FAK goes nuclear to control anti-tumor immunity – a new target in cancer immuno-therapy

Evading the antitumor immune response is important for the survival and progression of cancer. Recently, we identified an unexpected role for nuclear Focal Adhesion Kinase (FAK) activity in the control of tumor Treg levels and immune evasion by regulating chemokine and cytokine transcription in cancer cells. We proposed a potentially new purpose for FAK kinase inhibitors, which can cause immune-mediated tumor regression

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 nonreceptor 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

Nuclear FAK and Runx1 cooperate to regulate IGFBP3, cell cycle progression and tumor growth

Abstract Nuclear focal adhesion kinase (FAK) is a potentially important regulator of gene expression in cancer, impacting both cellular function and the composition of the surrounding tumor microenvironment. Here, we report in a murine model of skin squamous cell carcinoma (SCC) that nuclear FAK regulates Runx1-dependent transcription of insulin-like growth factor binding protein 3 (IGFBP3), and that this regulates SCC cell-cycle progression and tumor growth in vivo. Furthermore, we identified a novel molecular complex between FAK and Runx1 in the nucleus of SCC cells and showed that FAK interacted with a number of Runx1-regulatory proteins, including Sin3a and other epigenetic modifiers known to alter Runx1 transcriptional function through posttranslational modification. These findings provide important new insights into the role of FAK as a scaffolding protein in molecular complexes that regulate gene transcription. Cancer Res; 77(19); 5301–12. ©2017 AACR.</jats:p

Imaging Drug Uptake by Bioorthogonal Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy in tandem with bioorthogonal Raman labelling strategies is set to revolutionise the direct visualisation of intracellular drug uptake. Rational evaluation of a series of Raman-active labels has allowed the identification of highly active labels which have minimal perturbation on the biological efficacy of the parent drug. Drug uptake has been correlated with markers of cellular composition and cell cycle status, and mapped across intracellular structures using dual-colour and multi-modal imaging. The minimal phototoxicity and low photobleaching associated with SRS microscopy has enabled real-time imaging in live cells. These studies demonstrate the potential for SRS microscopy in the drug development process

FAK promotes stromal PD-L2 expression associated with poor survival in pancreatic cancer

BACKGROUND: Pancreatic Cancer is one of the most lethal cancers, with less than 8% of patients surviving 5 years following diagnosis. The last 40 years have seen only small incremental improvements in treatment options, highlighting the continued need to better define the cellular and molecular pathways contributing to therapy response and patient prognosis. METHODS: We combined CRISPR, shRNA and flow cytometry with mechanistic experiments using a Kras(G12D)p53(R172H) mouse model of pancreatic cancer and analysis of publicly available human PDAC transcriptomic datasets. RESULTS: Here, we identify that expression of the immune checkpoint, Programmed Death Ligand 2 (PD-L2), is associated with poor prognosis, tumour grade, clinical stage and molecular subtype in patients with Pancreatic Ductal Adenocarcinoma (PDAC). We further show that PD-L2 is predominantly expressed in the stroma and, using an orthotopic murine model of PDAC, identify cancer cell-intrinsic Focal Adhesion Kinase (FAK) signalling as a regulator of PD-L2 stromal expression. Mechanistically, we find that FAK regulates interleukin-6, which can act in concert with interleukin-4 secreted by CD4 T-cells to drive elevated expression of PD-L2 on tumour-associated macrophages, dendritic cells and endothelial cells. CONCLUSIONS: These findings identify further complex heterocellular signalling networks contributing to FAK-mediated immune suppression in pancreatic cancer

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 anti-metastatic 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 have used optical window chambers combined with the use of 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 3D tumor environment, and modulate cell-cell adhesion strength and endocytosis in vitro. This demonstrates 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 pre-clinical evaluation of chemotherapeutic agents, and shows that inhibition of the Src/FAK signaling axis may provide a strategy to prevent tumor cell spread by de-regulating E-cadherin-mediated cell-cell adhesions

In vivo imaging of the tumor and its associated microenvironment using combined CARS / 2-photon microscopy

The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment in vivo. However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche

High-Precision Photothermal Ablation using Biocompatible Palladium Nanoparticles and Laser Scanning Microscopy

Herein, we report a straightforward method for the scalable preparation of Pd nanoparticles (Pd-NPs) with reduced inherent cytotoxicity and high photothermal conversion capacity. These Pd-NPs are rapidly taken up by cells and able to kill labeled cancer cells upon short exposure to near-infrared (NIR) light. Following cell treatment with Pd-NPs, ablated areas were patterned with high precision by laser scanning microscopy, allowing one to perform cell migration assays with unprecedented accuracy. Using coherent Raman microscopy, cells containing Pd-NPs were simultaneously ablated and imaged. This novel methodology was combined with intravital imaging to mediate microablation of cancerous tissue in tumor xenografts in mice

FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

Objective: Immunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack of response. Here, we aimed to further investigate the immunoregulatory function of focal adhesion kinase (FAK) in PDAC, with specific emphasis on regulation of the type-II interferon response that is critical in promoting T-cell tumour recognition and effective immunosurveillance. Design: We combined CRISPR, proteogenomics and transcriptomics with mechanistic experiments using a KrasG12Dp53R172H mouse model of pancreatic cancer and validated findings using proteomic analysis of human patient-derived PDAC cell lines and analysis of publicly available human PDAC transcriptomics datasets. Results: Loss of PDAC cell-intrinsic FAK signalling promotes expression of the immunoproteasome and Major Histocompatibility Complex class-I (MHC-I), resulting in increased antigen diversity and antigen presentation by FAK-/- PDAC cells. Regulation of the immunoproteasome by FAK is a critical determinant of this response, optimising the physicochemical properties of the peptide repertoire for high affinity binding to MHC-I. Expression of these pathways can be further amplified in a STAT1-dependent manner via co-depletion of FAK and STAT3, resulting in extensive infiltration of tumour-reactive CD8 T-cells and further restraint of tumour growth. FAK-dependent regulation of antigen processing and presentation is conserved between mouse and human PDAC, but is lost in cells/tumours with an extreme squamous phenotype. Conclusion: Therapies aimed at FAK degradation may unlock additional therapeutic benefit for the treatment of PDAC through increasing antigen diversity and promoting antigen presentation