Christine Jean

FAK in cancer: mechanistic findings and clinical applications

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers. FAK promotes tumour progression and metastasis through effects on cancer cells, as well as stromal cells of the tumour microenvironment. The kinase-dependent and kinase-independent functions of FAK control cell movement, invasion, survival, gene expression and cancer stem cell self-renewal. Small molecule FAK inhibitors decrease tumour growth and metastasis in several preclinical models and have initial clinical activity in patients with limited adverse events. In this Review, we discuss FAK signalling effects on both tumour and stromal cell biology that provide rationale and support for future therapeutic opportunities.

VEGF-Induced Vascular Permeability Is Mediated by FAK

Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Endothelial cell focal adhesion kinase is a key intermediate between c-Src and the regulation of endothelial cell barrier function in the control of tumor metastasis.

Human Keratinocytes Acquire Cellular Cytotoxicity under UV-B Irradiation IMPLICATION OF GRANZYME B AND PERFORIN

Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.

UVA Induces Granzyme B in Human Keratinocytes through MIF IMPLICATION IN EXTRACELLULAR MATRIX REMODELING

In a previous study, we have described that UVB induces granzyme B (GrB) in human keratinocyte cells, and that confers potent cellular cytotoxicity against various cellular models, including immune cells (Hernandez-Pigeon, H., Jean, C., Charruyer, A., Haure, M. J., Titeux, M., Tonasso, L., Quillet-Mary, A., Baudouin, C., Charveron, M., and Laurent, G. (2006) J. Biol. Chem. 281, 13525–13532). Herein, we have found that, in contrast to UVB, UVA failed to enhance keratinocyte cellular cytotoxicity but was still able to trigger GrB production. We show that GrB is accumulated through a p38 MAPK-dependent transcriptional mechanism stimulated by redox-dependent migration inhibitory factor release. Moreover, GrB purified from UVA-treated cellular extracts was found to degrade fibronectin in vitro. Treatment with antisense oligonucleotide directed against GrB resulted in the inhibition of UVA-induced cell detachment and cell death and facilitated cell migration through fibronectin and vitronectin matrix upon UVA exposure. Altogether, these results suggest another function for GrB in the context of the UV response. Indeed, combined with our previous study, it appears that, whereas this enzyme mediates keratinocyte cellular cytotoxicity following UVB irradiation, GrB supports the capacity of keratinocyte to degrade extracellular matrix components following UVA irradiation. UV-mediated GrB production may thus have important consequences in photoaging and photocarcinogenesis.

FAK Inhibition Disrupts a β5 Integrin Signaling Axis Controlling Anchorage-Independent Ovarian Carcinoma Growth

Ovarian cancer ascites fluid contains matrix proteins that can impact tumor growth via integrin receptor binding. In human ovarian tumor tissue arrays, we find that activation of the cytoplasmic focal adhesion (FAK) tyrosine kinase parallels increased tumor stage, β5 integrin, and osteopontin matrix staining. Elevated osteopontin, β5 integrin, and FAK mRNA levels are associated with decreased serous ovarian cancer patient survival. FAK remains active within ovarian cancer cells grown as spheroids, and anchorage-independent growth analyses of seven ovarian carcinoma cell lines identified sensitive (HEY, OVCAR8) and resistant (SKOV3-IP, OVCAR10) cells to 0.1 μmol/L FAK inhibitor (VS-4718, formerly PND-1186) treatment. VS-4718 promoted HEY and OVCAR8 G0–G1 cell-cycle arrest followed by cell death, whereas growth of SKOV3-IP and OVCAR10 cells was resistant to 1.0 μmol/L VS-4718. In HEY cells, genetic or pharmacological FAK inhibition prevented tumor growth in mice with corresponding reductions in β5 integrin and osteopontin expression. β5 knockdown reduced HEY cell growth in soft agar, tumor growth in mice, and both FAK Y397 phosphorylation and osteopontin expression in spheroids. FAK inhibitor–resistant (SKOV3-IP, OVCAR10) cells exhibited anchorage-independent Akt S473 phosphorylation, and expression of membrane-targeted and active Akt in sensitive cells (HEY, OVCAR8) increased growth but did not create a FAK inhibitor–resistant phenotype. These results link osteopontin, β5 integrin, and FAK in promoting ovarian tumor progression. β5 integrin expression may serve as a biomarker for serous ovarian carcinoma cells that possess active FAK signaling. Mol Cancer Ther; 13(8); 2050–61. ©2014 AACR.

Epidermal Growth Factor Receptor/β-Catenin/T-Cell Factor 4/Matrix Metalloproteinase 1: A New Pathway for Regulating Keratinocyte Invasiveness after UVA Irradiation

Previous studies have established that UV irradiation results in epidermal growth factor receptor (EGFR) activation in keratinocytes. However, the signaling pathways and cellular effects related to this process remain incompletely elucidated. Herein, we describe for the first time that UVA-mediated EGFR activation results in beta-catenin tyrosine phosphorylation at the Y654 residue responsible for the dissociation of E-cadherin/alpha-catenin/beta-catenin complexes. Moreover, UVA induces an EGFR-dependent, but Wnt-independent, beta-catenin relocalization from the membrane to the nucleus followed by its association with T-cell factor 4 (TCF4). This newly formed beta-catenin/TCF4 complex binds to a specific site on matrix metalloproteinase 1 (MMP1) promoter and governs MMP1 gene and protein expression, as well as cell migration in collagen and gelatin. Altogether, these results suggest that UVA stimulates keratinocyte invasiveness through two coordinated EGFR-dependent processes: loss of cell-to-cell contact due to beta-catenin/E-cadherin/alpha-catenin dissociation and increased cell migration through extracellular matrix component degradation due to beta-catenin/TCF4-dependent MMP1 regulation. These events may represent an important step in epidermis repair following UVA injury and their abnormal regulation could contribute to photoaging and photocarcinogenesis.

