Jill K. Slack-Davis

Cellular Characterization of a Novel Focal Adhesion Kinase Inhibitor

Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC50 of 4 nm. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr397 with an IC50 of 30–100 nm. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr397 phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

Activation of the Ras–MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane–proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf–MEK1–MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells

The process of cell migration is initiated by protrusion at the leading edge of the cell, the formation of peripheral adhesions, the exertion of force on these adhesions, and finally the release of the adhesions at the rear of the cell. Focal adhesion kinase (FAK) is intimately involved in the regulation of this process, although the precise mechanism(s) whereby FAK regulates cell migration is unclear. We have used two approaches to reduce FAK expression in fibroblasts. Treatment of cells with FAK-specific siRNAs substantially reduced FAK expression and inhibited the spreading of fibroblasts in serum-free conditions, but did not affect the rate of spreading in the presence of serum. In contrast with the wild-type cells, the FAK siRNA-treated cells exhibited multiple extensions during cell spreading. The extensions appeared to be inappropriately formed lamellipodia as evidenced by the localization of cortactin to lamellipodial structures and the inhibition of such structures by expression of dominant-negative Rac. The wild-type phenotype was restored by reexpressing wild-type FAK in the knockdown cells, but not by expression of FAK containing a point mutation at the autophosphorylation site (FAK Y397F). In wound-healing assays, FAK knockdown cells failed to form broad lamellipodia, instead forming multiple leading edges. Similar results were obtained using primary mouse embryo fibroblasts from FAK-flox mice in which Cre-mediated excision was used to ablate the expression of FAK. These data are consistent with a role for FAK in regulating the formation of a leading edge during cell migration by coordinating integrin signaling to direct the correct spatial activation of membrane protrusion.

Mitogen-Activated Protein Kinase Feedback Phosphorylation Regulates MEK1 Complex Formation and Activation during Cellular Adhesion

ABSTRACT Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.

Vascular Cell Adhesion Molecule-1 Is a Regulator of Ovarian Cancer Peritoneal Metastasis

Ovarian cancers metastasize by attaching to and invading through the mesothelium, a single layer of mesothelial cells lining the peritoneal cavity. The presence of invasive peritoneal metastases is associated with a poor prognosis for ovarian cancer (5-year survival <25%). Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface receptor that mediates leukocyte attachment and extravasation across endothelial and mesothelial monolayers at sites of inflammation. Membranous VCAM-1 expression was observed on the mesothelium of 13 of 14 women with ovarian cancer compared with 6 of 15 who were cancer-free. Using a cell culture model system of mesothelial invasion, highly tumorigenic SKOV-3 and ES-2 cells were 2.5 to 3 times more efficient in transmigration through the mesothelial monolayer compared with poorly tumorigenic OVCAR-3 cells. Blocking antibodies to, or small interfering RNA knockdown of, VCAM-1 or its ligand alpha(4)beta(1) integrin significantly decreased, but did not completely inhibit, transmigration of SKOV-3 cells through mesothelial monolayers. Furthermore, using a mouse model of ovarian cancer metastasis, treatment with VCAM-1 function-blocking antibodies decreased tumor burden and increased survival. Together, these observations implicate VCAM-1-alpha(4)beta(1) integrin interactions in the regulation of ovarian cancer cell mesothelial invasion and metastatic progression and offer the possibility of novel therapeutic targets.

Focal Adhesion Kinase: Targeting Adhesion Signaling Pathways for Therapeutic Intervention

The tumor microenvironment plays a central role in cancer progression and metastasis. Within this environment, cancer cells respond to a host of signals including growth factors and chemotactic factors, as well as signals from adjacent cells, cells in the surrounding stroma, and signals from the extracellular matrix. Targeting the pathways that mediate many of these signals has been a major goal in the effort to develop therapeutics.

Differential requirement for focal adhesion kinase signaling in cancer progression in the transgenic adenocarcinoma of mouse prostate model

Increasing evidence indicates that adhesion signaling plays an important role in the tumor microenvironment, contributing to cancer progression, invasion, and metastasis. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that regulates adhesion-dependent cell signaling and has been implicated in mediating steps in cancer progression and metastasis in many human cancers, including prostate. We have investigated the role of FAK in the appearance of adenocarcinoma (atypical epithelial hyperplasia of T antigen) and neuroendocrine carcinomas in the transgenic adenocarcinoma of mouse prostate (TRAMP) model using either Cre-mediated recombination to genetically ablate FAK expression or pharmacologic inhibition of FAK activity with the small-molecule inhibitor, PF-562,271. We provide evidence that loss of FAK or its inhibition with PF-562,271 does not alter the progression to adenocarcinoma. However, continued FAK expression (and activity) is essential for the androgen-independent formation of neuroendocrine carcinoma. These data indicate that integrin signaling through FAK is an important component of cancer progression in the TRAMP model and suggest that treatment modalities targeting FAK may be an appropriate strategy for patients with castrate-resistant cancer. [Mol Cancer Ther 2009;8(8):2470–77]

