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Cancer stem cells (CSCs) has been a key target to cure cancer patients completely. Although many CSC markers have been identified, they are frequently cancer type-specific and those expressions are occasionally variable, which becomes an obstacle to elucidate the characteristics of the CSCs. Here we scrutinized the relationship between stemness elevation and geometrical features of single cells. The PAMPS hydrogel was utilized to create the CSCs from mouse myoblast C2C12 and its synovial sarcoma model cells. qRT-PCR analysis confirmed the significant increase in expression levels of Sox2, Nanog, and Oct3/4 on the PAMPS gel, which was higher in the synovial sarcoma model cells. Of note, the morphological heterogeneity was appeared on the PAMPS gel, mainly including flat spreading, elongated spindle, and small round cells, and the Sox2 expression was highest in the small round cells. To examine the role of morphological differences in the elevation of stemness, over 6,400 cells were segmented along with the Sox2 intensity, and 12 geometrical features were extracted at single cell level. A nonlinear mapping of the geometrical features by using uniform manifold approximation and projection (UMAP) clearly revealed the existence of relationship between morphological differences and the stemness elevation, especially for C2C12 and its synovial sarcoma model on the PAMPS gel in which the small round cells possess relatively high Sox2 expression on the PAMPS gel, which supports the strong relationship between morphological changes and the stemness elevation. Taken together, these geometrical features can be useful for morphological profiling of CSCs to classify and distinguish them for understanding of their role in disease progression and drug discovery.

Signaling adaptor protein Crk has been shown to play an important role in various human cancers. Crk links tyrosine kinases and guanine nucleotide exchange factors (GEFs) such as C3G and Dock180 to activate small G-proteins Rap and Rac, respectively. In pancreatic cancer, various molecular targeted therapies have provided no significant therapeutic benefit for the patients so far due to constitutive activation of KRAS by frequent KRAS mutation. Therefore, the establishment of novel molecular targeted therapy in KRAS-independent manner is required. Here, we investigated a potential of Crk as a therapeutic target in pancreatic cancer. Immunohistochemistry on human pancreatic cancer specimens revealed that the patients with high expression of Crk had a worse prognosis than those with low expression. We established Crk-knockdown pancreatic cancer cells by siRNA using PANC-1, AsPC-1, and MIA PaCa-2 cells, which showed decreased cell proliferation, invasion, and adhesion. In Crk-knockdown pancreatic cancer cells, the decrease of c-Met phosphorylation was observed. In the orthotopic xenograft model, Crk depletion prolonged survival of mice significantly. Thus, signaling adaptor protein Crk is involved in malignant potential of pancreatic cancer associated with decrease of c-Met phosphorylation, and Crk can be considered to be a potential therapeutic molecular target.

Negatively charged synthetic hydrogels have been known to facilitate various cellular responses including cell adhesion, proliferation, and differentiation; however, the molecular mechanism of hydrogel-dependent control of cell behavior remains unclear. Recently, we reported that negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induces chondrogenic differentiation of ATDC5 cells via novel protein reservoir function. In this study, we identified the cell adhesion molecules binding to PAMPS gels that act as mechanoreceptors. First, we performed a pull-down assay by particle gels using cell membrane proteins of ATDC5, and found that multiple membrane proteins bound to the PAMPS gel, whereas the uncharged poly(N,N'-dimethylacrylamide) gel as control did not bind to any membrane proteins. Western blot analysis indicated differential binding of integrin (ITG) isoforms to the PAMPS gel, in which the α4 isoform, but not α5 and αv, efficiently bound to the PAMPS gel. ITG α4 knockdown decreased cell spreading of ATDC5 on PAMPS gels, whereas the enhanced expression increased the behavior. Furthermore, ITG α4 depletion suppressed PAMPS gel-induced expression of bone morphogenic protein (BMP) 4 contributing to chondrogenic differentiation, in concordance with the reduction of ERK activation. These results demonstrated that membrane protein binding to PAMPS gels occurred in a charge-dependent manner, and that ITG α4 plays a crucial role in cell spreading on PAMPS gels and acts as a mechanoreceptor triggering cellular signaling leads to chondrogenic differentiation.

Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.

We have recently reported that transcription factor 8 (TCF8) negatively regulates pathological angiogenesis by regulating endothelial invasiveness by acting as a transcriptional attenuator of matrix metalloproteinase 1. TCF8 also modulates cell–matrix and cell–cell adhesion; however molecular mechanism of this TCF8 function remains obscure. Here, we provide evidence that TCF8 activates R-Ras, another class of angiogenic regulator, to suppress angiogenesis by a mechanism other than a transcriptional attenuator. Tube formation by human umbilical vein endothelial cells (HUVECs) facilitated by TCF8 suppression was significantly inhibited by the expression of constitutive active mutant of R-Ras. When we examined the mRNA expression levels of R-Ras regulators, no significant changes were observed to explain the R-Ras activation by TCF8. Interestingly, we found that TCF8 bound to CalDAG-GEFIII, an R-Ras activator, in the cytosol, indicating that TCF8 emanates signaling for R-Ras activation from cytosol to regulate angiogenesis negatively.

Signaling adaptor protein Crk has been shown to be involved in pathogenesis of human cancers including brain tumor where Crk was reported to be overexpressed. In this study, we addressed whether Crk is indispensable for malignant phenotype of brain tumor. In 20 surgical specimens of glioma, mRNA of both CrkI and CrkII was found to be elevated in malignant tumor. To define a precise role of Crk, we have established Crk-knockdown cell lines of glioblastoma KMG4 by siRNA, and early phase of cell adhesion to laminin was found to be suppressed. Wound healing assay revealed the decreased cell motility in Crk knockdown cells, and suppression of both anchorage-dependent and -independent growth were demonstrated in these cells. Furthermore, in vivo tumor forming potential was also markedly suppressed. These results suggest that Crk is required for early attachment to laminin, cell motility, and growth of glioblastoma cell line KMG4.

DOCK2, a CDM family protein exclusively found in hematopoietic cells, has been shown to play a role in lymphocyte migration by the regulation of actin cytoskeleton. Although DOCK2 has been shown to induce the activation of Rac1, the regulatory mechanism of Rac2, which is a hematopoietic cell-specific small GTPase, is still unknown. In this study, we examined the role of DOCK2 in the activation of Rac2 in hematopoietic cells. DOCK2 was found to associate with the zeta subunit of the CD3 complex of T cell receptors in Jurkat cells and to activate forced expressed Rac2 in 293T cells. In addition, the stable expression of DOCK2 in Jurkat cells exhibited the elevated activity of endogenous Rac2. Furthermore, the transcriptional activity of interleukin-2 (IL-2) was enhanced in DOCK2-expressing Jurkat cells and the dominant negative form of Rac2 suppressed its elevated IL-2 promoter activity. These results suggest that DOCK2 mediates TCR-dependent activation of Rac2, leading to the regulation of IL-2 promoter activity in T cells.