Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

<p>Abstract</p> <p>Background</p> <p>Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised.</p> <p>Results</p> <p>We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.</p> <p>Conclusion</p> <p>In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.</p

The MDM2-inhibitor Nutlin-3 synergizes with cisplatin to induce p53 dependent tumor cell apoptosis in non-small cell lung cancer

The p53/MDM2 interaction has been a well-studied target for new drug design leading to the development of the small molecule inhibitor Nutlin-3. Our objectives were to combine Nutlin-3 with cisplatin (CDDP), a well-known activator of the p53 pathway, in a series of non-small cell lung cancer cell lines in order to increase the cytotoxic response to CDDP. We report that sequential treatment (CDDP followed by Nutlin-3), but not simultaneous treatment, resulted in strong synergism. Combination treatment induced p53's transcriptional activity, resulting in increased mRNA and protein levels of MDM2, p21, PUMA and BAX. In addition we report the induction of a strong p53 dependent apoptotic response and induction of G2/M cell cycle arrest. The strongest synergistic effect was observed at low doses of both CDDP and Nutlin-3, which could result in fewer (off-target) side effects while maintaining a strong cytotoxic effect. Our results indicate a promising preclinical potential, emphasizing the importance of the applied treatment scheme and the presence of wild type p53 for the combination of CDDP and Nutlin-3

The Relevance of External Quality Assessment for Molecular Testing for ALK Positive Non-Small Cell Lung Cancer:Results from Two Pilot Rounds Show Room for Optimization

Molecular profiling should be performed on all advanced non-small cell lung cancer with non-squamous histology to allow treatment selection. Currently, this should include EGFR mutation testing and testing for ALK rearrangements. ROS1 is another emerging target. ALK rearrangement status is a critical biomarker to predict response to tyrosine kinase inhibitors such as crizotinib. To promote high quality testing in non-small cell lung cancer, the European Society of Pathology has introduced an external quality assessment scheme. This article summarizes the results of the first two pilot rounds organized in 2012-2013.Tissue microarray slides consisting of cell-lines and resection specimens were distributed with the request for routine ALK testing using IHC or FISH. Participation in ALK FISH testing included the interpretation of four digital FISH images.Data from 173 different laboratories was obtained. Results demonstrate decreased error rates in the second round for both ALK FISH and ALK IHC, although the error rates were still high and the need for external quality assessment in laboratories performing ALK testing is evident. Error rates obtained by FISH were lower than by IHC. The lowest error rates were observed for the interpretation of digital FISH images.There was a large variety in FISH enumeration practices. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing

Sensitive detection methods are key to identify secondary EGFR c.2369C&gt;T p.(Thr790Met) in non-small cell lung cancer tissue samples

Background: Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. Methods: Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for w

Cell-Free DNA From Metastatic Pancreatic Neuroendocrine Tumor Patients Contains Tumor-Specific Mutations and Copy Number Variations

Background: Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. So far, use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients.Methods: From 10 PNET patients, fresh frozen tumor tissue, buffy coat and plasma samples were collected. Whole-exome sequencing of primary tumor and germline DNA was performed to identify tumor-specific variants and copy number variations (CNVs). Subsequently, tumor-specific variants were quantified in plasma cfDNA with droplet digital PCR. In addition, CNV analysis of cfDNA was performed using shallow whole-genome sequencing.Results: Tumor-specific variants were detected in perioperative plasma samples of two PNET patients, at variant allele fractions (VAFs) of respectively 19 and 21%. Both patients had metastatic disease at time of surgery, while the other patients presented with localized disease. In the metastatic patients, CNV profiles of tumor tissue and cfDNA were significantly correlated. A follow-up plasma sample of a metastatic patient demonstrated an increased VAF (57%) and an increased chromosomal instability, in parallel with an increase in tumor burden.Conclusions: We are the first to report the presence of tumor-specific genetic alterations in cfDNA of metastatic PNET patients and their evolution during disease progression. Additionally, CNV analysis in cfDNA shows potential as a liquid biopsy

