A Computational Study to Identify TP53 and SREBF2 as Regulation Mediators of miR-214 in Melanoma Progression

In the complex world of post-transcriptional regulation, miR-214 is known to control in vitro tumor cell move- ment and survival to anoikis, as well as in vivo malignant cell extravasation from blood vessels and lung metastasis formation. miR-214 has also been found to be highly expressed in human melanomas, and to directly and indirectly regulate several genes involved in tumor progression and in the establishment of dis- tant metastases (Penna et al., 2011). In this work, we exploit a computational pipeline integrating data from multiple online data repositories to identify the presence of transcriptional or post-transcriptional regulatory modules involving miR-214 and a set of 73 previously identified miR-214 regulated genes. We identified 27 putative regulatory modules involving miR-214, NFKB1, SREBPF2, miR-33a and 9 out of the 73 miR-214 modulated genes (ALCAM, POSTN, TFAP2A, ADAM9, NCAM1, SEMA3A, PVRL2, JAG1, EGFR1). As a pre- liminary experimental validation we focused on 9 out of the 27 identified regulatory modules that involve two main players, miR-33a and SREBF2. The results confirm the importance of the predictions obtained with the presented computational approach

CyTRANSFINDER: a Cytoscape 3.3 plugin for three-component (TF, gene, miRNA) signal transduction pathway construction

Background: Biological research increasingly relies on network models to study complex phenomena. Signal Transduction Pathways are molecular circuits that model how cells receive, process, and respond to information from the environment providing snapshots of the overall cell dynamics. Most of the attempts to reconstruct signal transduction pathways are limited to single regulator networks including only genes/proteins. However, networks involving a single type of regulator and neglecting transcriptional and post-transcriptional regulations mediated by transcription factors and microRNAs, respectively, may not fully reveal the complex regulatory mechanisms of a cell. We observed a lack of computational instruments supporting explorative analysis on this type of three-component signal transduction pathways. Results: We have developed CyTRANSFINDER, a new Cytoscape plugin able to infer three-component signal transduction pathways based on user defined regulatory patterns and including miRNAs, TFs and genes. Since CyTRANSFINDER has been designed to support exploratory analysis, it does not rely on expression data. To show the potential of the plugin we have applied it in a study of two miRNAs that are particularly relevant in human melanoma progression, miR-146a and miR-214. Conclusions: CyTRANSFINDERsupportsthereconstructionofsmallsignaltransductionpathwaysamonggroupsof genes. Results obtained from its use in a real case study have been analyzed and validated through both literature data and preliminary wet-lab experiments, showing the potential of this tool when performing exploratory analysi

