Filopodia and stromal extracellular matrix as regulators of cancer cell invasion and growth

Bidirectional exchange of information between the cancer cells and their environment is essential for cancer to evolve. Cancer cells lose the ability to regulate their growth, gain the ability to detach from neighboring cells and finally some of the cells disseminate from the primary tumor and invade to the adjacent tissue. During cancer progression, cells acquire features that promote cancer motility and proliferation one of them being increased filopodia number. Filopodia are dynamic actin-rich structures extending from the leading edge of migrating cells and the main function of these structures is to serve as environmental sensors. It is nowadays widely appreciated, that not only the cancer cells, but also the surrounding of the tumor – the tumor microenvironment- contribute to cancer cell dissemination and tumor growth. Activated stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs) actively participate on tumor progression. CAFs are the most abundant cell type surrounding the cancer cells and they are the main cell type producing the extracellular matrix (ECM) within tumor stroma. CAFs secrete growth factors to promote tumor growth, direct cancer cell invasion as well as modify the stromal ECM architecture. The aim of this thesis was to investigate the function of filopodia, particularly the role of filopodia-inducing protein Myosin-X (Myo10), in breast cancer cell invasion and metastasis. We found that Myo10 is an important regulator of basal type breast cancer spreading downstream of mutant p53. In addition, I investigated the role of CAFs and their secreted matrix on tumor growth. According to the results, CAF-derived matrix has altered organization and stiffness which induces the carcinoma cell proliferation via epigenetic mechanisms. I identified histone demethylase enzyme JMJD1a to be regulated by the stiffness and to participate in stiffness induced growth control.Filopodiat ja strooman soluväliaine syöpäsolujen leviämisen ja kasvun säätelijöinä Normaalin kudoksen epiteelisolut ja sidekudos eivät ole suorassa kontaktissa, vaan niitä erottaa soluväliaineesta muodostunut tyvikalvo. Syövän muodostuessa ja sen edetessä syöpäsolut invasoivat tyvikalvon läpi sidekudokseen. Tyvikalvon alainen sidekudos sisältää pääosin sidekudossoluja (fibroblasteja) sekä niiden erittämää soluväliainetta. Soluväliaine on tärkeä solujen normaalin homeostasian säätelijä. Se säätelee solujen kiinnittymistä toisiinsa, solujen jakautumista sekä erilaistumista, eli prosesseja joiden säätely on eteenkin syövässä häiriintynyt. Syövän ympärillä on havaittu ns. syöpäfibroblasteja, joiden tiedetään erittävän solujen kasvua sekä uudisveristuonten muodostusta lisääviä kasvutekijöitä ja sitä kautta vaikuttavan aktiivisesti syövän etenemiseen. Tutkimukseni tarkoitus oli tutkia muuttuuko sidekudossolujen soluväliaineen rakenne normaalien fibroblastien aktivoituessa syöpäfibroblasteiksi. Havaitsimme, että syöpäfibroblastien tuottama soluväliaineen rakenne oli muuttunut sekä sen jäykkyys lisääntynyt. Nämä muutokset yhdessä johtivat syöpäsolujen lisääntyneeseen kasvuun syöpäfibroblastien tuottamalla soluväliaineella normaaliin soluväliaineeseen verrattuna. Havaitsimme tämän eron johtuvan epigeneettisiä muutoksia säätelevän JMJD1a-entsyymin muuntuneesta säätelystä eri soluväliaineilla. Syövän edetessä syöpäsolut leviävät lopulta veren- tai imusuonikierron kautta muualle elimistöön ja muodostavat etäpesäkkeitä. Solun sormimaissilla rakenteilla, filopodioilla, on todettu olevan tärkeä rooli etäpesäkkeiden muodostuksessa, sillä filopodioiden määrän on havaittu lisääntyvän syövässä ja vaikuttavan etenkin syöpäsolujen kykyyn liikkua. Myosin- X (Myo10) geenin tiedetään säätelevän filopodioiden muodostusta, mutta sen merkitystä syövän leviämisen säätelyssä ei ole aiemmin tutkittu. Havaitsimme, että Myo10 säätelee rintasyöpäsolujen liikkumista sekä solujen kykyä levitä ja muodostaa etäpesäkkeitä.Siirretty Doriast