Lobular Breast Cancer and Abdominal Metastases: A Retrospective Review and Impact on Survival

Background: The predominant breast cancer subtypes, invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC), have similar recurrence and survival rates but differing patterns of metastatic recurrence. Methods: A retrospective review of breast cancers treated at an academic medical center from 1999 to 2012 was performed. Demographic, pathologic, treatment, and follow-up data were collected for 179 ILC and 358 IDC patients (1:2 stage-matched). The median follow-up was 4.7 years. Results: The baseline characteristics were similar in the two groups. ILC was more likely to be hormone-receptor-positive/HER2-negative and mammographically occult. The number of surgical resections, breast conservation rate, systemic treatment, and taxane use was similar between the groups. The overall recurrence rate was the same. ILC recurred more often in the abdominal cavity (24.3% in ILC vs. 4.1% in IDC, p = 0.001). The disease-free survival and overall survival were equal. On multivariate analysis, age, stage of disease, hormone receptor status, and systemic therapy were associated with survival, but histology was not. Conclusions: Compared to ductal breast cancers, lobular breast cancers recur more often in the abdominal cavity. Both ILC and IDC have comparable surgical and medical treatment outcomes and survival. Our data suggest that enhanced surveillance and imaging might be useful in ILC.

Abstract 5407: T-type Ca2+ channel inhibitors sensitize ovarian cancer to carboplatin through downregulation of survivin gene expression

Recent studies by our group and others have shown that T-type Ca2+ channels play a reinforcing role in cancer cell proliferation, cell cycle progression and apoptosis evasion. We have demonstrated drug cooperativity between T-type Ca2+ channel inhibitor, mibefradil and carboplatin in vivo in ovarian cancer xenografts. To investigate the molecular mechanism by which T-type Ca2+ channels affect tumor growth, we used a panel of ovarian cancer cell lines together with mibefradil or small interfering RNA (siRNA) to silence the T-channels gene expression. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer with simultaneous increase in apoptosis and decrease in expression of the anti-apoptotic gene survivin (BIRC5). Combining carboplatin with mibefradil resulted in increased apoptosis. Analysis of intracellular signaling revealed T-type Ca2+ channel inhibitors induced changes in the levels of FOXO proteins, which regulate BIRC5 gene expression. Mibefradil treatment caused greater nuclear retention of FoxO1 and FoxO3a, two transcription factors that repress BIRC5 expression, while protein expression of the transcription factor that activates BIRC5 expression, FoxM1, decreased. Chromatin immunoprecipitation of FOXO proteins from mibefradil treated ovarian cancer cells demonstrated binding of FoxM1 and FoxO1 within the BIRC5 (survivin) promoter. Together, the data show that T-type Ca2+ channels support ovarian cancer cell cycle, proliferation and regulate pro-survival pathways through the FOXO-survivin signaling axis, which has been reported to be deregulated in ovarian cancer. Moreover, our results provide the rationale for future use of T-type antagonists as sensitizing agents in combination with standard chemotherapeutics. Citation Format: Barbara Dziegielewska, Eli V. Casarez, Wesley Z. Yang, Jaroslaw Dziegielewski, Jill K. Slack-Davis. T-type Ca2+ channel inhibitors sensitize ovarian cancer to carboplatin through downregulation of survivin gene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5407. doi:10.1158/1538-7445.AM2015-5407

Integrin Signaling: Cell Migration, Proliferation, and Survival

Publisher Summary Integrins are a family of heterodimeric, transmembrane receptors that mediate attachment of cells to the surrounding extracellular matrix (ECM). Integrins are responsible for sensing many aspects of the microenvironment, including the structure and composition of the ECM as well as biochemical signals generated following growth factor or cytokine stimulation. Integration of these complex signals contributes to the regulation of cellular migration, growth, and survival within an organism. A central function of integrins is to mediate a structural linkage between the dynamic intracellular cytoskeleton and the ECM that conveys both mechanical and chemical signals. Cell migration provides an exceptionally relevant model to study integrin signaling. Migration is a complex cellular process that involves the extension of lamellipodia; adhesion at sites within newly formed lamella, organization of force-generating adhesions, contraction and cell-body displacement, and detachment of the cell rear. The initial steps in cell migration require the formation of protrusive structures (lamellipodia) at the leading edge of the cell, and the stabilization of the protrusion by newly formed adhesion complexes. Cell proliferation is in dynamic balance with cell death. In cancer, increasing evidence indicates that integrins synergize with growth factor receptor signals to promote cell proliferation and to stimulate the migration of tumor cells from the primary site, and function to promote growth and survival at distant metastatic sites. Integrins play a major role in remodeling the tumor microenvironment, and are important regulators of migration and metastatic growth.