Molecular prognostic and predictive factors of breast cancer

Despite advances in the early detection and treatment of breast cancer, it remains a challenge to identify which patients may experience a poor prognosis or respond poorly to treatment. Familial predisposition is a major risk factor of breast cancer and, perhaps, a modifier of patients’ survival. There is also evidence suggesting that hereditary factors may impact a patient’s response to treatment. However, the magnitude of the effect, and the molecular mechanism behind it, is largely unknown. In Finland, over 4,000 women on average are annually diagnosed with breast cancer and the majority of them undergo chemotherapy; they may or may not respond to the treatment. It is challenging to identify germline markers and tumor molecular profiles, which objectively predict prognosis and treatment response, and translate this information into cancer therapy. The aim of this thesis was to identify prognostic and predictive markers in breast cancer by investigating cancer-related networks as well as candidate genes of regulatory networks in invasive breast cancer cases. Taking both a network analysis approach as well as a candidate gene study, clinicopathological and survival association analyses were performed to (I) study the association of germline variations in TP53 network genes with breast cancer patients’ survival and treatment outcome; (II) investigate the impact of two-SNP interaction of NF-κB signaling network on predicting patients’ survival; (III) evaluate the association of NQO1 protein expression and NF-κB activation with clinicopathological features of the tumors, patients’ survival, and treatment outcome; and (IV) study the role of the miR-30 family in breast cancer patients’ survival and drug response. The germline variations were studied in collaboration with the Breast Cancer Association Consortium (BCAC). In Study I, the variations were initially analyzed in a set of DNA samples from 925 invasive breast cancer cases from Helsinki Breast Cancer Study (HEBCS) included in BCAC, and were further analyzed in pooled data of 4,701 cases from four independent studies (including HEBCS) contributing to BCAC. In Study II, the germline variations were studied in extensive pooled data of 30,431 cases from 24 independent studies participating in BCAC. In Studies III and IV, the tumor samples for immunohistochemical and miRNA in situ hybridization of 1,240 cases were from two series of 884 unselected Finnish invasive breast cancer patients and an additional 542 familial cases. Gene expression analysis was performed using microarray data of total RNA from 187 fresh frozen primary breast cancer tumors. Drug sensitivity screening tested the influence of miR-30 family members on the response of human breast cancer cell lines to two drugs, doxorubicin and lapatinib. In Study I, a significant interaction effect was found between germline variations in TP53-related genes, PRKAG2 (rs4726050) and MDM2 SNP309, with PRKAG2 (rs4726050) rare G allele showing a dose-dependent impact for superior breast cancer survival only among the MDM2 SNP309 rare G allele carriers. Also, PPP2R2B (rs10477313) rare A allele predicted increased survival after hormonal therapy. Further studies are warranted to clarify the impact of PRKAG2 and PPP2R2B on patients’ survival. In Study II, the SNP-SNP interaction test in the NF-κB activating pathway found two interacting SNP pairs, rs5996080-rs7973914 and rs17243893-rs57890595, which was associated with patients’ survival under recessive and dominant models of inheritance, respectively. While rs5996080 and rs7973914 were included in the study for representing the haplotype block harboring NF-κB activating genes, BAFFR and TNFR1/3, they physically reside in SREBF2 and SCNN1A, thus, the interacting effect found between these two loci may represent either of the genes. The dominant SNP pair, rs17243893 and rs57890595, represented TRAF2 and TRAIL-R4. Based on the published function of the interacting genes, and the in silico analysis of this study, the survival association of the identified SNP pairs