Keefe T. Chan

FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion.

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.

Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.

Regulation of Adhesion Dynamics by Calpain-mediated Proteolysis of Focal Adhesion Kinase (FAK)

The coordinated and dynamic regulation of adhesions is required for cell migration. We demonstrated previously that limited proteolysis of talin1 by the calcium-dependent protease calpain 2 plays a critical role in adhesion disassembly in fibroblasts (Franco, S. J., Rodgers, M. A., Perrin, B. J., Han, J., Bennin, D. A., Critchley, D. R., and Huttenlocher, A. (2004) Nat. Cell Biol. 6, 977–983). However, little is known about the contribution of other calpain substrates to the regulation of adhesion dynamics. We now provide evidence that calpain 2-mediated proteolysis of focal adhesion kinase (FAK) regulates adhesion dynamics in motile cells. We mapped the preferred calpain cleavage site between the two C-terminal proline-rich regions after Ser-745, resulting in a C-terminal fragment similar in size to the FAK-related non-kinase (FRNK). We generated mutant FAK with a point mutation (V744G) that renders FAK resistant to calpain proteolysis but retains other biochemical properties of FAK. Using time-lapse microscopy, we show that the dynamics of green fluorescent protein-talin1 are impaired in FAK-deficient cells. Expression of wild-type but not calpain-resistant FAK rescues talin dynamics in FAK-deficient cells. Taken together, our findings suggest a novel role for calpain proteolysis of FAK in regulating adhesion dynamics in motile cells.

Unraveling the enigma: Progress towards understanding the coronin family of actin regulators

Coronins are a conserved family of actin cytoskeleton regulators that promote cell motility and modulate other actin-dependent processes. Although these proteins have been known for 20 years, substantial progress has been made in the past 5 years towards their understanding. In this review, we examine this progress, place it into the context of what was already known, and pose several questions that remain to be addressed. In particular, we cover the emerging consensus about the role of Type I coronins in coordinating the function of Arp2/3 complex and ADF/cofilin proteins. This coordination plays an important role in leading-edge actin dynamics and overall cell motility. Finally, we discuss the roles played by the more exotic coronins of the Type II and III classes in cellular processes away from the leading edge.

Integrins in cell migration.

Integrins are cell-surface adhesion receptors that play a central role in regulating cell migration by mediating interactions between the extracellular matrix and the actin cytoskeleton. Substantial progress has been made in understanding the mechanisms by which the formation and breakdown of adhesions are regulated. Here we describe general methods used to study integrin-mediated cell migration. Furthermore, we outline detailed procedures to examine focal adhesion assembly and disassembly using time-lapse fluorescent microscopy. Finally, we provide methods for the analysis of podosomes, which are highly dynamic adhesive structures that form in immune cells and invasive cancer cells.

Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

β2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion

IntroductionFor efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of β-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion.MethodsTo characterize the molecular and cellular mechanisms of β-adrenergic signaling on the invasive properties of breast cancer cells, we used functional cellular assays to quantify invadopodia formation and to evaluate cell invasion in two-dimensional and three-dimensional environments. The functional significance of β-adrenergic regulation of invadopodia was investigated in an orthotopic mouse model of spontaneous breast cancer metastasis.Resultsβ-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the β2-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate invadopodia formation. Increased invadopodia occurred at the expense of focal adhesion formation, resulting in a switch to increased tumor cell invasion through three-dimensional extracellular matrix. β2-adrenoceptor signaling increased invasion of tumor cells from explanted primary tumors through surrounding extracellular matrix, suggesting a possible mechanism for the observed increased spontaneous tumor cell dissemination in vivo. Selective antagonism of β2-adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination.ConclusionThese findings provide insight into conditions that control tumor cell invasion by identifying signaling through β2-adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using β-blockers to target β2-adrenoceptors to limit tumor cell dissemination and metastasis.

Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin

Multiphoton microscopy is a powerful tool that enables the visualization of fluorescently tagged tumor cells and their stromal interactions within tissues in vivo. We have developed an orthotopic model of implanting multicellular melanoma tumor spheroids into the dermis of the mouse ear skin without the requirement for invasive surgery. Here, we demonstrate the utility of this approach to observe the primary tumor, single cell actin dynamics, and tumor-associated vasculature. These methods can be broadly applied to investigate an array of biological questions regarding tumor cell behavior in vivo.

IL2 Inducible T-cell Kinase, a Novel Therapeutic Target in Melanoma.

Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation. Experimental Design: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined. Results: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0–G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Ptennull/BrafV600E) melanomas. Conclusions: We conclude that ITK, formerly considered an immune cell–specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanoma model supports this possibility. Clin Cancer Res; 21(9); 2167–76. ©2015 AACR.

Abstract B206: Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers.

Protein kinases play key roles in defining the transformation of many solid tumors, including renal cell carcinoma (RCC), and have become attractive drug targets. The Ror-family receptor tyrosine kinases (RTKs) are transmembrane proteins with putative tyrosine kinase activities that play crucial roles during the development of various organs and tissues. One of these receptors, the RTK-like orphan receptor 2 (Ror2) is known as a developmentally regulated receptor that enhances tumor cell migration and tumor invasiveness. Recently, our lab reported on the expression of Ror2 in human RCC tumors and cell lines, and that its expression is correlated with invasive growth in culture. In mammals, Ror2 has been shown to act as a receptor or co-receptor for Wnt5a, a member of the Wnt family, inducing a noncanonical Wnt signaling cascade. We have found that Ror2 is expressed in various cell lines, including 786-O, HEK293, HeLa, SaOS2, and U2OS. Cell migration analysis using single cell tracking confirmed that Ror2 promotes cell migration, further enhanced by Wnt5a stimulation. Separately, we have shown that Ror2 expression correlates with enhanced canonical Wnt-signaling through an increased pool of downstream stable β-catenin in RCC and activation of canonical targets. However, the kinase activity of Ror2 has been controversial. Using 786-O Ror2 overexpressing cells (786-O/Ror2), we detected that Ror2 becomes phosphorylated upon Wnt5a treatment. Based on a report of antibody induced homodimerization of Ror2 necessary for stimulation, we treated 786-O/Ror2 cells with Ror2 antibody and verified a significantly enhanced phosphorylation. Based on these findings, we hypothesize that receptor dimerization via Wnt ligand engagement or antibody treatment is necessary for effective signal transduction. We have thus utilized the PathHunter Ror2 activity assay developed by DiscoveRX, to use blockade of dimerization as an assay for Ror2 targeted drug development. This system utilizes an EGFR/Ror2 chimera cell line that expresses the cytosolic portion of ROR2 containing the kinase domain tagged with a ProLink tag at the C-terminus and fused to the extracellular and transmembrane domains of EGFR. Receptor activation is mediated by EGF addition, which results in a dose-dependent increase in signal caused by complementation of the SH2 tagged with the complementary EA enzyme fragment binding to the phosphorylated receptor. Thus, activation reads out in dimerization and phosphorylation which in turn, results in enzyme fragment complementation in this assay. Our data using this EGFR/Ror2 chimera U2OS cell line show that stimulation of these cells with EGF induces phosphorylation to a great extent both by IP/western, and chimeric signal. We believe these tools are useful in screening compounds in search of Ror2 inhibitors for RCC or other cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B206. Citation Format: Zufan Debebe, Melissa Porter, Emily E. Hull-Ryde, Neal Rasmussen, Adam Sendor, Jacqueline Norris-Drouin, Keefe Chan, James E. Bear, William P. Janzen, Kimryn Rathmell. Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B206.