Marta Canel

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.

Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.

The ability of tumor cells to invade and metastasize requires deregulation of interactions with adjacent cells and the extracellular matrix. A major challenge of cancer biology is to observe the dynamics of the proteins involved in this process in their functional and physiologic context. Here, for the first time, we have used photobleaching and photoactivation to compare the mobility of cell adhesion and plasma membrane probes in vitro and in tumors grown in mice (in vivo). We find differences between in vitro and in vivo recovery dynamics of two key molecules, the tumor suppressor E-cadherin and the membrane-targeting sequence of H-Ras. Our data show that E-cadherin dynamics are significantly faster in vivo compared with cultured cells, that the ratio of E-cadherin stabilized in cell-cell junctions is significantly higher in vivo, and that E-cadherin mobility correlates with cell migration. Moreover, quantitative imaging has allowed us to assess the effects of therapeutic intervention on E-cadherin dynamics using dasatinib, a clinically approved Src inhibitor, and show clear differences in the efficacy of drug treatment in vivo. Our results show for the first time the utility of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals. Furthermore, this work highlights critical differences in molecular dynamics in vitro and in vivo, which have important implications for the use of cultured disease models as surrogates for living tissue.

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.

Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

Src/FAK-mediated regulation of E-cadherin as a mechanism for controlling collective cell movement: insights from in vivo imaging.

Recent advances in confocal and multi-photon microscopy, together with fluorescent probe development, have enabled cancer biology studies to go beyond the culture dish and interrogate cancer-associated processes in the complex in vivo environment. Regulation of the tumor suppressor protein E-cadherin plays an important role in cancer development and progression and may contribute to the decision between ‘single cell’ and ‘collective invasion’ in vivo. Mounting evidence from in vitro and in vivo experiments places the two non-receptor protein tyrosine kinases Src and Focal Adhesion Kinase, at the heart of E-cadherin regulation, and the crosstalk between integrins and cadherins. Here we discuss recent insights, attained using high resolution fluorescent in vivo imaging, into the regulation of E-cadherin and collective invasion. We focus on the regulatory crosstalk between the Src/FAK signaling axis and E-cadherin in vivo.

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.

p140Cap dual regulation of E-cadherin/EGFR cross-talk and Ras signalling in tumour cell scatter and proliferation

The adaptor protein p140Cap/SNIP is a novel Src-binding protein that regulates Src activation through C-terminal Src kinase (Csk). Here, by gain and loss of function approaches in breast and colon cancer cells, we report that p140Cap immobilizes E-cadherin at the cell membrane and inhibits EGFR and Erk1/2 signalling, blocking scatter and proliferation of cancer cells. p140Cap-dependent regulation of E-cadherin/EGFR cross-talk and cell motility is due to the inhibition of Src kinase. However, rescue of Src activity is not sufficient to restore Erk1/2 phosphorylation and proliferation. Indeed, p140Cap also impairs Erk1/2 phosphorylation by affecting Ras activity, downstream to the EGFR. In conclusion, p140Cap stabilizes adherens junctions and inhibits EGFR and Ras signalling through the dual control of both Src and Ras activities, thus affecting crucial cancer properties such as invasion and growth. Interestingly, p140Cap expression is lost in more aggressive human breast cancers, showing an inverse correlation with EGFR expression. Therefore, p140Cap mechanistically behaves as a tumour suppressor that inhibits signalling pathways leading to aggressive phenotypes.

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

The Src family tyrosine kinases are key modulators of cancer cell invasion and metastasis and a number of Src kinase inhibitors are currently in clinical development for the treatment of solid tumours. However, there is growing evidence that Src is also upregulated at very early stages of epithelial cancer development. We have investigated the role of Src in mouse skin, which is one of the most tractable models of epithelial homoeostasis and tumorigenesis. We found that Src protein expression and activity was regulated during the normal hair cycle and was increased specifically during the proliferative anagen phase and also in response to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). AZD0530, a selective Src inhibitor, prevented the TPA-induced proliferation of basal keratinocytes both in vivo and in vitro. Moreover, treatment with AZD0530 reduced papilloma formation following the well-established 7,12-dimethylbenz(a)anthracene/TPA skin carcinogenesis protocol but did not inhibit the subsequent proliferation of the papillomas. Furthermore, AZD0530 did not alter the malignant conversion of papillomas to squamous cell carcinoma suggesting a role for Src in early tumour development in the skin carcinogenesis model, rather than at later stages of tumour progression. Src expression and activity were also seen in human actinic keratoses that are hyperproliferative pre-malignant skin lesions, indicating that Src may also play a role in the early stages of human skin tumour development. Thus, Src inhibitors such as AZD0530 may therefore have chemopreventative properties in patients with hyperproliferative epidermal disorders.

Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

The dynamic control of E-cadherin is critical for establishing and maintaining cell-cell junctions in epithelial cells. The concentration of E-cadherin molecules at adherens junctions (AJs) is regulated by lateral movement of E-cadherin within the plasma membrane and endocytosis. Here we set out to study the interplay between these processes and their contribution to E-cadherin dynamics. Using photoactivation (PA) and fluorescence recovery after photobleaching (FRAP) we were able to monitor the fate of E-cadherin molecules within the plasma membrane. Our results suggest that the motility of E-cadherin within, and away from, the cell surface are not exclusive or independent mechanisms and there is a fine balance between the two which when perturbed can have dramatic effects on the regulation of AJs.

Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo

Despite our advanced understanding of primary cancer development and progression, metastasis and the systemic spread of the disease to secondary sites remains the leading cause of cancer-associated death. The metastatic process is therefore a major potential therapeutic target area for cancer researchers and elucidating the key steps that are susceptible to therapeutic intervention will be critical to improve our treatment strategies. Recent advances in intravital imaging are rapidly improving our insight into this process and are helping in the design of stage-specific drug regimes for the treatment of metastatic cancer. Here we discuss current developments in intravital imaging and our recent use of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals to assess the efficacy of therapeutic intervention on early stages of tumour cell metastasis.