br We demonstrated that SRC regulates
We demonstrated that SRC-2 regulates the expression of Lyn, which is generally considered an EMT promoter and its expression positively correlates with the TNBC molecular phenotype [21,39,40]. Interest-ingly, lipid rafts, parts of cell membrane where active Lyn can be found [41,42], are formed and maintained by caveolin 1 (CAV1) and caveolin 2 (CAV2) , two basal/stemness markers, which we found to be downregulated by SRC-2 and SRC-3 depletion. Lyn exists in two protein isoforms, the longer p56Lyn and shorter p53Lyn isoform (marked here as Lyn56 and Lyn53), as a consequence of alternative splicing . The dynamic activity of two Lyn isoforms may play a role in the survival of metastatic mesenchymal cancer cells, but their precise biological role has not yet been identified. Detachment of HeLa Epirubicin HCl from the surface decreases the total protein level of Lyn56 and redistributes the rest into the lipid rafts, leaving Lyn53 sequestered in low density membrane fractions, such as endosomes, which rescues the cells from anoikis . Here we show that the depletion of SRC-2 in MCF-7 cells induces op-posing changes in the repressive phosphorylation of the two Lyn iso-forms, decreasing the abundance of the Tyr507 phosphorylated (basal or repressed) Lyn56 isoform, as compared to the control cells, and in-creasing the level of repressed Lyn53 isoform. Considering SRC-2-de-pleted MCF-7 cells show an epithelial phenotype, characterized by in-creased cell-to-cell and cell-to-basal membrane contact, an increase in the protein level of Lyn, as well as a decrease in the level of activated Lyn53 isoform may indicate the involvement of SRC-2 in regulation of cell survival during the metastatic process.
In the present study we report that knock-down of Lyn in shSRC-2 cell spheroids inhibited the development of hollow acini, pointing to a SRC-2-specific involvement of Lyn in acinar development. This seems to be in contrast to another report on mesenchymal breast cancer lines, suggesting that knockdown of Lyn inhibits cell migration and invasion . However, the knowledge of Lyn in epithelial breast cancer cells is limited and, as far as we know, this is the first study of Lyn in MCF-7 spheroids that are considered as a mixed epithelial-mesenchymal cell model.
In conclusion, we show that, in addition to the genes commonly
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regulated by SRC-2 and SRC- 3, SRC-2 also has a separate and unique transcriptional niche in MCF-7 cells, compared to SRC-3, and exhibits a diﬀerent epithelial morphology in 3D cultures. While both coactivators seem to be involved in promoting the EMT program, SRC-2 specifically regulates the protein level of Lyn kinase. Finally, we report that SRC-2 partially regulates EMT by suppressing the expression of Lyn kinase.
Conflicts of interest
This work was supported by grants from the Norwegian Cancer Society and Western Norway Regional Health Authority.
The authors thank the Norwegian Microarray Consortium, University of Bergen for performing the microarray assay and Dr. Christine Stansberg for her expert assistance in data analysis. The flow cytometry was performed at the Flow Cytometry Core Facility, University of Bergen and we particularly thank Brith Bergum and Dr. Richard Davies for their help in flow cytometry data analysis. We are grateful to Divya Sri Priyanka Tallapragada for the help with the GSEA analysis interpretation. This study was supported by the Norwegian Cancer Society and Western Norway Regional Health Authority.
Appendix A. Supplementary data
 S. Kotiyal, S. Bhattacharya, Breast cancer stem cells, EMT and therapeutic targets, Journal of Steroid Biochemistry and Molecular Biology 185 (2019) 57–70