Periodic Reporting for period 1 - INTEGRIN REGULATION (Functional analysis of the kinome and phosphatome as determinants of integrin phosphorylation in cancer)
Reporting period: 2019-08-01 to 2021-07-31
Cancer dissemination from a primary tumour is a complex process that is mediated by cell-cell and cell-ECM (extracellular matrix) interactions. Integrin adhesion receptors are key signalling molecules that mediate the extracellular binding of ECM by cells, and the respective intracellular signalling resultant from that interaction. As such, they have been implicated in every step of cancer progression, as well as in diseases such as fibrosis and autoimmunity. While the importance of these receptors has been known for some time, targeting their extracellular domains has been largely unsuccessful in the clinic. Hence, new avenues for the therapeutic modulation of integrin activity are required in order to target their role in several diseases, especially in cancer therapies that are ham-strung by resistance to chemotherapeutics, anoikis and metastatic dissemination.
Given that the cytoplasmic domains of integrin adhesion receptors are essential hubs for protein-protein interactions, and that these domains are responsible for the cellular response to the extracellular environment, there remains a surprising lack in known regulators for integrin phosphorylation. Phosphorylation on specific residues is a reversible and highly dynamic process that can modulate the activity and protein-protein complex formation of many proteins within the cell. This process is bidirectionally regulated by kinases and phosphatases, which are themselves often druggable targets for targeted cancer therapies. As a proof-of-concept adhesion receptor, the fellow chose integrin beta1, as it is central to adhesion to the largest number of ECM ligands and there is experimental evidence for phosphorylation of this receptor on two tyrosine residues at the NPxY(783/795) sites within the cytoplasmic domain. Thus INTEGRIN REGULATION had three specific research objectives (SOs) that were focused on understanding the regulation and role of phosphorylation in the intracellular domains of integrin beta1.
SO1: Identification of novel regulatory kinases and/or phosphatases by unbiased screening approaches.
SO2: Confirmation of interactions using super-resolution imaging.
SO3: Functional assessment of identified kinases and/or phosphatases.
Given that the cytoplasmic domains of integrin adhesion receptors are essential hubs for protein-protein interactions, and that these domains are responsible for the cellular response to the extracellular environment, there remains a surprising lack in known regulators for integrin phosphorylation. Phosphorylation on specific residues is a reversible and highly dynamic process that can modulate the activity and protein-protein complex formation of many proteins within the cell. This process is bidirectionally regulated by kinases and phosphatases, which are themselves often druggable targets for targeted cancer therapies. As a proof-of-concept adhesion receptor, the fellow chose integrin beta1, as it is central to adhesion to the largest number of ECM ligands and there is experimental evidence for phosphorylation of this receptor on two tyrosine residues at the NPxY(783/795) sites within the cytoplasmic domain. Thus INTEGRIN REGULATION had three specific research objectives (SOs) that were focused on understanding the regulation and role of phosphorylation in the intracellular domains of integrin beta1.
SO1: Identification of novel regulatory kinases and/or phosphatases by unbiased screening approaches.
SO2: Confirmation of interactions using super-resolution imaging.
SO3: Functional assessment of identified kinases and/or phosphatases.
The realisation of the image-based screening approach from SO1 involved the development and application of a Förster resonance energy transfer (FRET) biosensor for integrin beta1 phosphorylation. We applied this biosensor in an RNA interference (RNAi) screen to identify regulatory phosphatases from the 107 known protein tyrosine phosphatases (PTPs) in the human genome. From this screen, we identified several potential regulatory PTPs, validating Shp2 and PTP-PEST as direct PTPs for integrin beta1. Following a literature search, we were also able to identify and validate two kinases (Src and Arg) for integrin beta1, enabling us to build a clearer regulatory picture for the reversible phosphorylation of this essential adhesion receptor.
In SO2 we further validated the regulatory landscape of integrin beta1 phosphorylation by performing interaction screens using mass spectrometry (Bimolecular complementation affinity purification). This allowed us to identify the proteins that are recruited to the phosphorylated integrin complex, and we are currently investigating the role of this complex during cancer invasion; particularly for the formation of invadopodia, which are used by cancer cells as part of the invasive process to escape the primary tumour.
