. By degrading intra- by Sangon Biotech Co. The lentivirus particles of shAtg5 cellular organelles and proteins, autophagy provides cells LV-ATG5-RNAi(9513-1) were purchased from GENE- with biochemical reaction substrates for the maintenance CHEM. The sequence for the sh-RNAs targeting S100A8 of homeostasis under nutrient deprivation or other was 5′-CCUGAAGAAAUUGCUAGAGTT-3′ and the – stressful conditions24 27. Both the anti- and pro- lentivirus particles of shS100A8 were prepared by metastatic roles of autophagy have been reported and GENECHEM. – appear to be context and stage-dependent28 31: in the initial stage of metastasis, autophagy may inhibit metas- Autophagy inhibitors and activator tasis by promoting the release of anti-metastatic immu- 3-MA (M9281) was purchased from Sigma. Baf-A1 – nomodulatory factors29,32 34. Once tumor cells enter (S1413) and Rapamycin (S1039) were purchased from hematogenous circulation, autophagy may promote Selleck. metastasis by protecting the circulating tumor cells from – anoikis29,35 37. During colonization at the metastatic site, Reverse transcription and quantitative real-time the role of autophagy becomes intricate. On the one hand, polymerase chain reaction (RT-qPCR) autophagy keeps the extravasated tumor cells in the Total RNAs were isolated using Trizol (Takara, Japan) dormancy stage thus prevents proliferation and coloni- and reverse-transcribed into cDNA using PrimeScript RT zation29,38,39. On the other hand, once micro-metastases Master Mix (Takara, Japan). RT-qPCR was performed are established, autophagy switches to promote macro- using SYBR Green Real-time PCR Master Mix kit (Takara, metastases via helping tumor cells adapt to the foreign Japan) according to the manufacturer’s instructions. The microenvironment29,40,41. following PCR condition was used on the Light Cycler: 39 In the present study, we focus on uncovering cycles of 95 °C for 30 s, 95 °C for 5 s, followed by 60 °C for mechanisms underlying autophagic regulation of the 30 s in a 10 µl reaction volume. Relative expression was colonization step of metastasis. We employed a paired of normalized to that of GAPDH internal control. The for- lung-derived oligometastatic cell line (OL) and the ward primer sequences and reverse primer sequences of homologous polymetastatic cell line (POL) from human Sec23a, Atg5, S100A8,
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