Melanoma stimulates the proteolytic activity of HaCaT keratinocytes
Abstract 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...
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| Format: | Article |
| Language: | English |
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BMC
2022-09-01
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| Series: | Cell Communication and Signaling |
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| Online Access: | https://doi.org/10.1186/s12964-022-00961-w |
| _version_ | 1811264877277216768 |
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| author | Justyna Mazurkiewicz Aleksandra Simiczyjew Ewelina Dratkiewicz Magdalena Kot Katarzyna Pietraszek-Gremplewicz Dominika Wilk Marcin Ziętek Rafał Matkowski Dorota Nowak |
| author_facet | Justyna Mazurkiewicz Aleksandra Simiczyjew Ewelina Dratkiewicz Magdalena Kot Katarzyna Pietraszek-Gremplewicz Dominika Wilk Marcin Ziętek Rafał Matkowski Dorota Nowak |
| author_sort | Justyna Mazurkiewicz |
| collection | DOAJ |
| description | Abstract 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 |
| first_indexed | 2024-04-12T20:12:14Z |
| format | Article |
| id | doaj.art-2117950ab02546d4a09a257a9f23b444 |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-04-12T20:12:14Z |
| publishDate | 2022-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-2117950ab02546d4a09a257a9f23b4442022-12-22T03:18:12ZengBMCCell Communication and Signaling1478-811X2022-09-0120111710.1186/s12964-022-00961-wMelanoma stimulates the proteolytic activity of HaCaT keratinocytesJustyna Mazurkiewicz0Aleksandra Simiczyjew1Ewelina Dratkiewicz2Magdalena Kot3Katarzyna Pietraszek-Gremplewicz4Dominika Wilk5Marcin Ziętek6Rafał Matkowski7Dorota Nowak8Department of Cell Pathology, Faculty of Biotechnology, University of WroclawDepartment of Cell Pathology, Faculty of Biotechnology, University of WroclawDepartment of Cell Pathology, Faculty of Biotechnology, University of WroclawDepartment of Cell Pathology, Faculty of Biotechnology, University of WroclawDepartment of Cell Pathology, Faculty of Biotechnology, University of WroclawDepartment of Cell Pathology, Faculty of Biotechnology, University of WroclawDepartment of Oncology and Division of Surgical Oncology, Wroclaw Medical UniversityDepartment of Oncology and Division of Surgical Oncology, Wroclaw Medical UniversityDepartment of Cell Pathology, Faculty of Biotechnology, University of WroclawAbstract 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 abstracthttps://doi.org/10.1186/s12964-022-00961-wHaCaTMelanomaTumor microenvironmentProteolysisMigrationInvasion |
| spellingShingle | Justyna Mazurkiewicz Aleksandra Simiczyjew Ewelina Dratkiewicz Magdalena Kot Katarzyna Pietraszek-Gremplewicz Dominika Wilk Marcin Ziętek Rafał Matkowski Dorota Nowak Melanoma stimulates the proteolytic activity of HaCaT keratinocytes Cell Communication and Signaling HaCaT Melanoma Tumor microenvironment Proteolysis Migration Invasion |
| title | Melanoma stimulates the proteolytic activity of HaCaT keratinocytes |
| title_full | Melanoma stimulates the proteolytic activity of HaCaT keratinocytes |
| title_fullStr | Melanoma stimulates the proteolytic activity of HaCaT keratinocytes |
| title_full_unstemmed | Melanoma stimulates the proteolytic activity of HaCaT keratinocytes |
| title_short | Melanoma stimulates the proteolytic activity of HaCaT keratinocytes |
| title_sort | melanoma stimulates the proteolytic activity of hacat keratinocytes |
| topic | HaCaT Melanoma Tumor microenvironment Proteolysis Migration Invasion |
| url | https://doi.org/10.1186/s12964-022-00961-w |
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