These mechanistic insights into integrin phosphorylation were then fed into SO3, which involves investigating the functional role of integrin phosphorylation during cancer progression. To this end, the fellow generated breast cancer cell lines with fluorescently-tagged integrin beta1, where the phosphorylatable tyrosine residues in the NPxY(783/795) sites were mutated to non-phosphorylatable phenylalanines (YYFF). Using these cell lines, we have shown that the cells expressing the YYFF non-phosphorylatable mutant integrin beta1 are defective in their ability to invade into three-dimensional (3D) organotypic matrices or to form invadopodia on fluorescent gelatin, suggesting that integrin phosphorylation dynamics are critical for efficient cancer cell invasion.
In SO2 we further validated the regulatory landscape of integrin beta1 phosphorylation by performing interaction screens using mass spectrometry (Bimolecular complementation affinity purification). This allowed us to identify the proteins that are recruited to the phosphorylated integrin complex, and we are currently investigating the role of this complex during cancer invasion; particularly for the formation of invadopodia, which are used by cancer cells as part of the invasive process to escape the primary tumour.
These mechanistic insights into integrin phosphorylation were then fed into SO3, which involves investigating the functional role of integrin phosphorylation during cancer progression. To this end, the fellow generated breast cancer cell lines with fluorescently-tagged integrin beta1, where the phosphorylatable tyrosine residues in the NPxY(783/795) sites were mutated to non-phosphorylatable phenylalanines (YYFF). Using these cell lines, we have shown that the cells expressing the YYFF non-phosphorylatable mutant integrin beta1 are defective in their ability to invade into three-dimensional (3D) organotypic matrices or to form invadopodia on fluorescent gelatin, suggesting that integrin phosphorylation dynamics are critical for efficient cancer cell invasion.
The clear anti-invasive effect linked to the loss of the phosphorylatable residues in the integrin beta1 cytoplasmic domain has led the fellow to test inhibitors against the PTP Shp2 and Src kinase, both of which show an anti-invasive effect against the invasive and metastatic breast cancer cells used in INTEGRIN REGULATION. This suggests that dynamic changes in integrin phosphorylation are important for the regulation of invasive progression, where adhesion complexes need to be rapidly assembled and disassembled as the cell moves through a complex extracellular environment. The importance of phosphorylation dynamics is often missed when using standard single-endpoint biochemical approaches; however, using the FRET biosensor for integrin phosphorylation is helping us to understand where and when the phosphorylation is important for cancer invasion. Key to this investigation is that kinase or phosphatase inhibitors that are able to disrupt the dynamics of integrin phosphorylation may have the added benefit of acting as anti-invasives and further supports the use of targeted therapeutics as part of combination therapies. This is especially important in the light of cancer treatments that fail due to the inability to effectively target metastasis. Hence, the use of anti-invasive compounds to contain the primary tumour prior to metastatic dissemination is increasingly gaining traction in ongoing clinical trials.
In order to maximise the impact of INTEGRIN REGULATION, the fellow has secured a further Academy of Finland postdoctoral fellowship to ensure that the work is published in a high impact journal that will reach the widest audience possible. This will ensure that the novel approaches and concepts applied in INTEGRIN REGULATION are fully disseminated and that the field can benefit from the findings. Currently, the fellow is evaluating the clinical relevance of the hits from the RNAi FRET screen, which will then be assessed by immunohistochemistry staining of large breast cancer cohorts, along with functional validation using combination therapies aimed at assessing the potential of inhibiting integrin beta1 phosphorylation as an anti-metastatic in breast cancer models. These aspects will provide an essential link from the mechanistic insights to improving patient outcomes from INTEGRIN REGULATION.
In order to maximise the impact of INTEGRIN REGULATION, the fellow has secured a further Academy of Finland postdoctoral fellowship to ensure that the work is published in a high impact journal that will reach the widest audience possible. This will ensure that the novel approaches and concepts applied in INTEGRIN REGULATION are fully disseminated and that the field can benefit from the findings. Currently, the fellow is evaluating the clinical relevance of the hits from the RNAi FRET screen, which will then be assessed by immunohistochemistry staining of large breast cancer cohorts, along with functional validation using combination therapies aimed at assessing the potential of inhibiting integrin beta1 phosphorylation as an anti-metastatic in breast cancer models. These aspects will provide an essential link from the mechanistic insights to improving patient outcomes from INTEGRIN REGULATION.