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The breast milk composition includes a multitude of bioactive factors such as viable cells, lipids and proteins. Measuring the levels of specific proteins in breast milk plasma can be challenging because of the large dynamic range of protein concentrations and the presence of interfering substances. Therefore, most proteomic studies of breast milk have been able to identify under 1000 proteins. Optimised procedures and the latest separation technologies used in milk proteome research could lead to more precise knowledge of breast milk proteome. This study (n = 53) utilizes three different protein quantification methods, including direct DIA, library-based DIA method and a hybrid method combining direct DIA and library-based DIA. On average we identified 2400 proteins by hybrid method. By applying these methods, we quantified body mass index (BMI) associated variation in breast milk proteomes. There were 210 significantly different proteins when comparing the breast milk proteome of obese and overweight mothers. In addition, we analysed a small cohort (n = 5, randomly selected from 53 samples) by high field asymmetric waveform ion mobility spectrometry (FAIMS). FAIMS coupled with the Orbitrap Fusion Lumos mass spectrometer, which led to 41.7% higher number of protein identifications compared to Q Exactive HF mass spectrometer., (© 2024 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)

Mitochondrial glycolysis and hyperactivity of the phosphatidylinositol 3-kinase-protein kinase B (AKT) pathway are hallmarks of malignant brain tumors. However, kinase inhibitors targeting AKT (AKTi) or the glycolysis master regulator pyruvate dehydrogenase kinase (PDKi) have failed to provide clinical benefits for brain tumor patients. Here, we demonstrate that heterogeneous glioblastoma (GB) and medulloblastoma (MB) cell lines display only cytostatic responses to combined AKT and PDK targeting. Biochemically, the combined AKT and PDK inhibition resulted in the shutdown of both target pathways and priming to mitochondrial apoptosis but failed to induce apoptosis. In contrast, all tested brain tumor cell models were sensitive to a triplet therapy, in which AKT and PDK inhibition was combined with the pharmacological reactivation of protein phosphatase 2A (PP2A) by NZ-8-061 (also known as DT-061), DBK-1154, and DBK-1160. We also provide proof-of-principle evidence for in vivo efficacy in the intracranial GB and MB models by the brain-penetrant triplet therapy (AKTi + PDKi + PP2A reactivator). Mechanistically, PP2A reactivation converted the cytostatic AKTi + PDKi response to cytotoxic apoptosis, through PP2A-elicited shutdown of compensatory mitochondrial oxidative phosphorylation and by increased proton leakage. These results encourage the development of triple-strike strategies targeting mitochondrial metabolism to overcome therapy tolerance in brain tumors., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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 tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 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 tumour 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., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

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.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically., Competing Interests: Declaration of interests The Johnstone laboratory receives funding support from Roche, BMS, Astra Zeneca, and MecRx. R.W.J. is a shareholder in MecRx and receives consultancy payments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies., (© 2020 Kauko et al.)

Disease relapse from standard chemotherapy in acute myeloid leukemia (AML) is poorly understood. The importance of protein phosphatase 2A (PP2A) as an AML tumor suppressor is emerging. Therefore, here, we examined the potential role of endogenous PP2A inhibitor proteins as biomarkers predicting AML relapse in a standard patient population by using three independent patient materials: cohort1 ( n = 80), cohort2 ( n = 48) and The Cancer Genome Atlas Acute Myeloid Leukemia (TCGA LAML) dataset ( n = 160). Out of the examined PP2A inhibitors (CIP2A, SET, PME1, ARPP19 and TIPRL), expression of ARPP19 mRNA was found to be independent of the current AML risk classification. Functionally, ARPP19 promoted AML cell viability and expression of oncoproteins MYC, CDK1, and CIP2A. Clinically, ARPP19 mRNA expression was significantly lower at diagnosis ( p = 0.035) in patients whose disease did not relapse after standard chemotherapy. ARPP19 was an independent predictor for relapse both in univariable ( p = 0.007) and in multivariable analyses ( p = 0.0001) and gave additive information to EVI1 expression and risk group status (additive effect, p = 0.005). Low ARPP19 expression was also associated with better patient outcome in the TCGA LAML cohort ( p = 0.019). In addition, in matched patient samples from diagnosis, remission and relapse phases, ARPP19 expression was associated with disease activity ( p = 0.034), indicating its potential usefulness as a minimal residual disease (MRD) marker. Together, these data demonstrate the oncogenic function of ARPP19 in AML and its risk group independent role in predicting AML patient relapse tendency.