may be a result of interplay between these gene pairs and their downstream influence on the dynamic of canonical and non-canonical NF-κB pathways. In Study III, the immunohistochemical staining analysis of NQO1 expression and NF-κB nuclear localization (inferred activity) did not find significant association between either of the proteins and patients’ survival or treatment outcome. However, an inverse correlation between NQO1 expression and NF-κB activity was observed in breast cancer tumors. The NQO1/NF-κB inverse correlation was also reflected in their association with ER status, as well as their correlation with gene expression. In Study IV, a significant association was found between the high expression of miR-30d and longer metastasis-free survival, particularly in subgroups of patients with high proliferative tumors, ER negativity, HER2 positivity, and among those who received chemotherapy. However, the high expression of miR-30 appeared to also correlate with the characteristics of aggressive tumors, i.e. higher grade, positive nodal status, and high proliferation (estimated by high Ki67). In a drug sensitivity screening test of all miR-30 family members, miR-30a–e sensitized the human breast cancer cell lines to doxorubicin. Also, in the HER2-positive HCC1954 cell line, miR-30d sensitized the cells to lapatinib. The pathway enrichment analysis of miR-30 family members in the METABRIC gene expression dataset revealed that high levels of miR-30 family occurred simultaneously with low expressions of genes involved in cell movements, consistent with the observed association with longer metastasis-free survival. The result of this work suggests prognostic/predictive potentials for candidate genes in cancer-related networks (TP53 and NF-κB), as well as regulatory networks (microRNAs), which warrant further investigations.Suomessa rintasyöpä diagnosoidaan keskimäärin yli 4000 naisella vuosittain ja suurin osa heistä saa kemoterapiaa; he saattavat reagoida hoitoon tai eivät. On haastavaa löytää geneettinen merkki, joka ennustaisi ennusteen sekä hoitovasteen. Yhtä haastavaa on soveltaa näitä tietoja syöpähoidossa. Tämän väitöskirjan tarkoituksena oli löytää uusia rintasyövän geneettisiä markkereita tutkimalla syöpään liittyviä geenejä kliinisellä, patologisella ja potilaiden eloonjääntianalyysillä. Tässä opinnäytetyössä käytetty aineisto sisälsi 1240 kasvainnäytettä Suomesta ja 30 431 DNA-näytettä Breast Cancer Association Consortiumilta (BCAC). Merkittävä vuorovaikutus havaittiin TP53: een liittyvien geenien, PRKAG2:n (rs4726050) ja MDM2 SNP309, mikä liittyi potilaan parempaan eloonjäämiseen. PPP2R2B (rs10477313) ennusti myös pidemmän eloonjäämisen hormonaalisen hoidon jälkeen. Lisätutkimuksia tarvitaan tutkimaan PRKAG2:n ja PPP2R2B:n vaikutusta potilaan eloonjäämiseen. NFkB-pathway tutkimuksessa SNP-SNP-vuorovaikutustesti löysi kaksi SNP-paria, rs5996080-rs7973914 ja rs17243893-rs57890595, jotka liittyivät potilaiden eloonjäämiseen. Nämä SNP: t osoittavat BAFFR: n, TNFR1 / 3: n, SREBF2: n, SCNN1A: n, TRAF2: n tai TRAIL-R4: n mahdollisen yhteyden potilaan eloonjäämiseen. Tunnistettujen SNP-parien selviytymisyhteys voi johtua näiden geeniparien välisestä vuorovaikutuksesta ja niiden alavirran vaikutuksesta kanonisten ja ei-kanonisten NF-KB-reittien dynamiikkaan. NQO1- ja NF-KB-ilmentymisen immunohistokemiallinen analyysi ei löytänyt merkittävää yhteyttä näiden proteiinien ilmentymisen ja potilaan eloonjäämisen tai hoidon lopputuloksen välillä. Rintasyöpäkasvaimissa havaittiin kuitenkin käänteinen korrelaatio NQO1-ilmentymisen ja NF-KB-aktiivisuuden välillä. miR-30d:n ilmeneminen assosioitui potilaiden parempaan eloonjäämiseen, erityisesti sellaisten potilaiden alaryhmissä, joilla on korkea Ki67, ER-negatiivisuus, HER2-positiivisuus, ja kemoterapiaa saaneiden potilaiden joukossa. MiR-30a-e herkistää myös ihmisen rintasyövän solulinjat doksorubisiinille ja lapatinibille. Tämän työn tulos viittaa ennustepotentiaaliin geeneihin syöpään liittyvissä verkoissa (TP53 ja NF-KB) sekä säätelyverkoissa (mikroRNA: t). Näiden tulosten vahvistamiseksi tarvitaan lisää tutkimuksia

