Morphology, Motility, and Cytoskeletal Architecture of Breast Cancer Cells Depend on Keratin 19 and Substrate

Abstract

Cancer cells gain motility through events that accompany modulation of cell shape and include altered expression of keratins. However, the role of keratins in change of cancer cell architecture is not well understood. Therefore, we ablated the expression of keratin 19 (K19) in breast cancer cells of the MDA-MB-231 cell line and found that cells lacking K19 become more elongated in culture, with morphological reversion toward the parental phenotype upon transduction of KRT19. Also, the number of actin stress fibers and focal adhesions were significantly reduced in KRT19 knockout (KO) cells. The altered morphology of KRT19 KO cells was then characterized quantitatively using digital holographic microscopy (DHM), which not only confirmed the phenotypic change of KRT19 KO cells but also identified that the K19-dependent morphological change is dependent on the substrate type. A new quantitative method of single cell analysis from DHM, via average phase difference maps, facilitated evaluation of K19-substrate interactive effects on cell morphology. When plated on collagen substrate, KRT19 KO cells were less elongated and resembled parental cells. Assessing single cell motility further showed that while KRT19 KO cells moved faster than parental cells on a rigid surface, this increase in motility became abrogated when cells were plated on collagen. Overall, our study suggests that K19 inhibits cell motility by regulating cell shape in a substrate-dependent manner. Thus, this study provides a potential basis for the altered expression of keratins associated with change in cell shape and motility of cancer cells.

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

“…In this study, we demonstrate the role of K19 in maintaining rounded epithelial cell morphology of the MCF7 breast cancer cell line, which is of estrogen-positive luminal subtype where predominant expression of K19 can be found [35][36][37][38][39]. Interestingly, altered cell shape due to changed K19 expression was also observed in transformed mammary stem/progenitor cells [40] and triple negative breast cancer cell lines BT549 [23] and MDA-MB -231 [41], suggesting that K19 plays a crucial role in maintaining the architecture of breast cancer cells across different differentiation stages and molecular subtypes. Although functions of K19 in normal settings have not been fully resolved, K19 is likely involved in maintaining cell architecture through desmosomes as a member of the keratin family of proteins.…”

“…The holograms were captured by a 1.3 MP CMOS camera (Lumenera Corporation, Inc., Ontario, Canada). Detailed information about the set up was published in [34,41,62]. Cell phase-derived pseudoheight maps, calculated directly from phase maps by assuming a cell index of refraction of 1.381, of parental and KRT19 KO2 cells cultured on glass were collected with a sample size of n = 259 and 173, respectively.…”

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

“…In this study, we demonstrate the role of K19 in maintaining rounded epithelial cell morphology of the MCF7 breast cancer cell line, which is of estrogen-positive luminal subtype where predominant expression of K19 can be found [39][40][41][42][43] . Interestingly, altered cell shape due to changed K19 expression was also observed in transformed mammary stem/progenitor cells 44 and triple negative breast cancer cell lines BT549 23 and MDA-MB-231 45 , suggesting that K19 plays a crucial role in maintaining the architecture of breast cancer cells across different differentiation stages and molecular subtypes. Although functions of K19 in normal settings have not been fully resolved, K19 is likely involved in maintaining cell architecture through desmosomes as a member of the keratin family of proteins.…”

“…The holograms were captured by a 1.3 MP CMOS camera (Lumenera Corporation, Inc., Ontario, Canada). Detailed information about the set up was published in 45,68,69 . Cell phase maps of parental and KRT19 KO2 cells cultured on glass were collected with a sample size of n = 259 and 173, respectively.…”

Tracking Single Cells Motility on Different Substrates

“…We also describe a method to track random cell migration using wrMTrck [39] plugin in ImageJ [40]. This technique can easily be used to study cell motility at a single cell level on different substrates such as glass and collagen hydrogels [41]. A protocol using MDA-MB-231 cells is described here but various adherent cell types can be analyzed using this technique.…”