Analysis of H3K4me3 and H3K27me3 bivalent promotors in HER2+ breast cancer cell lines reveals variations depending on estrogen receptor status and significantly correlates with gene expression

Background: The role of histone modifications is poorly characterized in breast cancer, especially within the major subtypes. While epigenetic modifications may enhance the adaptability of a cell to both therapy and the surrounding environment, the mechanisms by which this is accomplished remains unclear. In this study we focus on the HER2 subtype and investigate two histone trimethylations that occur on the histone 3; the trimethylation located at lysine 4 (H3K4me3) found in active promoters and the trimethylation located at lysine 27 (H3K27me3) that correlates with gene repression. A bivalency state is the result of the co-presence of these two marks at the same promoter.Methods: In this study we investigated the relationship between these histone modifications in promoter regions and their proximal gene expression in HER2+ breast cancer cell lines. In addition, we assessed these patterns with respect to the presence or absence of the estrogen receptor (ER). To do this, we utilized ChIP-seq and matching RNA-seq from publicly available data for the AU565, SKBR3, MB361 and UACC812 cell lines. In order to visualize these relationships, we used KEGG pathway enrichment analysis, and Kaplan-Meyer plots.Results: We found that the correlation between the three types of promoter trimethylation statuses (H3K4me3, H3K27me3 or both) and the expression of the proximal genes was highly significant overall, while roughly a third of all genes are regulated by this phenomenon. We also show that there are several pathways related to cancer progression and invasion that are associated with the bivalent status of the gene promoters, and that there are specific differences between ER+ and ER- HER2+ breast cancer cell lines. These specific differences that are differentially trimethylated are also shown to be differentially expressed in patient samples. One of these genes, HIF1AN, significantly correlates with patient outcome.Conclusions: This study highlights the importance of looking at epigenetic markings at a subtype specific level by characterizing the relationship between the bivalent promoters and gene expression. This provides a deeper insight into a mechanism that could lead to future targets for treatment and prognosis, along with oncogenesis and response to therapy of HER2+ breast cancer patients.</p

Placental DNA methylation marks are associated with maternal depressive symptoms during early pregnancy

Maternal depressive symptoms during pregnancy are a significant risk factor for adverse developmental and health outcomes of the offspring. The molecular mechanisms mediating the long-term effects of this exposure are not well understood. Previous studies have found association between prenatal exposure to maternal psychological distress and placental DNA methylation of candidate genes, which can influence placental barrier function and development of the fetus. Our objective in this study was to determine epigenome wide association of maternal depressive symptoms in early pregnancy with the placental DNA methylation. For this purpose we examined DNA methylomes of 92 placental samples by using reduced representation bisulfite sequencing. The placental samples were collected after deliveries of 39 girls and 59 boys, whose mothers had Edinburgh Postnatal Depression Score ranging from 0 to 19 at gestational week 14. According to our results maternal depressive symptoms are associated with DNA methylation of 2833 CpG sites, which are particularly over-represented in genic enhancers. The genes overlapping or nearest to these sites are functionally enriched for development of neurons and show expression enrichment in several regions of developing brain. The genomic regions harboring the DNA methylation marks are enriched for single nucleotide polymorphisms associated with mental disease trait class. Potential cellular signaling cascades mediating the effects include inflammatory and hormonal pathways. As a conclusion our results suggest that maternal depressive symptoms during early pregnancy are associated with DNA methylation marks in placenta in genes, which are important for the development and long-term health of the brain. Whether similar marks can be detected in exposed children remains to be elucidated in further studies.</p

Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal-not cancer-stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/ TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression.Peer reviewe

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia

Tukiliikepankki CrossFit-harjoitteluun. Opas CrossFit-harrastajille.

