The EMBO Journal

ALYREF condensation stabilizes m5C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

Abstract The role of epigenetic regulation of RNAs in the tumorigenesis remains incompletely understood. This study uncovers a critical function of the 5-methylcytosine (m 5 C) RNA modification reader protein ALYREF (also termed, ALY; BEF) in ovarian cancer. ALYREF is elevated in ovarian cancer patient samples, and its depletion reduces ovarian tumorigenesis and metastasis in mice in a m 5 C-dependent manner. Mechanistically, ALYREF binds to the m 5 C-modified mRNA of ADP-ribosyltransferase PARP10, competing with exosome complex component MTR4, and enhancing the stability and nuclear export of PARP10 mRNA. Further, ALYREF forms condensates in the nucleus of ovarian cancer cells, and depletion or mutation of ALYREF’s intrinsically disordered regions rescues its control on PARP10 mRNA nucleoplasmic distribution and stability, reduces tumor growth and is required for promotion of ovarian cancer aggressiveness and proliferation. Finally, ALYREF and PARP10 expression correlate with poor prognosis in ovarian cancer patients. Together, these findings suggest that ALYREF phase separation facilitates the malignant progression of ovarian cancer by promoting PARP10 expression and thereby enhancing PARP10-dependent proliferative pathways in a m 5 C-dependent manner.

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)P 3 ‐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.

Stable expansion of high‐grade serous ovarian cancer organoids requires a low‐Wnt environment

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low-Wnt environment for long-term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss-of-function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.