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.

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.

Interaction between LIS1 and PDE4, and its role in cytoplasmic dynein function.

LIS1, a WD40 repeat scaffold protein, interacts with components of the cytoplasmic dynein motor complex to regulate dynein-dependent cell motility. Here, we reveal that cAMP-specific phosphodiesterases (PDE4s) directly bind PAFAH1B1 (also known as LIS1). Dissociation of LIS1-dynein complexes is coupled with loss of dynein function, as determined in assays of both microtubule transport and directed cell migration in wounded monolayers. Such loss in dynein functioning can be achieved by upregulation of PDE4, which sequesters LIS1 away from dynein, thereby uncovering PDE4 as a regulator of dynein functioning. This process is facilitated by increased intracellular cAMP levels, which selectively augment the interaction of long PDE4 isoforms with LIS1 when they become phosphorylated within their regulatory UCR1 domain by protein kinase A (PKA). We propose that PDE4 and dynein have overlapping interaction sites for LIS1, which allows PDE4 to compete with dynein for LIS1 association in a process enhanced by the PKA phosphorylation of PDE4 long isoforms. This provides a further example to the growing notion that PDE4 itself may provide a signalling role independent of its catalytic activity, exemplified here by its modulation of dynein motor function.