Fat1 deletion promotes hybrid EMT state, tumour stemness and metastasis.

FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.

Clinical, neuroimaging and molecular characteristics of PPP2R5D-related neurodevelopmental disorders: an expanded series with functional characterisation and genotype-phenotype analysis.

BACKGROUND: Variants in PPP2R5D, affecting the regulatory B56δ subunit of protein phosphatase 2A (PP2A), have been identified in individuals with neurodevelopmental abnormalities. However, the molecular and clinical spectra remain incompletely understood. METHODS: Individuals with PPP2R5D variants were enrolled through Simons Variation in Individuals Project/Simons Searchlight. Data were collected from medical history interviews, medical record review, online validated instruments and neuroimaging review. Genetic variants were biochemically characterised. RESULTS: We studied 76 individuals with PPP2R5D variants, including 68 with pathogenic de novo variants, four with a variant of uncertain significance (VUS) and four siblings with a novel dominantly inherited pathogenic variant. Among 13 pathogenic variants, eight were novel and two (p.Glu198Lys and p.Glu200Lys) were highly recurrent. Functional analysis revealed impaired PP2A A/C-subunit binding, decreased short linear interaction motif-dependent substrate binding or both-with the most severe phenotypes associated with variants that completely retained one of these binding characteristics and lost the other-further supporting a dominant-negative disease mechanism. p.Glu198Lys showed the highest C-binding defect and a more severe clinical phenotype. The inherited p.Glu197Gly variant had a mild substrate binding defect, and three of four VUS had no biochemical impact. Common clinical phenotypes were language, intellectual or learning disabilities (80.6%), hypotonia (75.0%), macrocephaly (66.7%), seizures (45.8%) and autism spectrum disorder (26.4%). The mean composite Vineland score was 59.8, and most participants were in the 'moderate to low' and 'low' adaptive levels in all domains. CONCLUSION: Our study delineates the most common features of PPP2R5D-related neurodevelopmental disorders, expands the clinical and molecular spectrum and identifies genotype-phenotype correlations.

Human pancreatic cancer patients with Epithelial-to-Mesenchymal Transition and an aggressive phenotype show a disturbed balance in Protein Phosphatase Type 2A expression and functionality.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a low survival, its incidence is rising and little therapeutic improvements are expected in the near future. It has been observed that Epithelial-to-Mesenchymal transition (EMT) contributes (including in PDAC) to a more aggressive cancer phenotype. Additionally, largely unexplored, studies indicate a mechanistic interplay between Protein Phosphatase Type 2A (PP2A) enzymes and EMT that could offer treatment opportunities. The aim was to investigate the relation of a PP2A expression signature (encompassing all PP2A subunits, endogenous inhibitors and activators) with EMT and aggressive pancreatic cancer, and to discuss possible implications. METHODS: We retrieved different PDAC expression datasets from NCBI to capture the variation in patients, and analyzed these using datamining, survival analysis, differential gene and protein expression. We determined genes highly associated with aggressive PDAC. For in vitro evaluation, Panc-1 cells were treated with the pharmacologic PP2A inhibitor Okadaic Acid (OA). Additionally, two OA-resistant Panc-1 clones were developed and characterized. RESULTS: In patients, there is a strong correlation between EMT and aggressive PDAC, and between aggressive PDAC and PP2A, with a significant upregulation of PP2A inhibitor genes. Several PP2A genes significantly correlated with decreased survival. In vitro, short-term exposure to OA induced EMT in Panc-1 cells. This shift towards EMT was further pronounced in the OA-resistant Panc-1 clones, morphologically and by pathway analysis. Proteomic analysis and gene sequencing showed that the advanced OA-resistant model most resembles the clinical PDAC presentation (with EMT signature, and with several specific PP2A genes upregulated, and others downregulated). CONCLUSIONS: We demonstrated a strong association between EMT, altered PP2A expression and aggressive PDAC in patients. Also, in vitro, PP2A inhibition induces EMT. Overall, statistics suggests the mechanistic importance of PP2A dysregulation for PDAC progression. Translationally, our observations indicate that pharmacologic restoration of PP2A activity could be an attractive therapeutic strategy to block or reverse progression.

De Novo Mutations Affecting the Catalytic Cα Subunit of PP2A, PPP2CA, Cause Syndromic Intellectual Disability Resembling Other PP2A-Related Neurodevelopmental Disorders.

Type 2A protein phosphatases (PP2As) are highly expressed in the brain and regulate neuronal signaling by catalyzing phospho-Ser/Thr dephosphorylations in diverse substrates. PP2A holoenzymes comprise catalytic C-, scaffolding A-, and regulatory B-type subunits, which determine substrate specificity and physiological function. Interestingly, de novo mutations in genes encoding A- and B-type subunits have recently been implicated in intellectual disability (ID) and developmental delay (DD). We now report 16 individuals with mild to profound ID and DD and a de novo mutation in PPP2CA, encoding the catalytic Cα subunit. Other frequently observed features were severe language delay (71%), hypotonia (69%), epilepsy (63%), and brain abnormalities such as ventriculomegaly and a small corpus callosum (67%). Behavioral problems, including autism spectrum disorders, were reported in 47% of individuals, and three individuals had a congenital heart defect. PPP2CA de novo mutations included a partial gene deletion, a frameshift, three nonsense mutations, a single amino acid duplication, a recurrent mutation, and eight non-recurrent missense mutations. Functional studies showed complete PP2A dysfunction in four individuals with seemingly milder ID, hinting at haploinsufficiency. Ten other individuals showed mutation-specific biochemical distortions, including poor expression, altered binding to the A subunit and specific B-type subunits, and impaired phosphatase activity and C-terminal methylation. Four were suspected to have a dominant-negative mechanism, which correlated with severe ID. Two missense variants affecting the same residue largely behaved as wild-type in our functional assays. Overall, we found that pathogenic PPP2CA variants impair PP2A-B56(δ) functionality, suggesting that PP2A-related neurodevelopmental disorders constitute functionally converging ID syndromes.