Role of miRNAs in Cancer

MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome

The role of SREBP1 in hormone-dependent breast cancer

Despite significant progress in diagnostics and treatments, such as targeted endocrine therapy, 30% of patients with hormone-dependent breast cancer eventually develop disease recurrence predominantly due to drug resistance. Resistance to hormone deprivation therapy is multifactorial and involves several molecular events. An increasing body of research has identified an emerging hallmark of cancer describing the capability of modifying and reprogramming cellular metabolism in order to fuel neoplastic proliferation. Our group has previously uncovered how the specific type of treatment plays a significant role in this process. In particular, breast cancer (BCa) cells developing resistance to aromatase inhibitors (AI) endogenously trigger cholesterol biosynthesis (CB) through sterol regulatory element binding protein 1 (SREBP1) regulation leading to a sustained oestrogen independent, oestrogen receptor alpha (ERα) activation. Cellular lipid metabolism is controlled by SREBP1. The altered lipid metabolism, also known as “lipogenic phenotype”, has been linked with prostate cancer (PCa) pathogenesis: the expression of SREBP1 in prostate cancer is strongly correlated with Gleason grade (pathological grade) and its overexpression is sufficient to increase tumorigenicity and invasion of prostate cancer cells. Furthermore, de novo lipid biosynthesis has been associated with cancer progression, poorer prognosis and shorter patient survival. Considering what is known about the pathobiology of lipids in cancer, it is plausible that invading cells evolve mechanisms to bypass the tight homeostatic regulation of intracellular cholesterol adapting to their new environmental conditions. With this idea in mind, we sought to identify the molecular mechanisms of activation of SREBP1 as key regulator of de novo cholesterol biosynthesis in hormone-dependent cancers resistant to endocrine therapy. Firstly, we wanted to investigate SREBP1 regulation in hormone-dependent cancer cells. We found that SREBP1-driven lipogenesis is consistently upregulated after long-term steroid deprivation, thus when cells become hormone independent. In vivo immunohistochemistry (IHC) data support the hypothesis that SREBP1 might be pivotal in driving de novo cholesterol biosynthesis in endocrine therapy resistant BCa cells. Moreover, the switch of metabolic dependency upon resistance development identified by metabolic profiling, is associated with increased de novo cholesterol and fatty acid synthesis. In order to examine whether cholesterol biosynthesis may be upregulated by modulating SREBP1 signalling, we investigated SREBP1 recruitment to the chromatin. Optimization of ChIP protocol allowed for a genome-wide profiling of SREBP1 binding in BCa and PCa cancer cell lines. Downstream analysis showed a difference in SREBP1 recruitment between parental and long-term starved derived cell lines in MCF7. Furthermore, SREBP1 binding profiles distinguished cancer cells based on the tissue of origin (breast versus prostate cancer). Our data also confirmed a significant co-occurrence between AR and SREBP1 binding sites in PCa and suggested a possible crosstalk between SREBP1 and the ERα in BCa on chromatin. Thirdly, we asked what the targets of SREBP1 are and if they can promote invasive potential. ChIP-seq differential binding analysis unexpectedly revealed non-canonical targets for SREBP1. In particular, we showed that cells acquiring resistance to AI undergo active cytoskeleton re-organisation via Keratin 80 (KRT80) and actin remodelling. This process is driven by epigenetic reprogramming at the type II keratin locus dependent on de novo SREBP1 binding to a single enhancer that is activated upon chronic AI treatment and leading to KRT80 upregulation. Our data strongly suggest that therapy plays a direct role in shaping the biophysical properties and invasive potential of breast cancer cells, by inducing epigenetic rearrangements leading to KRT80 upregulation and concomitant cytoskeletal reorganization. In summary, our study investigates the role of SREBP1 as a key player in endogenous cholesterol accumulation and autonomous activation of the nuclear receptor signalling, leading to the hormone independent tumour proliferation and invasion via global cytoskeletal re-arrangements in hormone-dependent cancers.Open Acces

Ceramide synthase 4: a novel metabolic regulator of oncogene-induced senescence

Senescence is a cell stress program characterized by a stable cell cycle arrest and thus aims to protect against replication of potentially harmful cells. In oncogene-induced senescence (OIS) the cell cycle arrest is brought about by activation of an oncogene. This in turn initiates a DNA damage response and subsequently, the DDR induces p53-p21 and RB tumour suppressor pathways. The metabolism of senescent cells is highly altered, notably there is increased secretion of proteins and increased functional activity of certain metabolic enzymes. There have been many recent studies investigating the role of specific metabolic pathways in OIS and how they may be targeted for therapeutic benefit. This thesis aims to identify novel metabolic regulators of OIS, by combining high throughput RNAi screening and LC-MS based methods. This thesis has identified and validated 17 essential OIS metabolic genes; in this list, there was enrichment for genes involved in lipid biosynthetic processes. Lipid metabolism was an attractive focus for this thesis as it has not been extensively studied in current literature. Next, ceramide synthase 4 (CERS4) was extensively validated as a key enzyme for both OIS and replicative senescence. Using LC-MS based lipidomics, CERS4-driven rewiring of lipid metabolism in OIS was revealed and this corresponded with an accumulation of ceramides due to increased de novo ceramide synthesis. It was then confirmed OIS-related ceramide is mechanistically linked to cell cycle via the PP1-RB-E2F axis. Ceramide activates PP1, which physically binds to RB in a CERS4-dependent manner. PP1 is then able to dephosphorylate and activate RB, which inhibits transcription of E2F targets (cell cycle genes). Overall, this thesis identifies a metabolic checkpoint that links altered lipid metabolism with OIS