A novel Src kinase inhibitor reduces tumour formation in a skin carcinogenesis model

The Src family tyrosine kinases are key modulators of cancercell invasion and metastasis and a number of Src kinase inhibitorsare currently in clinical development for the treatment of solidtumours. However, there is growing evidence that Src is alsoupregulated at very early stages of epithelial cancer development.We have investigated the role of Src in mouse skin, which isone of the most tractable models of epithelial homoeostasisand tumorigenesis. We found that Src protein expression andactivity was regulated during the normal hair cycle and wasincreased specifically during the proliferative anagen phaseand also in response to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate(TPA). AZD0530, a selective Src inhibitor, prevented the TPA-inducedproliferation of basal keratinocytes both in vivo and in vitro.Moreover, treatment with AZD0530 reduced papilloma formationfollowing the well-established 7,12-dimethylbenz(a)anthracene/TPAskin carcinogenesis protocol but did not inhibit the subsequentproliferation of the papillomas. Furthermore, AZD0530 did notalter the malignant conversion of papillomas to squamous cellcarcinoma suggesting a role for Src in early tumour developmentin the skin carcinogenesis model, rather than at later stagesof tumour progression. Src expression and activity were alsoseen in human actinic keratoses that are hyperproliferativepre-malignant skin lesions, indicating that Src may also playa role in the early stages of human skin tumour development.Thus, Src inhibitors such as AZD0530 may therefore have chemopreventativeproperties in patients with hyperproliferative epidermal disorders. Abbreviations: DMBA, 7,12-dimethylbenz(a)anthracene; PBS, phosphate-buffered saline; SFK, Src family kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate Received July 2, 2008; revised December 3, 2008; accepted December 3, 2008.     

Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.     

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

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Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior

Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr(407), Tyr(576), Tyr(577), and Tyr(861), which are considered to be Src kinase substrates. This Src kinase-independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes-deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr(925) of FAK was not evident in SrcMF- or Src251-expressing cells, and lack of Src kinase-dependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors.     

p120-catenin is required for the collective invasion of squamous cell carcinoma cells via a phosphorylation-independent mechanism

Loss of E-cadherin-mediated cell-cell junctions has been correlated with cancer cell invasion and poor patient survival. p120-catenin has emerged as a key player in promoting E-cadherin stability and adherens junction integrity and has been proposed as a potential invasion suppressor by preventing release of cells from the constraints imposed by cadherin-mediated cell-cell adhesion. However, it has been proposed that tyrosine phosphorylation of p120 may contribute to cadherin-dependent junction disassembly during invasion. Here, we use small interfering RNA (siRNA) in A431 cells to show that knockdown of p120 promotes two-dimensional migration of cells. In contrast, p120 knockdown impairs epidermal growth factor-induced A431 invasion into three-dimensional matrix gels or in organotypic culture, whereas re-expression of siRNA-resistant p120, or a p120 isoform that cannot be phosphorylated on tyrosine, restores the collective mode of invasion employed by A431 cells in vitro. Thus, p120 promotes A431 cell invasion in a phosphorylation-independent manner. We show that the collective invasion of A431 cells depends on the presence of cadherin-mediated (P- and E-cadherin) cell-cell contacts, which are lost in cells where p120 expression is knocked down. Furthermore, membranous p120 is maintained in invasive squamous cell carcinomas in tumours suggesting that p120 may be important for the collective invasion of tumours cells in vivo.     

Quantitative in vivo imaging of the effects of inhibiting integrin signaling via Src and FAK on cancer cell movement: effects on E-cadherin dynamics

Most cancer-related deaths are due to the development of metastatic disease, and several new molecularly targeted agents in clinical development have the potential to prevent disease progression. However, it remains difficult to assess the efficacy of antimetastatic agents in the clinical setting, and an increased understanding of how such agents work at different stages of the metastatic cascade is important in guiding their clinical use. We used optical window chambers combined with photobleaching, photoactivation, and photoswitching to quantitatively measure (a) tumor cell movement and proliferation by tracking small groups of cells in the context of the whole tumor, and (b) E-cadherin molecular dynamics in vivo following perturbation of integrin signaling by inhibiting focal adhesion kinase (FAK) and Src. We show that inhibition of Src and FAK suppresses E-cadherin-dependent collective cell movement in a complex three-dimensional tumor environment, and modulates cell-cell adhesion strength and endocytosis in vitro. This shows a novel role for integrin signaling in the regulation of E-cadherin internalization, which is linked to regulation of collective cancer cell movement. This work highlights the power of fluorescent, direct, in vivo imaging approaches in the preclinical evaluation of chemotherapeutic agents, and shows that inhibition of the Src/FAK signaling axis may provide a strategy to prevent tumor cell spread by deregulating E-cadherin-mediated cell-cell adhesions.     

Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

Focal adhesion kinase (FAK ) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397 , Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase‐dead (KD ) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb‐iCre^ert ‐driven endothelial cell (EC )‐specific, tamoxifen‐inducible homozygous mutant mouse lines: FAK wild‐type (WT ), FAK KD , and FAK double mutant (DM ), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre +;FAK^{WT /WT} , ECCre +;FAK^{KD /KD} and ECCre +;FAK^{DM /DM} mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre +;FAK^{WT /WT} and ECCre ?;FAK^{WT /WT} control mice. In contrast, tumour growth and vessel density were decreased in ECCre +;FAK^{KD /KD} and ECCre +;FAK^{DM /DM} mice, as compared with Cre ? littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre +;FAK^{KD /KD} and ECCre +;FAK^{DM /DM} mice. Furthermore, although ECCre +;FAK^{KD /KD} mice showed reduced blood vessel leakage, ECCre +;FAK^{DM /DM} and ECCre ?;FAK^{DM /DM} mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated Y397 autophosphorylation was reduced in Cre+;FAK^{KD /KD} ECs as compared with Cre+;FAK^{WT /WT} cells, with no change in phosphorylation of the known Src targets FAK‐Y577 , FAK‐Y861 , FAK‐Y925 , paxillin‐Y118 , p130Cas‐Y410. Cre+;FAK^{DM /DM} ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE ‐cadherin‐pY658 levels in Cre+;FAK^{KD /KD} ECs were rescued in Cre+FAK^{DM /DM} ECs , corresponding with the rescue in vessel leakage in the ECCre +;FAK^{DM /DM} mice. We show that EC ‐specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAK^DM/DM mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAK^KD/KD mice in vivo . This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The role of focal adhesion kinase catalytic activity on the proliferation and migration of squamous cell carcinoma cells

Focal adhesion kinase (FAK) is upregulated in several epithelial tumours and there has been considerable interest in developing small molecule kinase inhibitors of FAK. However, FAK also has important adaptor functions within the cell, integrating signals from both integrins and growth factors. To investigate the role of FAKs kinase domain, we generated fak-deficient squamous cell carcinoma (SCC) cell lines. Re-expression of a wild type or kinase dead FAK allowed us to delineate its kinase dependent functions. In addition, we used the novel FAK kinase inhibitor PF-562,271. The kinase activity of FAK was important for tumour cell migration and polarity but more striking was its requirement for the anchorage independent 3 dimensional (3D) proliferation of SCC cells and their growth as xenografts in mice. Inhibition of FAK activity and prevention of growth in 3D correlated with Src inhibition. We further identified a mechanism whereby FAK regulates proliferation in 3D via regulation of the kinase activity of Src. This was dependent on the kinase activity of FAK and its resulting phosphorylation on Y397 that provides a high affinity binding site for Src. These data support the further development of FAK kinase inhibitors as agents that have the potential to inhibit both tumour cell migration and proliferation.     

Involvement of focal adhesion kinase in cellular invasion of head and neck squamous cell carcinomas via regulation of MMP-2 expression

Focal adhesion kinase (FAK) is considered intimately involved in cancer progression. Our previous research has demonstrated that overexpression of FAK is an early and frequent event in squamous cell carcinomas of the supraglottic larynx, and it is associated with the presence of metastases in cervical lymph nodes. The purpose of this study was to examine the functional role of FAK in the progression of head and neck squamous cell carcinomas (HNSCC). To this end, expression of FAK-related nonkinase (FRNK) or small interfering RNA (siRNA) against FAK was used to disrupt the FAK-induced signal transduction pathways in the HNSCC-derived SCC40 and SCC38 cell lines. Similar phenotypic effects were observed with the two methodological approaches in both cell lines. Decreased cell attachment, motility and invasion were induced by FRNK and FAK siRNA, whereas cell proliferation was not impaired. In addition, increased cell invasion was observed upon FAK overexpression in SCC cells. FRNK expression resulted in a downregulation of MMP-2 and MMP-9 expression. Interestingly, MMP-2 overexpression in FRNK-expressing cells rescued FRNK inhibition of cell invasion. This is the first demonstration of a direct rescue of impaired cell invasion by the re-expression of MMP-2 in a tumour cell type with decreased expression of functional FAK. Collectively, these data reported here support the conclusion that FAK enhances invasion of HNSCC by promoting both increased cell motility and MMP-2 production, thus providing new insights into possible therapeutic intervention strategies.