Kinase inhibitor resistance constitutes a major unresolved clinical challenge in cancer. Furthermore, the role of serine/threonine phosphatase deregulation as a potential cause for resistance to kinase inhibitors has not been thoroughly addressed. We characterize protein phosphatase 2A (PP2A) activity as a global determinant of KRAS-mutant lung cancer cell resistance across a library of >200 kinase inhibitors. The results show that PP2A activity modulation alters cancer cell sensitivities to a large number of kinase inhibitors. Specifically, PP2A inhibition ablated mitogen-activated protein kinase kinase (MEK) inhibitor response through the collateral activation of AKT/mammalian target of rapamycin (mTOR) signaling. Combination of mTOR and MEK inhibitors induced cytotoxicity in PP2A-inhibited cells, but even this drug combination could not abrogate MYC up-regulation in PP2A-inhibited cells. Treatment with an orally bioavailable small-molecule activator of PP2A DT-061, in combination with the MEK inhibitor AZD6244, resulted in suppression of both p-AKT and MYC, as well as tumor regression in two KRAS-driven lung cancer mouse models. DT-061 therapy also abrogated MYC-driven tumorigenesis. These data demonstrate that PP2A deregulation drives MEK inhibitor resistance in KRAS-mutant cells. These results emphasize the need for better understanding of phosphatases as key modulators of cancer therapy responses., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Propagation of transient signals requires coordinated suppression of antagonistic phosphatase activity. Protein phosphatase 2A (PP2A) is a broad specificity serine/threonine phosphatase that functions as an antagonist of many signaling pathways associated with growth and proliferation, and endogenous inhibitory mechanisms suppress PP2A activity in response to mitogenic stimuli. These inhibitory mechanisms, including expression and activation of endogenous inhibitor proteins and phosphoregulation of PP2A subunits, are also engaged by aberrant constitutive activation of mitogenic pathways in cancer. Inhibition of PP2A activity has been shown to promote malignant transformation and endogenous inhibitory mechanisms of PP2A have been associated with malignant progression and prognosis in a wide range of cancers. Despite existence of recurrent mutations and other genetic and gene regulatory alterationsin PP2A genes, they collectively appear at relatively low frequency, and in only some cancer types. The non-genomic inhibition of PP2A activity by increased expression of endogenous PP2A inhibitor proteins greatly exceeds the frequency of genetic mutations of PP2A genes in human cancers. This feature makes PP2A an untypical tumor suppressor, and may have influenced its recognition as one of the critical human cell transformation mechanisms. We propose that non-genetic inhibition is the dominant mechanism causing loss of PP2A tumor suppressor function in cancer cells, possibly because these mechanisms do not elicit genomic instability associated with genetic loss of function of specific PP2A subunits., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 and Implementation: The phosphonormalizer package is freely available under GPL ( > =2) license from Bioconductor (https://bioconductor.org/packages/phosphonormalizer)., Contact: sohrab.saraei@utu.fi or laura.elo@utu.fi., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Ribosome biogenesis regulates animal growth and is controlled by nutrient-responsive mTOR signaling. How ribosome biogenesis is regulated during the developmental growth of animals and how nutrient-responsive signaling adjusts ribosome biogenesis in this setting have remained insufficiently understood. We uncover PWP1 as a chromatin-associated regulator of developmental growth with a conserved role in RNA polymerase I (Pol I)-mediated rRNA transcription. We further observed that PWP1 epigenetically maintains the rDNA loci in a transcription-competent state. PWP1 responds to nutrition in Drosophila larvae via mTOR signaling through gene expression and phosphorylation, which controls the nucleolar localization of dPWP1. Our data further imply that dPWP1 acts synergistically with mTOR signaling to regulate the nucleolar localization of TFIIH, a known elongation factor of Pol I. Ribosome biogenesis is often deregulated in cancer, and we demonstrate that high PWP1 levels in human head and neck squamous cell carcinoma tumors are associated with poor prognosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two-hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N-terminal CIP2A fragment (amino acids 1-560) at 3.0 Å resolution, and by subsequent structure-based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N-terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure-function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy., (© 2017 The Authors.)

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.

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy., (©2013 AACR)

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., (Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.

Objectives: To develop a quantitative, internally standardized real-time RT-PCR assay for prostate cancer antigen 3 (PCA3), a non-translated gene found to be prostate-specific and highly overexpressed in cancer, and examine the role of PCA3 in peripheral blood with a small sample cohort., Design and Methods: The RT-PCR assay for PCA3 is based on target-specific lanthanide probes. Peripheral blood from 91 prostatic cancer/disorder patients and healthy controls was assayed for PCA3 and prostate-specific antigen (PSA) expression., Results: The dynamic range of the assay reaches over eight orders of magnitude and the limit of quantification is 800 copies per milliliter blood. Peripheral blood from 2/9 patients with metastasized cancers were PCA3 positive, whereas all the other samples were negative. Eight samples were PSA positive., Conclusions: The degree of PCA3 positivity in circulating cells from prostate cancer patients is low compared to that of PSA. In contrast to some previous reports, we found no PCA3 expression in healthy individuals.