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.

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.

PPP2R4 dysfunction promotes KRAS-mutant lung adenocarcinoma development and mediates opposite responses to MEK and mTOR inhibition.

KRAS-mutant lung adenocarcinomas represent the largest molecular subgroup of non-small cell lung cancers (NSCLC) and are notorious for their dismal survival perspectives. To gain more insights in etiology and therapeutic response, we focused on the tumor suppressor Protein Phosphatase 2A (PP2A) as a player in KRAS oncogenic signaling. We report that the PP2A activator PTPA (encoded by PPP2R4) is commonly affected in NSCLC by heterozygous loss and low-frequent loss-of-function mutation, and this is specifically associated with poorer overall survival of KRAS-mutant lung adenocarcinoma patients. Reduced or mutant PPP2R4 expression in A549 cells increased anchorage-independent growth in vitro and xenograft growth in vivo, correlating with increased Ki67 and c-MYC expression. Moreover, KrasG12D-induced lung tumorigenesis was significantly accelerated in Ppp2r4 gene trapped mice as compared to Ppp2r4 wild-type. A confined kinase inhibitor screen revealed that PPP2R4-depletion induced resistance against selumetinib (MEK inhibitor), but unexpectedly sensitized cells for temsirolimus (mTOR inhibitor), in vitro and in vivo. Our findings underscore a clinically relevant role for PTPA loss-of-function in KRAS-mutant NSCLC etiology and kinase inhibitor response.

Increased LGR6 Expression Sustains Long-Term Wnt Activation and Acquisition of Senescence in Epithelial Progenitors in Chronic Lung Diseases.

Chronic lung diseases (CLDs) represent a set of disorders characterized by the progressive loss of proper lung function. Among severe CLDs, the incidence of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) has grown over the last decades, mainly in the elderly population. Several studies have highlighted an increased expression of senescence-related markers in the resident progenitor cells in COPD and IPF, possibly undermining epithelial integrity and contributing to the progression and the aggravation of both diseases. Recently, the chronic activation of the canonical Wnt/ß-catenin pathway was shown to induce cellular senescence. Here, we investigated the localization and the expression of leucin-rich repeat-containing G-protein-coupled receptor 6 (LGR6), a protein that activates and potentiates the canonical Wnt signalling. Through immunohistochemical analyses, we identified a lesion-associated rise in LGR6 levels in abnormal lung epithelial progenitors in COPD and IPF when compared to histologically normal tissues. Moreover, in areas of aberrant regeneration, chronic damage and fibrosis, LGR6-expressing epithelial progenitors displayed a major increase in the expression of senescence-associated markers. Our study suggests the involvement of LGR6 in the chronic activation of the Wnt/ß-catenin pathway, mediating the impairment and exhaustion of epithelial progenitors in COPD and IPF.

Tumor suppressive protein phosphatases in human cancer: Emerging targets for therapeutic intervention and tumor stratification.

Aberrant protein phosphorylation is one of the hallmarks of cancer cells, and in many cases a prerequisite to sustain tumor development and progression. Like protein kinases, protein phosphatases are key regulators of cell signaling. However, their contribution to aberrant signaling in cancer cells is overall less well appreciated, and therefore, their clinical potential remains largely unexploited. In this review, we provide an overview of tumor suppressive protein phosphatases in human cancer. Along their mechanisms of inactivation in defined cancer contexts, we give an overview of their functional roles in diverse signaling pathways that contribute to their tumor suppressive abilities. Finally, we discuss their emerging roles as predictive or prognostic markers, their potential as synthetic lethality targets, and the current feasibility of their reactivation with pharmacologic compounds as promising new cancer therapies. We conclude that their inclusion in clinical practice has obvious potential to significantly improve therapeutic outcome in various ways, and should now definitely be pushed forward.

PP2A Inactivation Mediated by PPP2R4 Haploinsufficiency Promotes Cancer Development.

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4 Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825-37. ©2017 AACR.