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine

Physiological function of the PR130 B" subunit of protein phosphatase 2A

Omkeerbare fosforylering is een uiterst belangrijk mechanisme dat tussenkomt in de regulatie van vele cellulaire processen. Deze modificatie wordt bewerkstelligd door proteïne kinasen en fosfatasen. Proteïne fosfatase 2A (PP2A) is verantwoordelijk voor een groot deel van de serine/threonine defosforylerende activiteit in een cel en komt zo tussen in tal van signalisatie transductiewegen, waaronder celcyclus regulatie, cytoskelet organisatie, gen transcriptie en proteïne synthese. PP2A komt hoofdzakelijk voor als een heterotrimeer, opgebouwd uit een katalytische C subeenheid, een structurele A subeenheid en een regulerende B subeenheid. Tot op heden zijn er twee A subeenheden, twee C subeenheden en meer dan 20 B subeenheden geïdentificeerd. Bijgevolg bestaan er een groot aantal verschillende PP2A complexen, elk met een mogelijk specifieke functie. In deze studie hebben we onze aandacht gevestigd op de PP2A complexen die PR130 als regulerende B subeenheid bevatten (PP2AT130). PR130 is het grootste proteïne van de PR72/B familie van regulerende subeenheden. De PR72/B leden bevatten allen een goed geconserveerd centraal gedeelte met daarin twee Ca2+-bindende EF hand motieven.In het eerste gedeelte van dit werk hebben we de sequentie en genomische organisatie van de verschillende PR72/B isovormen afgeleid bij de muis en dit als basis gebruikt om een aangepaste naamgeving voor de PR72/B familie te introduceren. We vonden 4 transcripten (PR130/B a1,a3; PR72/B a2,a4) afgeleid van Ppp2r3a (waarvan PR130/B a1 en PR72/B a2 de belangrijkste zijn), 4 transcripten (PR59/B d1,2,3,4) afgeleid van Ppp2r3d (waarvan PR59/B d3 niet kan binden met PP2A en PR59/B d4 in heel geringe mate aanwezig is) en 1 transcript (G5PR/B g) afgeleid van Ppp2r3c. Alhoewel we PR70 isovormen terugvinden bij de mens, hond en kikker, is er bij de muis geen PR70 ortholoog aanwezig. Ook vonden we geen humaan ortholoog van de verschillende muize PR59/B d isovormen. Hieruit kunnen we afleiden dat de PR72/B familie, in vergelijking met PR55/B en PR61/B , een grotere diversiteit vertoont op evolutionair vlak. Via Northern blotting hebben we de weefselspecificiteit en de embryonale expressie van de belangrijkste muize PR72/B isovormen onderzocht. Allen komen hoog tot expressie in het hartweefsel. Mogelijk hebben zij daar een belangrijke functie. Immunohistochemische analyse van hart en skeletspier toonde een gestreept expressiepatroon, de zogenaamde A band, aan voor PR130 en PR72. Daarnaast zagen we een epitheliale expressie voor PR130 in de blaas en nier. Voorts hebben we ook de subcellulaire lokalisatie en de impact op de celcyclus voor de verschillende B /PR72 isovormen bepaald. Deze vertoonden zowel verschillen als gelijkenissen. Bovenstaande studie verstrekt waardevolle informatie voor de generatie en karakterisatie van toekomstige PR72/B knock-out muizen.In het tweede gedeelte van dit werk hebben we geprobeerd meer inzicht te krijgen in de fysiologische functies die door PP2AT130 beïnvloed worden. In een eerste luik hebben we de capaciteit van IQ-1 om PP2AT130 en PP2AT72 in vitro en in vivo te inhiberen, nagekeken. Na IQ-1 toevoeging zagen we een reductie van de protamine gestimuleerde PP2AT130/72 activiteit, niettegenstaande we geen verschil in binding van PR130/72 met PP2A zagen. Verdere analyse is dus nodig vooraleer we deze stof kunnen gebruiken om in vivo specifiek PP2AT130 te inhiberen. In een tweede luik hebben we PR130-interagerende proteinen geïdentificeerd via de gist dubbel-hybride methode met PR130N als aas en via massaspectrometrische identificatie van proteïnen die coïmmunoprecipiteren met PR130. Beide strategieën hebben geleid tot de identificatie van LPP, een proteïne belangrijk voor de communicatie tussen celadhesieplaatsen en de kern. Hoewel LPP en PR130 zich beide bevinden in focale contacten en aan de frontale celmembraan van migrerende cellen, is enkel LPP aanwezig in focale adhesies. We konden aantonen dat PR130 een negatieve invloed heeft op cel-matrix adhesie en dat het LPP-PP2AT130 complex noodzakelijk is voor een efficiënte celmigratie. Daarnaast zagen we, afhankelijk van het celtype, een dynamische expressie van PR130 in de kern. De functie(s) van PR130 in de kern zijn voorlopig nog onbekend. SHIP2, een inositol en fosfoinositide 5 -fosfatase werd ook geïdentificeerd als een PR130-interagerend proteïne. Dit proteïne bindt ondermeer aan de EGF receptor (EGFR) en heeft een negatief effect op EGFR degradatie. We hebben kunnen aantonen dat ook PR130 associeert met de EGFR en een negatieve invloed heeft op de EGF-geïnduceerde EGFR degradatie. Bovendien translokeert PR130 net als SHIP2 naar de celmembraan na EGF stimulatie. Momenteel zijn we de hypothese aan het onderzoeken dat PR130 een brug vormt tussen SHIP2 en de EGFR en zo SHIP2 helpt om de EGFR degradatie te beïnvloeden.Uit bovenstaande gegevens kunnen we besluiten dat PR130 een negatieve invloed heeft op cel adhesie en EGFR degradatie en een positieve rol speelt tijdens cel migratie. Cel adhesie en migratie spelen een belangrijke rol tijdens de ontwikkeling van kanker. Daarnaast is geweten dat EGFR hyperactivatie correleert met verschillende soorten kanker. Bijgevolg suggereren deze data dat PR130 mogelijks een tumor promoverende functie vervult.status: publishe

