PTEN deficiency exposes a requirement for an ARF GTPase module for integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.

The ARF GTPase regulatory network in collective invasion and metastasis.

The ability to remodel and move cellular membranes, and the cargoes regulated by these membranes, allows for specialised functions to occur in distinct regions of the cell in a process known as cellular polarisation. The ability to collectively co-ordinate such polarisation between cells allows for the genesis of multicellularity, such as the formation of organs. During tumourigenesis, the rules for such tissue polarisation become dysregulated, allowing for collective polarity rearrangements that can drive metastasis. In this review, we focus on how membrane trafficking underpins collective cell invasion and metastasis in cancer. We examine this through the lens of the ADP-ribosylation factor (ARF) subfamily of small GTPases, focusing on how the ARF regulatory network - ARF activators, inactivators, effectors, and modifications - controls ARF GTPase function.

The small GTPase ARF3 controls invasion modality and metastasis by regulating N-cadherin levels.

ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.

The Use of Three-Dimensional Cell Culture to Study Apicobasal Polarization and Lumen Formation.

The three-dimensional culture of epithelial cells allows the characterization of processes required for collective epithelial polarization, such as formation of an epithelial lumen. Madin-Darby Canine Kidney (MDCK) cells have been instrumental in pioneering 3-Dimensional culture analysis methods. Here we describe methods for MDCK cell three-dimensional culture, generation of stable engineered cell lines, immunolabeling, and imaging approaches that allow for analysis of apical-basal polarity during lumen formation in this model.

RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis.

RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

The MSP-RON axis stimulates cancer cell growth in models of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage-stimulating protein (MSP) and its tyrosine kinase receptor, Recepteur d'origine nantais (RON), are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1. After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14-Cre;Brca1F/F ;Trp53F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates serine/threonine kinase 1 and extracellular regulated MAPK activation as well as cancer cell growth in cell lines derived from the two mouse models, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki-67. Analysis of human gene expression data confirmed that the genes encoding MSP and RON are robustly expressed in human TNBC as well as other subsets of breast cancer. Our findings uncover a mouse model where MSP expression and RON expression are naturally increased, and they provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of breast cancer.

The phospholipid PI(3,4)P2 is an apical identity determinant.

Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P2 localization and function is distinct from the basolateral determinant PI(3,4,5)P3. PI(3,4)P2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P2 is a determinant of apical membrane identity.

A functional genomics screen reveals a strong synergistic effect between docetaxel and the mitotic gene DLGAP5 that is mediated by the androgen receptor.

Based on a molecular classification of prostate cancer using gene expression pathway signatures, we derived a set of 48 genes in critical pathways that significantly predicts clinical outcome in all tested patient cohorts. We tested these genes in a functional genomics screen in a panel of three prostate cancer cell lines (LNCaP, PC3, DU145), using RNA interference. The screen revealed several genes whose knockdown caused strong growth inhibition in all cell lines. Additionally, we tested the gene set in the presence of docetaxel to see whether any gene exhibited additive or synergistic effects with the drug. We observed a strong synergistic effect between DLGAP5 knockdown and docetaxel in the androgen-sensitive line LNCaP, but not in the two other androgen-independent lines. We then tested whether this effect was connected to androgen pathways and found that knockdown of the androgen receptor by si-RNA attenuated the synergy significantly. Similarly, androgen desensitized LNCaP-AI cells had a higher IC50 to docetaxel and did not exhibit the synergistic interaction. Short-term exposure to enzalutamide did not significantly alter the behaviour of parental LNCaP cells. An immunofluorescence analysis in LNCaP cells suggests that under the double insult of DLGAP5 knockdown and docetaxel, cells predominantly arrest in metaphase. In contrast, the knockdown of the androgen receptor by siRNA appears to assist cells to progress through metaphase in to anaphase, even in the presence of docetaxel. Our data suggest that DLGAP5 has a unique function in stabilizing spindle formation and surviving microtubule assault from docetaxel, in an androgen-regulated cell cycle system.

Fibroblast-derived HGF drives acinar lung cancer cell polarization through integrin-dependent RhoA-ROCK1 inhibition.

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate ß1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways.

Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks.

Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described 'spatial rheostat' controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin one and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.

ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival.

Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis.

p70S6K is regulated by focal adhesion kinase and is required for Src-selective autophagy.

