A Systems Biology Analysis of PP2A Functions in Cancer Cells

Cancer is characterized by aberrant activation of phosphorylation signalling cascades. However, despite the critical role of phosphatases in protein phosphorylation, their contribution to cancer cell signalling is only emerging. Notably, Protein phosphatase 2A (PP2A) has a well-established tumor suppressor function but it is poorly understood which of its many targets are relevant for this function. This is partly due to the wide range of activities that PP2A participates in and partly due to the fact that PP2A activity regulation, as well as the deregulation in cancer, occurs via many auxiliary subunits and endogenous inhibitor proteins. In this MD-PhD thesis, we have used various systems biology approaches, including phosphoproteomics, high throughput drug sensitivity screening, and transcriptomics to study the functions of the most frequently mutated PP2A subunit, PPP2R1A, as well as three of its endogenous inhibitor proteins, CIP2A, PME1, and SET in cancer cells. This study demonstrates that PP2A reactivation is poorly tolerated by several types of cancer cells and results in downregulation of multiple oncogenic pathways, as well as induction of senescence. Specifically, CIP2A is a regulator of MYC transactivation in basal type breast cancers and our results indicate multiple cooperative mechanisms by which PP2A regulates MYC. Analysis of PP2A dephosphorylome also provided novel insights into general organization of phosphorylation signalling and emphasized the role of PP2A inhibition in the nucleus. By combining the phosphoprotemics data with cancer cell responses to over 300 drugs, we have identified mechanistically distinct types of interactions between drug sensitivity and PP2A activity. We further validated that inhibition of PP2A in KRAS mutant lung cancers confers resistance to MAPK pathway inhibitors including the combination of Raf and MEK inhibitors. Together, these findings provide new evidence to support PP2A reactivation as cancer therapeutic strategy and to support evaluating PP2A activity as a predicitive marker for cancer therapy responses.Syövälle ominainen piirre on fosforylaation perustuvien signaalipolkujen poikkeava aktivoituminen. Huolimatta fosfataasien keskeisestä tehtävästä proteiinien fosforylaatiossa, niiden merkityksestä syöpäsolujen signlaalinvälityksessä on vasta vähän tietoa. Erityisesti Proteiinifosfataasi 2A:lla (PP2A) on selkeästi osoitettu olevan kasvunestäjproteiini-ominaisuuksia, mutta se mitkä PP2A:n monista kohdeproteiineista ovat tärkeitä syövän kannalta tunnetaan huonosti. Osittain tämä johtuu PP2A:n toimintojen moninaisuudesta ja osittain siitä, että PP2A:n säätely, sekä syövissä esiintyvät säätelyn häiriöt, tapahtuvat ylimääräisten alayksiköiden ja inhibiiittoriproteiinien kautta Tässä väitöskirjatutkimuksessa olemme tutkineet PP2A:n yleisimmin mutatoituneen alayksikön, PPP2R1A:n, sekä kolmen inhibiittoriproteiinin, CIP2A:n, PME-1:n ja SETin, toimintoja syöpäsoluissa käyttäen erilaisia systeemibilogisia lähestymistapoja, mukaan lukien fosfoproteomiikkaa, lääkeherkkyysseulontaa ja transkriptomi-analyysiä. Tämä tutkimus osoittaa, että monet syöpäsolut sietävät huonosti PP2A:n uudelleen aktivoimista, joka johtaa useiden onkogeenisten signaalipolkujen estymiseen ja senesenssin käynnistymiseen. Tuloksemme viittaavat siihen, että PP2A säätelee MYC-onkogeeniä useilla toisiaan tukevilla mekanismeilla, ja CIP2A:lla on merkitystä MYCin transaktivaation säätelyssä basaalityypin rintasyövässä. PP2A:n defosforylomista saatu tieto auttaa myös ymmärtämään yleisellä tasolla fosforylaatiosignaloinnin järjestäytymistä soluissa ja osoittaa, että PP2A:n inhibitiolla on keskeinen merkitys tumassa. Yhdistämällä fosfoproteomiikan ja syöpäsolujen vasteet yli 300 lääkkeelle olemme tunnistaneet useita mekanismeiltaan erilaisia yhteisvaikutuksia PP2A:n aktiivisuuden ja lääkeherkkyyksien välillä. PP2A:n estämisestä aiheutuva resistenssin MAPK-signaalipolun inhbiittoreille KRAS-mutaatioita kantasvissa syöpäsoluissa vahvistettiin lisätutukimuksilla. PP2A:n estäminen teki solut resistenteiksi myös MEK ja RAF inhibiittorien yhdistelmälle. Yhdessä nämä tulokset puoltavat PP2A:n reaktivaatiota syövän hoitostrategiana ja PP2A:n aktiivisuuden määrittämistä syöpähoitojen ennustekijänä.Siirretty Doriast

