Pten function in zebrafish: anything but a fish story.

Zebrafish is an excellent model system for the analysis of gene function. We and others use zebrafish to investigate the function of the tumor suppressor, Pten, in tumorigenesis and embryonic development. Zebrafish have two pten genes, ptena and ptenb. The recently identified N-terminal extension of human PTEN that may facilitate cell membrane transfer, appears not to be conserved in zebrafish Ptena or Ptenb. Mutants that retain a single wild type pten allele develop tumors, predominantly hemangiosarcomas. Homozygous double mutants are embryonic lethal. Zebrafish embryos lacking functional Pten display enhanced proliferation of endothelial cells, resulting in hyperbranching of blood vessels. In addition, ptena-/-ptenb-/- mutant embryos display enhanced proliferation of hematopoietic stem and progenitor cells and concomitant arrest of differentiation, although Pten-deficient cells commit to all blood cell lineages. Zebrafish is an ideal model for intravital imaging and future work using ptena-/-ptenb-/- mutants will enhance our understanding of the function of Pten in vivo.

Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program 'iTHER'.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

Improvement of clinical management and outcome in hepatocellular carcinoma nowadays compared with historical cohorts.

OBJECTIVES: Therapeutic approaches in the treatment of hepatocellular carcinoma (HCC) depend on tumour stage, liver function and patient comorbidities. The aim of this study was to investigate the influence of tumour stage and therapeutic approach on overall survival in HCC. MATERIALS AND METHODS: Two hundred and fourteen patients with HCC diagnosed between December 2012 and May 2017 were assessed retrospectively for tumour stage [Barcelona Clinic Liver Cancer (BCLC)], liver function (Child-Pugh score), therapeutic approach and outcome (mean survival time). The results were compared to two historical cohorts from our centre diagnosed between 1999 and 2013 and 1988 and 1999, respectively. RESULTS: Nowadays, HCC is diagnosed in earlier tumour stages and with better liver function compared with the historical cohorts (P<0.001). Survival times depend on both BCLC stages and liver function for all therapeutic approaches. The 1-year survival rate in the present cohort was 79.4% compared with 58.6% in the historical cohort.In terms of BCLC stages, therapeutic approaches followed HCC guidelines in 43.9% of cases.Whereas the percentage of patients receiving resection or ablation did not change between the historical and the present cohort, there was a tendency towards a decrease in transarterial chemoembolization, with a shift towards selective internal radiotherapy, accompanied by an increase in systemic therapy with sorafenib.Also, the percentage of patients receiving single instead of multiple therapies was significantly higher in the present cohort compared with the historical cohort (P=0.016). In 62/83 patients receiving single therapy (64.7%), tumour remission was maintained during the period of follow-up. CONCLUSION: HCC is increasingly being diagnosed in earlier stages, so that single therapy is often sufficient. Besides BCLC stages, therapy in HCC must consider liver function, tumour location, local expertise and patients' comorbidities and preferences. Further research is needed to evaluate the benefit of early multimodal concepts. Therapeutic approaches in HCC remain individual decisions.

Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development.

The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten.

Fine-Tuning of Pten Localization and Phosphatase Activity Is Essential for Zebrafish Angiogenesis.

The lipid- and protein phosphatase PTEN is an essential tumor suppressor that is highly conserved among all higher eukaryotes. As an antagonist of the PI3K/Akt cell survival and proliferation pathway, it exerts its most prominent function at the cell membrane, but (PIP3-independent) functions of nuclear PTEN have been discovered as well. PTEN subcellular localization is tightly controlled by its protein conformation. In the closed conformation, PTEN localizes predominantly to the cytoplasm. Opening up of the conformation of PTEN exposes N-terminal and C-terminal regions of the protein that are required for both interaction with the cell membrane and translocation to the nucleus. Lack of Pten leads to hyperbranching of the intersegmental vessels during zebrafish embryogenesis, which is rescued by expression of exogenous Pten. Here, we observed that expression of mutant PTEN with an open conformation rescued the hyperbranching phenotype in pten double homozygous embryos and suppressed the increased p-Akt levels that are characteristic for embryos lacking Pten. In addition, in pten mutant and wild type embryos alike, open conformation PTEN induced stalled intersegmental vessels, which fail to connect with the dorsal longitudinal anastomotic vessel. Functional hyperactivity of open conformation PTEN in comparison to wild type PTEN seems to result predominantly from its enhanced recruitment to the cell membrane. Enhanced recruitment of phosphatase inactive mutants to the membrane did not induce the stalled vessel phenotype nor did it rescue the hyperbranching phenotype in pten double homozygous embryos, indicating that PTEN phosphatase activity is indispensable for its regulatory function during angiogenesis. Taken together, our data suggest that PTEN phosphatase activity needs to be carefully fine-tuned for normal embryogenesis and that the control of its subcellular localization is a key mechanism in this process.

A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.