CrossFit on Greg Glassmanin kehittelemä korkean intensiteetin urheilulaji, jonka suosio kuntoliikkujien keskuudessa on kasvanut viime vuosina. Laji yhdistelee voimistelua, painon- ja voimanostoa sekä aerobista harjoittelua. Lajin jatkuvasti nousevan suosion vuoksi on äärimmäisen tärkeää tutkia lajiharjoittelun ja urheiluvammojen riskitekijöitä ja ennaltaehkäisykeinoja. Lajiharjoittelu on jo itsessään monipuolista, mutta sen lisäksi tarvitaan tukiliikeharjoittelua vahvistamaan kehoa monipuolisesti lajin vaatimuksia ajatellen. Puolierojen korjaaminen, kehonhallinnan kehittäminen ja lajiharjoittelussa vähemmälle jäävien lihasten vahvistaminen ovat tukiliikeharjoittelun tavoitteita. Yleisimmin lajissa loukkaantuvia anatomisia rakenteita ovat olkapää, lanneranka ja polvi. Tämän opinnäytetyön tarkoituksena oli luoda toimeksiantajan käyttöön tukiliikepankki CrossFit-harjoitteluun. Tämän opinnäytetyön tavoitteena on antaa lajin harrastajille ja valmentajille tietoa lajin loukkaantumisriskeistä, yleisimmistä vammaprofiileista sekä siitä, miten yleisimpiä CrossFit-harjoitteluun liitettyjä urheiluvammoja voidaan ehkäistä tukiliikeharjoittelun avulla. Tavoitteena on kannustaa lajin harrastajia toteuttamaan tukiliikeharjoittelua myös itsenäisesti. Työn toimeksiantajana toimi CrossFit & Lifting Varkaus Oy, jonka asiakkaille tämän opinnäytetyön produktiivisena tuotteena kehitettiin PDF-muotoinen tulostettava tai sähköisesti käytettävä tukiliikepankki. Kehittämistarve tunnistettiin aiemmin opintojen aikana tehtyjen oppimistehtävien perusteella. Ideointivaiheen lopputuloksena päädyttiin siihen, että opinnäytetyön tuloksena kehitetään kuvallinen tukiliikeopas. Luonnosteluvaiheeseen sisältyi laaja tiedonhakuprosessi, jossa käytettiin teoriapohjana oppaalle mahdollisimman tuoretta tutkimustietoa. Oppaan kehittelyssä on otettu huomioon hyvän oppaan kriteerit sekä se, että opas vastaa sisällöllisesti ja visuaalisesti toimeksiantajaa. Viimeistelyvaiheessa oppaasta pyydettiin palautetta sen tulevilta käyttäjiltä, ja opasta viimeisteltiin palautteiden perusteella olemassa olevien resurssien puitteissa. Tukiliikeopas sisältää johdannon ja teoriaosuuden tukiliikeharjoittelusta, sekä kirjalliset ja kuvalliset ohjeet tukiliikeharjoitteluun sopivista liikkeistä

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PtdIns(3,4,5)P3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia-inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas

Analysis of H3K4me3 and H3K27me3 bivalent promotors in HER2+ breast cancer cell lines reveals variations depending on estrogen receptor status and significantly correlates with gene expression.

