p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties

The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes

The potential therapeutic benefit of targeting S-phase kinase-associated protein 2 (SKP2) in neuroblastoma

PhD ThesisS-phase kinase-associated protein 2 (SKP2) is the substrate recognition subunit of the SCF E3 ubiquitin ligase complex which monitors the G1/S transition of the cell cycle. SKP2 is a positive regulator of cell cycle progression targeting tumour suppressor proteins for degradation, primarily the cyclin-dependent kinase inhibitor p27KIP1. An oncogenic protein, SKP2 is frequently overexpressed in human cancers and contributes to malignant progression. SKP2 has previously been identified as a possible MYCN target gene in neuroblastoma and based on these reports it is hypothesised that SKP2 is a potential therapeutic target in MYCN amplified disease. In this study a positive correlation between MYCN expression and SKP2 mRNA expression was shown in the SHEP-Tet21N MYCN-regulatable cell line and in a panel of MYCN amplified and non-amplified neuroblastoma cell lines. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to E-box DNA binding motifs within the SKP2 promoter, and induced transcriptional activity which was decreased by the removal of MYCN and mutation of the E-boxes. SKP2 knockdown induced cell cycle arrest and apoptosis in non-MYCN amplified neuroblastoma cell lines independent of the p53 pathway. The G1 arrest induced was rescued in-part by the knockdown of p27KIP1 confirming the importance of the SKP2/p27 axis in cell cycle progression in neuroblastoma. Structure-activity relationship analysis identified a sub-set of putative SKP2 inhibitors which inhibited growth and suppressed SKP2-mediated p27 degradation in HeLa cells. Additionally, treatment of the MYCN regulatable SHEP-Tet21N cell line with commercially available direct or indirect modulators of SKP2 activity identified a MYCN-dependent sensitivity. In conclusion these data show that SKP2 is a direct transcriptional target of MYCN and suggests that SKP2 is a potential therapeutic target in neuroblastoma.Cancer Research U

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

Cyclic AMP modulation and its effects on chemo-resistant colon cancer cell proliferation and survival

One of the major problems associated with colorectal cancer is resistance to cytotoxic chemotherapeutic agents. New strategies are therefore required to inhibit colon cancer proliferation and survival. Here I use modulators of cAMP pathways, including inhibitors of phosphodiesterase 4 (PDE4) enzymes, which are under clinical development for other disease states, to inhibit the breakdown of cAMP and to assess the effects of raising intracellular cAMP on colon cancer proliferation and survival. I found that some chemo-resistant cancer cells are addicted to keeping low cAMP in PDE4 regulated compartments, and modulation of this pool causes G1/S-phase arrest and apoptosis. I also show that PDE4 controlled cAMP negatively regulates the PI 3-Kinase/Akt pathway, which some cells are addicted to for survival. Furthermore, I investigated the expression and role of PDE4 enzymes in metastatic colon cancer cells and assessed the effects of modulating their expression on survival. Also, I used a clinically relevant analogue of forskolin, an agonist of adenylyl cyclase, to examine the general effect on growth of epithelial cancer cell lines. This work might provide new strategies for the treatment of advanced colon cancer

Myeloid Leukemia

The current book comprises a series of chapters from experts in the field of myeloid cell biology and myeloid leukemia pathogenesis. It is meant to provide reviews about current knowledge in the area of basic science of acute (AML) and chronic myeloid leukemia (CML) as well as original publications covering specific aspects of these important diseases. Covering the specifics of leukemia biology and pathogenesis by authors from different parts of the World, including America, Europe, Africa, and Asia, this book provides a colorful view on research activities in this field around the globe