Analyses of merlin/NF2 connection to FAK inhibitor responsiveness in serous ovarian cancer.

OBJECTIVE Focal adhesion kinase (FAK) is overexpressed in serous ovarian cancer. Loss of merlin, a product of the neurofibromatosis 2 tumor suppressor gene, is being evaluated as a biomarker for FAK inhibitor sensitivity in mesothelioma. Connections between merlin and FAK in ovarian cancer remain undefined. METHODS Nine human and two murine ovarian cancer cell lines were analyzed for growth in the presence of a small molecule FAK inhibitor (PF-271, also termed VS-6062) from 0.1 to 1 μM for 72 h. Merlin was evaluated by immunoblotting and immunostaining of a human ovarian tumor tissue array. Growth of cells was analyzed in an orthotopic tumor model and evaluated in vitro after stable shRNA-mediated merlin knockdown. RESULTS Greater than 50% inhibition of OVCAR8, HEY, and ID8-IP ovarian carcinoma cell growth occurred with 0.1 μM PF-271 in anchorage-independent (p<0.001) but not in adherent culture conditions. PF-271-mediated reduction in FAK Y397 phosphorylation occurred independently of growth inhibition. Suspended growth of OVCAR3, OVCAR10, IGROV1, IGROV1-IP, SKOV3, SKOV3-IP, A2780, and 5009-MOVCAR was not affected by 0.1 μM PF-271. Merlin expression did not correlate with serous ovarian tumor grade or stage. PF-271 (30 mg/kg, BID) did not inhibit 5009-MOVCAR tumor growth and merlin knockdown in SKOV3-IP and OVCAR10 cells did not alter suspended cell growth upon PF-271 addition. CONCLUSIONS Differential responsiveness to FAK inhibitor treatment was observed. Intrinsic low merlin protein level correlated with PF-271-mediated anchorage-independent growth inhibition, but reduction in merlin expression did not induce sensitivity to FAK inhibition. Merlin levels may be useful for patient stratification in FAK inhibitor trials.

FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth

IntroductionFocal adhesion kinase (FAK) controls cell growth and survival downstream of integrin-matrix receptors. Upon adhesion loss or FAK inhibition, FAK can translocate to the nucleus. The nucleolus is a non-membrane nuclear structure that regulates ribosome biogenesis and cell proliferation. Nucleostemin (NS), a nucleolar-localized protein, modulates cell cycle progression, stemness, and three-dimensional tumor spheroid formation. The signaling pathways that regulate NS levels in tumors remain undefined.MethodsHuman breast carcinoma cells were evaluated for growth in culture (adherent and anchorage-independent spheroid) and as orthotopic tumors. FAK signaling was evaluated by pharmacological FAK inhibitor addition (PF-271, IC50 ~ 0.1 μM) and by small hairpin RNA (shRNA) knockdown followed by re-expression of FAK wildtype (WT) or a kinase-dead (KD, K454R) FAK point mutant. Immunoblotting was used to evaluate FAK, NS, nucleolar phosphoprotein B23, and nucleolin levels. Total and phosphospecific antibody imunoblotting were used to detect changes in FAK, Akt kinase (Akt also known as protein kinase B), and 4E-binding protein 1 (4E-BP1) phosphorylation, a translation repressor protein and target of the mammalian target of rapamycin (mTOR) complex. Immunohistochemical, co-immunoprecipitation, and cellular fractionation analyses were used to evaluate FAK association with nucleoli.ResultsPharmacological (0.1 μM PF-271) or genetic inhibition of FAK activity prevents MDA-MB-231 and 4T1L breast carcinoma growth as spheroids and as orthotopic tumors. FAK inhibition triggers proteasome-mediated decreased NS levels but no changes in other nucleolar proteins such as B23 (nucleophosmin) or nucleolin. Active FAK was associated with purified nucleoli of anchorage-independent cells and present within nucleoli of human invasive ductal carcinoma tumor samples. FAK co-immunoprecipitated with B23 that binds NS and a complex between FAK, NS, Akt, and mTOR was detected. Constitutively-active Akt kinase promoted tumor spheroid growth, stabilized NS levels, and promoted pS65 4E-BP1 phosphorylation in the presence of inhibited FAK. Rapamycin lowered NS levels and inhibited pS65 4E-BP1 phosphorylation in cells with activated Akt-mTOR signaling.ConclusionsFAK signaling occurs in the nucleolus, active FAK protects NS, and Akt-mTOR pathway regulates NS protein stability needed for breast carcinoma spheroid and tumor growth.

Anti-tumor activity of obinutuzumab and rituximab in a follicular lymphoma 3D model

Follicular lymphomas (FLs) account for 35–40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody (MAb) rituximab (RTX). The development of the type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here, using FL cells we show that RTX and GA101 display a similar activity on RL cells cultured in 2D. However, 2D culture cannot mimic tumor spatial organization and conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a non-Hodgkins lymphoma (NHL) 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and used it to compare RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared with 2D culture. Moreover, we observed that in the 3D model GA101 was more effective than RTX both in inhibiting MALC growth through induction of (lysosomal) cell death and senescence and in inhibiting intracellular signaling pathways, such as mammalian target of rapamycin, Akt, PLCgamma (Phospholipase C gamma) and Syk. Altogether, our study demonstrates that spatial organization strongly influences the response to antibody treatment, supporting the use of 3D models for the testing of therapeutic agents in NHL.