Protein Phosphatase 2A (PP2A) mutations in brain function, development, and neurologic disease.

By removing Ser/Thr-specific phosphorylations in a multitude of protein substrates in diverse tissues, Protein Phosphatase type 2A (PP2A) enzymes play essential regulatory roles in cellular signalling and physiology, including in brain function and development. Here, we review current knowledge on PP2A gene mutations causally involved in neurodevelopmental disorders and intellectual disability, focusing on PPP2CA, PPP2R1A and PPP2R5D. We provide insights into the impact of these mutations on PP2A structure, substrate specificity and potential function in neurobiology and brain development.

An okadaic acid fragment analogue prevents nicotine-induced resistance to cisplatin by recovering PP2A activity in non-small cell lung cancer cells.

We herein report the design, synthesis, and functional impact of an okadaic acid (OA) small analogue, ITH12680, which restores the activity of phosphoprotein phosphatase 2A (PP2A), whose deficient activity has been implicated in nicotine-mediated tumor progression and chemoresistance in non-small cell lung cancer (NSCLC). For its design, we paid attention to the structure of the PP2A-OA complex, where the C16-C38 OA fragment confers PP2A affinity and selectivity, but it is not involved in the inhibitory effect. Confirming this hypothesis, PP2A activity was not inhibited by ITH12680. By contrast, the compound partially restored OA-exerted PP2A inhibition in vitro. Moreover, flow cytometry and immunoblotting experiments revealed that ITH12680 reversed nicotine-induced cisplatin resistance in NSCLC cells, as it prevented nicotine-induced reduction of Bax expression and inhibited nicotine-mediated activation of cell survival and proliferation kinases, Akt and ERK1/2. Our findings suggest that the rescue of nicotine-inhibited PP2A activity could diminish the resistance to cisplatin treatment observed in NSCLC patients who continue smoking.

PP2A binds to the LIM domains of lipoma-preferred partner through its PR130/B″ subunit to regulate cell adhesion and migration.

Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn²âº-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP-PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130-LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics.

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells.

Phosphoinositides, particularly phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], are recognized by SHIP2 (also known as INPPL1) a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the preferred SHIP2 substrate is PI(3,4,5)P3, PI(4,5)P2 can also be dephosphorylated by this enzyme to phosphatidylinositol 4-phosphate (PI4P). Through depletion of SHIP2 in the glioblastoma cell line 1321 N1, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In 1321 N1 cells, the PI(4,5)P2-binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls 1321 N1 cell migration through the organization of focal adhesions. Thus, our results reveal a new role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma 1321 N1 cells.

PP2A T61 epsilon is an inhibitor of MAP4K3 in nutrient signaling to mTOR.

The mammalian target of rapamycin (mTOR) pathway is activated by a variety of stimuli, including nutrients such as glucose and amino acids. The Ste20 family kinase MAP4K3 is regulated by amino acids and acts upstream of mTORC1. Here we investigate how MAP4K3 activity is regulated by amino acid sufficiency. We identify a transautophosphorylation site in the MAP4K3 kinase activation segment (Ser170) that is required for MAP4K3 activity and its activation of mTORC1 signaling. Following amino acid withdrawal, Ser170 is dephosphorylated via PP2A complexed to PR61 epsilon, a PP2A-targeting subunit. Inhibition of PR61 epsilon expression prevents MAP4K3 Ser170 dephosphorylation and impairs mTORC1 inhibition during amino acid withdrawal. We propose that during amino acid sufficiency Ser170-phosphorylated MAP4K3 activates mTORC1, but that upon amino acid restriction MAP4K3 preferentially interacts with PP2A(T61 epsilon), promoting dephosphorylation of Ser170, MAP4K3 inhibition, and, subsequently, inhibition of mTORC1 signaling.

Targeted Therapies in Type II Endometrial Cancers: Too Little, but Not Too Late.

Type II endometrial carcinomas (ECs) are responsible for most endometrial cancer-related deaths due to their aggressive nature, late stage detection and high tolerance for standard therapies. However, there are no targeted therapies for type II ECs, and they are still treated the same way as the clinically indolent and easily treatable type I ECs. Therefore, type II ECs are in need of new treatment options. More recently, molecular analysis of endometrial cancer revealed phosphorylation-dependent oncogenic signalling in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways to be most frequently altered in type II ECs. Consequently, clinical trials tested pharmacologic kinase inhibitors targeting these pathways, although mostly with rather disappointing results. In this review, we highlight the most common genetic alterations in type II ECs. Additionally, we reason why most clinical trials for ECs using targeted kinase inhibitors had unsatisfying results and what should be changed in future clinical trial setups. Furthermore, we argue that, besides kinases, phosphatases should no longer be ignored in clinical trials, particularly in type II ECs, where the tumour suppressive phosphatase protein phosphatase type 2A (PP2A) is frequently mutated. Lastly, we discuss the therapeutic potential of targeting PP2A for (re)activation, possibly in combination with pharmacologic kinase inhibitors.