Protein phosphatase 2A PR130/B″α1 subunit binds to the SH2 domain-containing inositol polyphosphate 5-phosphatase 2 and prevents epidermal growth factor (EGF)-induced EGF receptor degradation sustaining EGF-mediated signaling

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Are anaplastic lymphoma kinase (ALK) and O6-methylguanine- DNA methyltransferase (MGMT) promoter methylation driver biomarkers of pulmonary neuroendocrine tumors (NETs) and carcinomas (NECs)?

Background: Novel targets in neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) are needed to improve outcome. The presence of O6- Methylguanine-DNA methyltransferase (MGMT) promoter methylation in NETs and NECs may act as a predictive marker for response on treatment with temozolomide. As anaplastic lymphoma kinase (ALK) plays an important role in the nervous system we hypothesized that ALK rearrangement can act as a biomarker in patients with NETs and NECs. Materials and Methods: We performed a retrospective analysis to establish the frequency of MGMT promoter methylation and ALK expression in tissue samples of patients with NETs and NECs. Results: 21% (14/67) of patients tested positive for MGMT promoter methylation. MGMT promoter methylation was present in 33% (3/9) patients with typical carcinoid, in 22% (2/9) patients with atypical carcinoid, in 22% (8/37) patients with small cell lung cancer and in 8% (1/12) patient with large cell neuroendocrine carcinoma. ALK- expression was present in 14% (10 of 70 patients). In all of these patients, no ALK-rearrangement nor ALK-mutation was revealed. Conclusions: Routine testing of NET and NEC samples for an ALK rearrangement is not recommended as ALK-expression is not associated with an ALK-rearrangement. Routine testing of NET and NEC samples for MGMT will detect a promoter hypermethylation in a sizable minority of patients who are eligible for a targeted treatment with temozolomide