Unveiling Epithelial Plasticity Regulation in Lung Cancer: Exploring the Crosstalk Among Tks4 Scaffold Protein Partners.

The epithelial to mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein,Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer (NSCLC). In this study, we used CRISPR/Cas9 method to knockout Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectroscopy on four different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.

Studying the Association of TKS4 and CD2AP Scaffold Proteins and Their Implications in the Partial Epithelial-Mesenchymal Transition (EMT) Process.

Colon cancer is a leading cause of death worldwide. Identification of new molecular factors governing the invasiveness of colon cancer holds promise in developing screening and targeted therapeutic methods. The Tyrosine Kinase Substrate with four SH3 domains (TKS4) and the CD2-associated protein (CD2AP) have previously been linked to dynamic actin assembly related processes and cancer cell migration, although their co-instructive role during tumor formation remained unknown. Therefore, this study was designed to investigate the TKS4-CD2AP interaction and study the interdependent effect of TKS4/CD2AP on oncogenic events. We identified CD2AP as a novel TKS4 interacting partner via co-immunoprecipitation-mass spectrometry methods. The interaction was validated via Western blot (WB), immunocytochemistry (ICC) and proximity ligation assay (PLA). The binding motif of CD2AP was explored via peptide microarray. To uncover the possible cooperative effects of TKS4 and CD2AP in cell movement and in epithelial-mesenchymal transition (EMT), we performed gene silencing and overexpressing experiments. Our results showed that TKS4 and CD2AP form a scaffolding protein complex and that they can regulate migration and EMT-related pathways in HCT116 colon cancer cells. This is the first study demonstrating the TKS4-CD2AP protein-protein interaction in vitro, their co-localization in intact cells, and their potential interdependent effects on partial-EMT in colon cancer.

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System.

Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.