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

Abstract

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 β1‐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 β1‐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

“…We emphasise that phenotypic screening, proteomic profiling of non-mutant GTPases, consideration of splice variants, and in vivo experimentation are important tools in the experimental arsenal, in addition to fundamental biochemical in vitro approaches. Collectively, a seeming resurgence in interest in ARF GTPases in recent years, such as the following non-exhaustive example list [ 13 , 21 , 33 , 42 , 43 , 47 , 59 ], may help to elucidate their function and potential targetability in human disease.…”

“…We recently used ARF6-TurboID (wild-type ARF6, not a GTP-preferential mutant) in high-grade serous ovarian cancer (HGSOC) cells to map the ARF6 interactome across an isogenic series of loss of TP53 and PTEN, alone or in combination [ 47 ]. In HGSOC, the loss of TP53 and PTEN results in aggressive tumourigenesis and some level of treatment resistance in both patients and murine models [ 48 ].…”

“…PTEN is frequently perturbed — mutated, down-regulated, or lost — in cancer [ 58 ], suggesting that an imbalance in this pathway is a core part of tumourigenesis. In our recent paper, we described that PTEN-null cells become dependent on a CYTH2-ARF6-AGAP1 module to regulate active β1-integrin recycling to promote invasion [ 47 ] ( Figure 3b ). PI(3,4,5)P 3 localised to the front of cellular protrusions to drive collective invasion.…”