PMID- 36123693 OWN - NLM STAT- MEDLINE DCOM- 20220922 LR - 20220929 IS - 1478-811X (Electronic) IS - 1478-811X (Linking) VI - 20 IP - 1 DP - 2022 Sep 19 TI - Melanoma stimulates the proteolytic activity of HaCaT keratinocytes. PG - 146 LID - 10.1186/s12964-022-00961-w [doi] LID - 146 AB - BACKGROUND: Keratinocytes constitute a major part of the melanoma microenvironment, considering their protective role towards melanocytes in physiological conditions. However, their interactions with tumor cells following melanomagenesis are still unclear. METHODS: We used two in vitro models (melanoma-conditioned media and indirect co-culture of keratinocytes with melanoma cells on Transwell inserts) to activate immortalized keratinocytes towards cancer-associated ones. Western Blotting and qPCR were used to evaluate keratinocyte markers and mediators of cell invasiveness on protein and mRNA expression level respectively. The levels and activity of proteases and cytokines were analysed using gelatin-FITC staining, gelatin zymography, chemiluminescent enzymatic test, as well as protein arrays. Finally, to further study the functional changes influenced by melanoma we assessed the rate of proliferation of keratinocytes and their invasive abilities by employing wound healing assay and the Transwell filter invasion method. RESULTS: HaCaT keratinocytes activated through incubation with melanoma-conditioned medium or indirect co-culture exhibit properties of less differentiated cells (downregulation of cytokeratin 10), which also prefer to form connections with cancer cells rather than adjacent keratinocytes (decreased level of E-cadherin). While they express only a small number of cytokines, the variety of secreted proteases is quite prominent especially considering that several of them were never reported as a part of secretome of activated keratinocytes' (e.g., matrix metalloproteinase 3 (MMP3), ADAM metallopeptidase with thrombospondin type 1 motif 1). Activated keratinocytes also seem to exhibit a high level of proteolytic activity mediated by MMP9 and MMP14, reduced expression of TIMPs (tissue inhibitor of metalloproteinases), upregulation of ERK activity and increased levels of MMP expression regulators-RUNX2 and galectin 3. Moreover, cancer-associated keratinocytes show slightly elevated migratory and invasive abilities, however only following co-culture with melanoma cells on Transwell inserts. CONCLUSIONS: Our study offers a more in-depth view of keratinocytes residing in the melanoma niche, drawing attention to their unique secretome and mediators of invasive abilities, factors which could be used by cancer cells to support their invasion of surrounding tissues. Video abstract. CI - (c) 2022. The Author(s). FAU - Mazurkiewicz, Justyna AU - Mazurkiewicz J AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. justyna.mazurkiewicz2@uwr.edu.pl. FAU - Simiczyjew, Aleksandra AU - Simiczyjew A AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. FAU - Dratkiewicz, Ewelina AU - Dratkiewicz E AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. FAU - Kot, Magdalena AU - Kot M AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. FAU - Pietraszek-Gremplewicz, Katarzyna AU - Pietraszek-Gremplewicz K AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. FAU - Wilk, Dominika AU - Wilk D AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. FAU - Zietek, Marcin AU - Zietek M AD - Department of Oncology and Division of Surgical Oncology, Wroclaw Medical University, Plac Hirszfelda 12, 53-413, Wroclaw, Poland. AD - Wroclaw Comprehensive Cancer Center, Plac Hirszfelda 12, 53-413, Wroclaw, Poland. FAU - Matkowski, Rafal AU - Matkowski R AD - Department of Oncology and Division of Surgical Oncology, Wroclaw Medical University, Plac Hirszfelda 12, 53-413, Wroclaw, Poland. AD - Wroclaw Comprehensive Cancer Center, Plac Hirszfelda 12, 53-413, Wroclaw, Poland. FAU - Nowak, Dorota AU - Nowak D AD - Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Video-Audio Media DEP - 20220919 PL - England TA - Cell Commun Signal JT - Cell communication and signaling : CCS JID - 101170464 RN - 0 (Cadherins) RN - 0 (Core Binding Factor Alpha 1 Subunit) RN - 0 (Culture Media, Conditioned) RN - 0 (Cytokines) RN - 0 (Galectin 3) RN - 0 (RNA, Messenger) RN - 0 (Thrombospondins) RN - 0 (Tissue Inhibitor of Metalloproteinases) RN - 68238-35-7 (Keratins) RN - 9000-70-8 (Gelatin) RN - EC 3.4.24.17 (Matrix Metalloproteinase 3) RN - EC 3.4.24.35 (Matrix Metalloproteinase 9) RN - EC 3.4.24.80 (Matrix Metalloproteinase 14) RN - I223NX31W9 (Fluorescein-5-isothiocyanate) SB - IM MH - Cadherins/metabolism MH - Core Binding Factor Alpha 1 Subunit MH - Culture Media, Conditioned/pharmacology MH - Cytokines MH - Fluorescein-5-isothiocyanate MH - Galectin 3 MH - Gelatin MH - Humans MH - Keratinocytes/pathology MH - Keratins MH - Matrix Metalloproteinase 14 MH - *Matrix Metalloproteinase 3 MH - Matrix Metalloproteinase 9/metabolism MH - *Melanoma/pathology MH - RNA, Messenger/metabolism MH - Thrombospondins MH - Tissue Inhibitor of Metalloproteinases PMC - PMC9484146 OTO - NOTNLM OT - HaCaT OT - Invasion OT - Melanoma OT - Migration OT - Proteolysis OT - Tumor microenvironment COIS- The authors declare that there is no competing interests. EDAT- 2022/09/20 06:00 MHDA- 2022/09/23 06:00 PMCR- 2022/09/19 CRDT- 2022/09/19 23:39 PHST- 2022/06/30 00:00 [received] PHST- 2022/08/20 00:00 [accepted] PHST- 2022/09/19 23:39 [entrez] PHST- 2022/09/20 06:00 [pubmed] PHST- 2022/09/23 06:00 [medline] PHST- 2022/09/19 00:00 [pmc-release] AID - 10.1186/s12964-022-00961-w [pii] AID - 961 [pii] AID - 10.1186/s12964-022-00961-w [doi] PST - epublish SO - Cell Commun Signal. 2022 Sep 19;20(1):146. doi: 10.1186/s12964-022-00961-w.