Fosfataasien avulla syövän kimppuun

Syöpäsolujen sisäisten viestiverkostojen toiminnan muuttuminen on tärkeä syövän syntymekanismi ja vaikuttaa monien syöpälääkkeiden vasteisiin. Tärkein viestiverkostojen toimintaa säätelevä biokemiallinen mekanismi on viestintään osallistuvien proteiinien fosforylaatio, joka muuttaa kohdeproteiinin aktiivisuutta. Proteiinien fosforylaatiota säätelee kinaasiproteiinien ja fosforylaatiota poistavien fosfataasien välinen tasapaino. Useita onkogeenisia kinaaseja vastaan on kehitetty lääkkeitä, mutta valitettavasti niiden kliininen teho jää useimmiten hyvinkin lyhytaikaiseksi. Sen sijaan useiden fosfataasien merkitys syövässä on toistaiseksi ollut huonosti tunnettu. Viimeaikaiset tutkimukset ovat osoittaneet, että fosfataaseilla on tärkeä merkitys syövässä ja että niiden aktiivisuuden lääkkeellinen muokkaaminen voisi tarjota täysin uusia mahdollisuuksia syövän hoitoon

A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites

Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness. A competitive CRISPR method discovers targets and phenotypic effects of the MYC oncogene.Peer reviewe

Association of transcript levels of 10 established or candidate-biomarker gene targets with cancerous versus non-cancerous prostate tissue from radical prostatectomy specimens

Objectives: The benefits of PSA (prostate specific antigen)-testing in prostate cancer remain controversial with a consequential need for validation of additional biomarkers. We used highly standardized reverse-transcription (RT)-PCR assays to compare transcript levels of 10 candidate cancer marker genes - BMP6, FGF-8b, KLK2, KLK3, KLK4, KLK15, MSMB, PCA3, PSCA and Trpm8 - in carefully ascertained non-cancerous versus cancerous prostate tissue from patients with clinically localized prostate cancer treated by radical prostatectomy. Design and methods: Total RNA was isolated from fresh frozen prostate tissue procured immediately after resection from two separate areas in each of 87 radical prostatectomy specimens. Subsequent histopathological assessment classified 86 samples as cancerous and 88 as histologically benign prostate tissue. Variation in total RNA recovery was accounted for by using external and internal standards and enabled us to measure transcript levels by RT-PCR in a highly quantitative manner. Results: Of the ten genes, there were significantly higher levels only of one of the less abundant transcripts, PCA3, in cancerous versus non-cancerous prostate tissue whereas PSCA mRNA levels were significantly lower in cancerous versus histologically benign tissue. Advanced pathologic stage was associated with significantly higher expression of KLK15 and PCA3 mRNAs. Median transcript levels of the most abundantly expressed genes (i.e. MSMB, KLK3, KLK4 and KLK2) in prostate tissue were up to 10(5)-fold higher than those of other gene targets. Conclusions: PCA3 expression was associated with advanced pathological stage but the magnitude of overexpression of PCA3 in cancerous versus non-cancerous prostate tissue was modest compared to previously reported data. (C) 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved

Phosphonormalizer: an R package for normalization of MS-based label-free phosphoproteomics

MOTIVATION: Global centering-based normalization is a commonly-used normalization approach in mass spectrometry-based label-free proteomics. It scales the peptide abundances to have the same median intensities, based on an assumption that the majority of abundances remain the same across the samples. However, especially in phosphoproteomics, this assumption can introduce bias, as the samples are enriched during sample preparation which can mask the underlying biological changes. To address this possible bias, phosphopeptides quantified in both enriched and non-enriched samples can be used to calculate factors that mitigate the bias.RESULTS: We present an R package phosphonormalizer for normalizing enriched samples in label-free mass spectrometry-based phosphoproteomics.AVAILABILITY: The phosphonormalizer package is freely-available under GPL ( > =2) license from Bioconductor ( https://bioconductor.org/packages/phosphonormalizer ).</h4

A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites

Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness. A competitive CRISPR method discovers targets and phenotypic effects of the MYC oncogene.</p

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.Peer reviewe

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.</p

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.publishedVersionPeer reviewe

PP2A methylesterase PME-1 suppresses anoikis and is associated with therapy relapse of PTEN-deficient prostate cancers

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumors with concomitant inhibition of two tumor suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having less than 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumor xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa