impedance-based-real-time-measurement-cancer-cell-migration

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

real-time-quantitative-measurement-tumor-cell-migration-invasion

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

Children's Mercy Research Institute, Children's Mercy Kansas City

University of Missouri Kansas City School of Medicine

Children’s Mercy Kansas City

University of Missouri-Kansas City School of Medicine

Specific cell shapes are fundamental to the organization and function of multicellular organisms. Fibroblast Growth Factor (FGF) signaling induces the elongation of lens fiber cells during vertebrate lens development. Nonetheless, exactly how this extracellular FGF signal is transmitted to the cytoskeletal network has previously not been determined. Here, we show that the Crk family of adaptor proteins, Crk and Crkl, are required for mouse lens morphogenesis but not differentiation. Genetic ablation and epistasis experiments demonstrated that  and  play overlapping roles downstream of FGF signaling in order to regulate lens fiber cell elongation. Upon FGF stimulation, Crk proteins were found to interact with Frs2, Shp2 and Grb2. The loss of Crk proteins was partially compensated for by the activation of Ras and Rac signaling. These results reveal that Crk proteins are important partners of the Frs2/Shp2/Grb2 complex in mediating FGF signaling, specifically promoting cell shape changes.

Crk proteins transduce FGF signaling to promote lens fiber cell elongation.

Natural killer cells are critical in the immune response to infection and malignancy. Prior studies have demonstrated that Crk family proteins can influence cell apoptosis, proliferation, and cell transformation. In this study, we investigated the role of Crk family proteins in mouse NK cell differentiation and host defense using a mouse CMV infection model. The number of NK cells, maturational state, and the majority of the NKR repertoire was similar in Crk x Crk-like (CrkL)-double-deficient and wild type NK cells. However, Crk family proteins were required for optimal activation, IFN-γ production, expansion, and differentiation of Ly49H NK cells, as well as host defense during mouse CMV infection. The diminished function of Crk x CrkL-double-deficient NK cells correlated with decreased phosphorylation of STAT4 and STAT1 in response to IL-12 and IFN-α stimulation, respectively. Together, our findings analyzing NK cell-specific Crk-deficient mice provide insights into the role of Crk family proteins in NK cell function and host defense.

Crk Adaptor Proteins Regulate NK Cell Expansion and Differentiation during Mouse Cytomegalovirus Infection.

CT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII, or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G-S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth.

Fibroblast Growth Requires CT10 Regulator of Kinase (Crk) and Crk-like (CrkL).

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Requirement for Crk and CrkL during postnatal lens development.

The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors. Here, we knocked down Crk, CrkL, or both using siRNAs in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells. These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.

Quantitative assessment of glioblastoma phenotypes in vitro establishes cell migration as a robust readout of Crk and CrkL activity.

Crk and CrkL are cellular counterparts of the viral oncoprotein v-Crk. Crk and CrkL are overexpressed in many types of human cancer, correlating with poor prognosis. Furthermore, gene knockdown and knockout of Crk and CrkL in tumor cell lines suppress tumor cell functions, including cell proliferation, transformation, migration, invasion, epithelial-mesenchymal transition, resistance to chemotherapy drugs, and in vivo tumor growth and metastasis. Conversely, overexpression of tumor cells with Crk or CrkL enhances tumor cell functions. Therefore, Crk and CrkL have been proposed as therapeutic targets for cancer treatment. However, it is unclear whether Crk and CrkL make distinct or overlapping contributions to tumor cell functions in various cancer types because Crk or CrkL have been examined independently in most studies. Two recent studies using colorectal cancer and glioblastoma cells clearly demonstrated that Crk and CrkL need to be ablated individually and combined to understand distinct and overlapping roles of the two proteins in cancer. A comprehensive understanding of individual and overlapping roles of Crk and CrkL in tumor cell functions is necessary to develop effective therapeutic strategies. This review systematically discusses crucial functions of Crk and CrkL in tumor cell functions and provides new perspectives on targeting Crk and CrkL in cancer therapy.

Crk and CrkL as Therapeutic Targets for Cancer Treatment.

CRK and CRKL encode cytoplasmic adaptors that contribute to the etiology of congenital heart disease. Neural crest cells (NCCs) are required for cardiac outflow tract (OFT) septation and aortic arch formation. The roles of Crk/Crkl in NCCs during mouse cardiovascular development remain unknown. To test this, we inactivated Crk and/or Crkl in NCCs. We found that the loss of Crk, rather than Crkl, in NCCs resulted in double outlet right ventricle, while loss of both Crk/Crkl in NCCs resulted in severe defects with earlier lethality due to failed OFT septation and severe dilation of the pharyngeal arch arteries (PAAs). We found that these defects are due to altered cell morphology resulting in reduced localization of NCCs to the OFT and failed integrity of the PAAs, along with reduced expression of Integrin signaling genes. Further, molecular studies identified reduced differentiation of vascular smooth muscle cells that may in part be due to altered Notch signaling. Additionally, there is increased cellular stress that leads to modest increase in apoptosis. Overall, this explains the mechanism for the Crk/Crkl phenotype.

ArticleTotal : 2	Year
Publication title	2020
Publication title	2023

Article	Year
Crk proteins transduce FGF signaling to promote lens fiber cell elongation. eLife\| PubMed ID: 29360039	2018
Crk Adaptor Proteins Regulate NK Cell Expansion and Differentiation during Mouse Cytomegalovirus Infection. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 29618525	2018
Fibroblast Growth Requires CT10 Regulator of Kinase (Crk) and Crk-like (CrkL). The Journal of biological chemistry\| PubMed ID: 27807028	2016
Requirement for Crk and CrkL during postnatal lens development. Biochemical and biophysical research communications\| PubMed ID: 32736680	2020
Quantitative assessment of glioblastoma phenotypes in vitro establishes cell migration as a robust readout of Crk and CrkL activity. The Journal of biological chemistry\| PubMed ID: 33561443	2021
Crk and CrkL as Therapeutic Targets for Cancer Treatment. Cells\| PubMed ID: 33801580	2021
Crk and Crkl have shared functions in neural crest cells for cardiac outflow tract septation and vascular smooth muscle differentiation. Human molecular genetics\| PubMed ID: 34686881	2022

Taeju Park

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