Karthikeyan Mythreye

Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

The type III TGF-β receptor regulates epithelial and cancer cell migration through β-arrestin2-mediated activation of Cdc42

Loss of expression of the TGF-β superfamily coreceptor, the type III TGF-β receptor (TβRIII or betaglycan), occurs in a broad spectrum of human cancers including breast, lung, ovarian, pancreatic, prostate, and renal cell cancer. TβRIII suppresses cancer progression in vivo, at least in part, by reducing cancer cell motility. However, the mechanism by which TβRIII regulates migration is unknown. Here, we demonstrate an unexpected TGF-β signaling independent role for TβRIII in activating Cdc42, altering the actin cytoskeleton and reducing directional persistence to inhibit random migration of both cancer and normal epithelial cells. Functionally, TβRIII through its interaction with the scaffolding protein β-arrestin2, activates Cdc42 and inhibits migration. These studies identify a TGF-β independent homeostatic function for TβRIII in regulating cell migration.

Proteoglycan Signaling Co–receptors: Roles in Cell Adhesion, Migration and Invasion

Signaling co-receptors are diverse, multifunctional components of most major signaling pathways, with roles in mediating and regulating signaling in both physiological and pathophysiological circumstances. Many of these signaling co-receptors, including CD44, glypicans, neuropilins, syndecans and TssRIII/betaglycan are also proteoglycans. Like other co-receptors, these proteoglycan signaling co-receptors can bind multiple ligands, promoting the formation of receptor signaling complexes and regulating signaling at the cell surface. The proteoglycan signaling co-receptors can also function as structural molecules to regulate adhesion, cell migration, morphogenesis and differentiation. Through a balance of these signaling and structural roles, proteoglycan signaling co-receptors can have either tumor promoting or tumor suppressing functions. Defining the role and mechanism of action of these proteoglycan signaling co-receptors should enable more effective targeting of these co-receptors and their respective pathways for the treatment of human disease.

The microtubule-based motor Kar3 and plus end–binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ∼8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.

Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae

Dicentric chromosomes undergo a breakage–fusion–bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis. Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore–microtubule or the kinetochore–DNA interface. Using a conditionally functional dicentric chromosome in vivo, we demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage. Mutations in chl4/mcm17/ctf17 segregate dicentric chromosomes through successive cell divisions without breakage, indicating that only one of the two centromeres is functional. Centromere DNA introduced into the cell is unable to promote kinetochore assembly in the absence of CHL4. In contrast, established centromeres retain their segregation capacity for greater than 25 generations after depletion of Chl4p. The persistent mitotic stability of established centromeres reveals the presence of an epigenetic component in kinetochore segregation. Furthermore, this study identifies Chl4p in the initiation and specification of a heritable chromatin state.

Endoglin mediates fibronectin/α5β1 integrin and TGF‐β pathway crosstalk in endothelial cells

Both the transforming growth factor β (TGF‐β) and integrin signalling pathways have well‐established roles in angiogenesis. However, how these pathways integrate to regulate angiogenesis is unknown. Here, we show that the extracellular matrix component, fibronectin, and its cellular receptor, α5β1 integrin, specifically increase TGF‐β1‐ and BMP‐9‐induced Smad1/5/8 phosphorylation via the TGF‐β superfamily receptors endoglin and activin‐like kinase‐1 (ALK1). Fibronectin and α5β1 integrin increase Smad1/5/8 signalling by promoting endoglin/ALK1 cell surface complex formation. In a reciprocal manner, TGF‐β1 activates α5β1 integrin and downstream signalling to focal adhesion kinase (FAK) in an endoglin‐dependent manner. α5β1 integrin and endoglin form a complex on the cell surface and co‐internalize, with their internalization regulating α5β1 integrin activation and signalling. Functionally, endoglin‐mediated fibronectin/α5β1 integrin and TGF‐β pathway crosstalk alter the responses of endothelial cells to TGF‐β1, switching TGF‐β1 from a promoter to a suppressor of migration, inhibiting TGF‐β1‐mediated apoptosis to promote capillary stability, and partially mediating developmental angiogenesis in vivo. These studies provide a novel mechanism for the regulation of TGF‐β superfamily signalling and endothelial function through crosstalk with integrin signalling pathways.

The type III transforming growth factor-β receptor inhibits proliferation, migration, and adhesion in human myeloma cells

Here we demonstrate that the transforming growth factor-β (TGF-β) superfamily coreceptor, the type III TGF-β receptor, functions to suppress multiple myeloma disease progression through inhibition of multiple myeloma cell proliferation, heterotropic cell–cell adhesion, and migration, in a manner largely independent of its ligand presentation role.

Type III TGF-β receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Comparison of mRNA from benign neuroblastic tumors and neuroblastomas revealed that expression of the type III TGF-β receptor (TGFBR3) decreases with advancing stage of neuroblastoma and this loss correlates with a poorer prognosis. Patients with MYCN oncogene amplification and low TGFBR3 expression were more likely to have an adverse outcome. In vitro, TβRIII expression was epigenetically suppressed by MYCN-mediated recruitment of histone deacetylases to regions of the TGFBR3 promoter. TβRIII bound FGF2 and exogenous FGFR1, which promoted neuronal differentiation of neuroblastoma cells. TβRIII and FGF2 cooperated to induce expression of the transcription factor inhibitor of DNA binding 1 via Erk MAPK. TβRIII-mediated neuronal differentiation suppressed cell proliferation in vitro as well as tumor growth and metastasis in vivo. These studies characterize a coreceptor function for TβRIII in FGF2-mediated neuronal differentiation, while identifying potential therapeutic targets and clinical biomarkers for neuroblastoma.

TβRIII/β-arrestin2 regulates integrin α5β1 trafficking, function, and localization in epithelial cells

The type III TGF-β receptor (TβRIII) is a ubiquitous co-receptor for TGF-β superfamily ligands with roles in suppressing cancer progression, in part through suppressing cell motility. Here we demonstrate that TβRIII promotes epithelial cell adhesion to fibronectin in a β-arrestin2 dependent and TGF-β/BMP independent manner by complexing with active integrin α5β1, and mediating β-arrestin2-dependent α5β1 internalization and trafficking to nascent focal adhesions. TβRIII-mediated integrin α5β1 trafficking regulates cell adhesion and fibronectin fibrillogenesis in epithelial cells, as well as α5 localization in breast cancer patients. We further demonstrate that increased TβRIII expression correlates with increased α5 localization at sites of cell-cell adhesion in breast cancer patients, while higher TβRIII expression is a strong predictor of overall survival in breast cancer patients. These data support a novel, clinically relevant role for TβRIII in regulating integrin α5 localization, reveal a novel crosstalk mechanism between the integrin and TGF-β superfamily signaling pathways and identify β-arrestin2 as a regulator of α5β1 trafficking.

TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion

Recent studies implicate a role for cell mechanics in cancer progression. Transforming growth factor β–induced epithelial-to-mesenchymal transition results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.