The Laminin‐Derived Peptide C16 Interacts With β1 Integrin, and Is Internalized by Vesicles Through Endocytic Pathway

Breast cancer is one of the leading causes of mortality worldwide. The tumor microenvironment plays a critical role in cancer progression. This microenvironment is composed of various cells embedded in the extracellular matrix (ECM). Laminin-111, a major ECM glycoprotein, produces bioactive peptides that influence tumor biology. We have shown that the laminin-derived peptide C16 (KAFDITYVRLKF), located in the short arm of the γ1 chain, regulates migration, invasion, and invadopodia formation in different cancer cells. Our findings suggest that the regulatory mechanisms underlying the effects of C16 are associated with β1 integrin. This prompted us to investigate the interaction between the C16 peptide and β1 integrin in breast cancer cells. We found that breast cancer cells bind to C16 peptide, and this attachment is inhibited by β1 integrin depletion via siRNA. Cellular localization of the C16 peptide was analyzed using transmission electron microscopy (TEM) and time-lapse fluorescence microscopy. TEM revealed that nanogold-conjugated C16 decorated the cell membrane and was localized in intracellular vesicles, indicating peptide endocytosis. Time-lapse confocal microscopy showed that C16 was internalized by breast cancer cells within 2 h of incubation, with this process increasing over time. Based on these observations, we hypothesized that the peptide is endocytosed and directed to the endosome-lysosome pathway for degradation. Time-lapse imaging demonstrated that part of the internalized peptide colocalized with lysosomes in breast cancer cells. This suggests that C16 may be involved in integrin recycling. Furthermore, rhodamine-labeled C16 colocalized with activated β1 integrins. Flow cytometry analysis showed that C16 increased β1 integrin activation starting at 1 h of treatment. In summary, our results suggest that after interacting with the cell membrane and activating β1 integrins, breast cancer cells internalize peptide C16, which plays a role in β1 integrin turnover.