Background: The role of histone modifications is poorly characterized in breast cancer, especially within the major subtypes. While epigenetic modifications may enhance the adaptability of a cell to both therapy and the surrounding environment, the mechanisms by which this is accomplished remains unclear. In this study we focus on the HER2 subtype and investigate two histone trimethylations that occur on the histone 3; the trimethylation located at lysine 4 (H3K4me3) found in active promoters and the trimethylation located at lysine 27 (H3K27me3) that correlates with gene repression. A bivalency state is the result of the co-presence of these two marks at the same promoter.Methods: In this study we investigated the relationship between these histone modifications in promoter regions and their proximal gene expression in HER2+ breast cancer cell lines. In addition, we assessed these patterns with respect to the presence or absence of the estrogen receptor (ER). To do this, we utilized ChIP-seq and matching RNA-seq from publicly available data for the AU565, SKBR3, MB361 and UACC812 cell lines. In order to visualize these relationships, we used KEGG pathway enrichment analysis, and Kaplan-Meyer plots.Results: We found that the correlation between the three types of promoter trimethylation statuses (H3K4me3, H3K27me3 or both) and the expression of the proximal genes was highly significant overall, while roughly a third of all genes are regulated by this phenomenon. We also show that there are several pathways related to cancer progression and invasion that are associated with the bivalent status of the gene promoters, and that there are specific differences between ER+ and ER- HER2+ breast cancer cell lines. These specific differences that are differentially trimethylated are also shown to be differentially expressed in patient samples. One of these genes, HIF1AN, significantly correlates with patient outcome.Conclusions: This study highlights the importance of looking at epigenetic markings at a subtype specific level by characterizing the relationship between the bivalent promoters and gene expression. This provides a deeper insight into a mechanism that could lead to future targets for treatment and prognosis, along with oncogenesis and response to therapy of HER2+ breast cancer patients.</p

The Regulation and Role of c-FLIP in Human Th Cell Differentiation

<div><p>The early differentiation of T helper (Th) cells is a tightly controlled and finely balanced process, which involves several factors including cytokines, transcription factors and co-stimulatory molecules. Recent studies have shown that in addition to the regulation of apoptosis, caspase activity is also needed for Th cell proliferation and activation and it might play a role in Th cell differentiation. The isoforms of the cellular FLICE inhibitory protein (c-FLIP) are regulators of CASPASE-8 activity and the short isoform, c-FLIP_S, has been shown to be up-regulated by IL-4, the Th2 driving cytokine. In this work, we have studied the expression and functional role of three c-FLIP isoforms during the early Th cell differentiation. Only two of the isoforms, c-FLIP_S and c-FLIP_L, were detected at the protein level although c-FLIP_R was expressed at the mRNA level. The knockdown of c-FLIP_L led to enhanced Th1 differentiation and elevated IL-4 production by Th2 cells, whereas the knockdown of c-FLIP_S diminished GATA3 expression and IL-4 production by Th2 cells. In summary, our results provide new insight into the role of c-FLIP proteins in the early differentiation of human Th cells.</p></div

Integrin endosomal signalling suppresses anoikis

Integrin containing focal adhesions (FAs) transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localises with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 (EEA1) and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage-independence and metastasis. Integrins are heterodimeric cell surface adhesion receptors functioning as integrators of the extra-cellular matrix (ECM) driven cues, the cellular cytoskeleton and the cellular signalling apparatus 1.Upon adhesion, integrins trigger the formation of plasma-membrane proximal large mechanosensing and signal-transmitting protein clusters depicted as “adhesomes” 2, 3. In addition, integrins undergo constant endocytic traffic to facilitate focal adhesion turnover, cell migration, invasion and cytokinesis 4. For other receptor systems it is well established that endocytic membrane traffic regulates bioavailability of cell-surface molecules and therefore the intensity and/or specificity of receptor-initiated signals 5, 6. Although active integrins and their ligands have been detected in endosomes 7–9 and increased integrin recycling to the plasma membrane contributes to enhanced signalling of co-trafficked receptor tyrosine kinases10, 11 it has remained unclear whether endocytosed active integrins signal in endosomes. Here, we demonstrate that integrin signalling is not restricted to focal adhesions as previously described and that endocytosis is necessary for full ECM-induced, integrin mediated ERK, AKT and FAK signalling. We find that FAK binds directly to and can become activated on purified endosomes. Moreover, the FERM-domain of FAK is able to bind purified integrin containing endosomes, suggesting the potential for integrin signalling complexes to assemble on endosomes after internalization of active integrins. Importantly, FAK is required for anchorage-independent growth and suppression of anoikis 12. Integrin endosomal signalling correlates with reduced anoikis sensitivity in normal cells and anchorage-independent growth and metastasis in breast cancer cells