Here we report that focal adhesion kinase (FAK) is required for optimal signalling to the Akt-p70S6K-S6 pathway in squamous cell carcinoma (SCC) cells. Specifically, in SCCs that are genetically deficient for FAK, there is reduced phosphorylation of Akt, p70S6K and S6, and signalling to Akt-p70S6K-S6 is more sensitive to inhibition by multiple agents that suppress the pathway. By contrast, mTOR is unaffected. Indeed, pharmacological agents that inhibit the Akt-p70S6K-S6 pathway, and PDK1 that lies upstream of Akt, also impair the autophagic targeting of activated c-Src (p-Src) in FAK deficient cells. This is associated with loss of a complex between p-Src and the autophagy protein LC3, a biochemical surrogate of impaired Src-selective autophagy. In keeping with a vital role for p70S6K, inhibition by a selective inhibitor and specific siRNA also impaired Src-selective autophagy. Finally, components of the PDK1-Akt-p70S6K signalling pathway were co-located with p-Src at autophagosomes, and Src and p70S6K co-exist in the same biochemical complex. We therefore deduce that the FAK-regulated signalling module PDK1-Akt-p70S6K that controls Src's intracellular trafficking operates at Src-containing autophagosomes.

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells.

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours.

Src-dependent autophagic degradation of Ret in FAK-signalling-defective cancer cells.

We have recently described that autophagic targeting of Src maintains cancer cell viability when FAK signalling is defective. Here, we show that the Ret tyrosine kinase is also degraded by autophagy in cancer cells with altered/reduced FAK signalling, preventing its binding to FAK at integrin adhesions. Inhibition of autophagy restores Ret localization to focal adhesions. Importantly, Src kinase activity is required to target Ret to autophagosomes and enhance Ret degradation. Src is thus a general mediator of selective autophagic targeting of adhesion-linked kinases, and Ret a second FAK-binding tyrosine kinase degraded through autophagy in cancer cells under adhesion stress. Src--by controlling not only its own degradation but also that of other FAK-binding partners--allows cancer cell survival, suggesting a new therapeutic strategy.

Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling.

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.

Signaling of the direction-sensing FAK/RACK1/PDE4D5 complex to the small GTPase Rap1.

We recently reported that a complex between focal adhesion kianse (FAK) and the molecular scaffold RACK1 controlled nascent integrin adhesion formation and cell polarization, via peripheral recruitment of the cAMP - degrading PDE4D5 isoform. Here we review and extend these studies by demonstrating that the FAK/RACK1/PDE4D5 'direction-sensing' complex likely functions by signaling, via the guanine nucleotide exchange factor EPAC , to its small GTPase target Rap1. Specifically, activating EPAC suppresses polarization of squamous cancer cells, while, in contrast, modulating PKA, the other major cAMP effector, has no effect. Moreover, FAK-deficient malignant keratinocytes re-expressing a FAK mutant that cannot bind to RACK1, namely FAK-E139A,D140A, display elevated Rap1 that is linked to impaired polarization. Thus, it is likely that the FAK/RACK1/PDE4D5 complex signals to keep Rap1 low at appropriate times and in a spatially-regulated manner as cells first sense their environment and make decisions about nascent adhesion stabilization and polarization. RACK1 is abundantly expressed in both normal and malignant keratinocytes, while FAK and PDE4D5 are both elevated in the cancer cells, suggesting that the FAK/RACK1/PDE4D5/Rap1 signaling axis may contribute to FAK's well documented role in tumor progression.

A complex between FAK, RACK1, and PDE4D5 controls spreading initiation and cancer cell polarity.

A fundamental question in cell biology concerns how cells respond to their environment by polarizing after sensing directional cues. This requires the differential localization of protein complexes in cells, and it is important to identify and understand how these complexes function. Here we describe a novel "direction-sensing" pathway that links the integrin effector focal adhesion kinase (FAK), the molecular scaffold protein RACK1, and activity of one of its client proteins, PDE4D5, a cAMP-degrading phosphodiesterase. The complex is recruited to nascent adhesions and promotes cell polarity. We identify FAK FERM domain residues whose mutation impairs RACK1 binding. When re-expressed in cancer cells in which endogenous fak is deleted by Cre-lox-mediated recombination, the RACK1-binding-impaired FAK mutant protein does not support formation of nascent actin adhesion structures as cells spread. These cancer cells, like FAK-deficient cells, cannot undergo directional responses, including wound-induced polarization or chemotactic invasion into three-dimensional matrix gels. We show that RACK1 serves as the molecular bridge linking FAK to the recruitment of PDE4D5. FAK/RACK1/PDE4D5 is a novel 'direction-sensing' complex that acts to recruit specific components of the cAMP second-messenger system to nascent integrin adhesions and to the leading edge of polarizing cells.