TRPV Protein Family—From Mechanosensing to Cancer Invasion

Biophysical cues from the cellular microenvironment are detected by mechanosensitive machineries that translate physical signals into biochemical signaling cascades. At the crossroads of extracellular space and cell interior are located several ion channel families, including TRP family proteins, that are triggered by mechanical stimuli and drive intracellular signaling pathways through spatio-temporally controlled Ca2+-influx. Mechanosensitive Ca2+-channels, therefore, act as critical components in the rapid transmission of physical signals into biologically compatible information to impact crucial processes during development, morphogenesis and regeneration. Given the mechanosensitive nature of many of the TRP family channels, they must also respond to the biophysical changes along the development of several pathophysiological conditions and have also been linked to cancer progression. In this review, we will focus on the TRPV, vanilloid family of TRP proteins, and their connection to cancer progression through their mechanosensitive nature

TRPV Protein Family—From Mechanosensing to Cancer Invasion

Biophysical cues from the cellular microenvironment are detected by mechanosensitive machineries that translate physical signals into biochemical signaling cascades. At the crossroads of extracellular space and cell interior are located several ion channel families, including TRP family proteins, that are triggered by mechanical stimuli and drive intracellular signaling pathways through spatio-temporally controlled Ca2+-influx. Mechanosensitive Ca2+-channels, therefore, act as critical components in the rapid transmission of physical signals into biologically compatible information to impact crucial processes during development, morphogenesis and regeneration. Given the mechanosensitive nature of many of the TRP family channels, they must also respond to the biophysical changes along the development of several pathophysiological conditions and have also been linked to cancer progression. In this review, we will focus on the TRPV, vanilloid family of TRP proteins, and their connection to cancer progression through their mechanosensitive nature

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.Noin joka neljäs suomalainen sairastuu elämänsä aikana syöpään. Syövän syntyyn vaikuttavat sekä perinnölliset seikat että altistuminen elintapojemme ja -ympäristön vaikutuksesta monenlaisille haitallisille tekijöille. Nämä haitalliset tekijät, kuten auringon UV-säteily, voivat aiheuttaa perimäainekseemme, DNA:han, mutaatioita. Soluissa olevat korjausmekanismit huolehtivat useimmiten näiden vaurioiden korjauksesta. Joskus kuitenkin nämä solun korjausmekanismit pettävät ja perimäainekseemme jää DNA-vaurioita solun jakautuessa. Kun nämä vauriot muuttuvat pysyviksi mutaatioiksi ne voivat pahimmassa tapauksessa johtaa syövän syntyyn, jos ne sijaitsevat esimerkiksi solun jakautumista kontrolloivissa geeneissä. Syövän kehitys on kuitenkin pitkän ajan prosessi ja se vaatii useita mutaatioita syntyäkseen. Yksi keskeisimmistä syövän synnyltä suojaavista tekijöistä soluissamme on kasvunrajoiteproteiini p53. p53:n merkityksestä syövän ehkäisyssä kertoo osaltaan se että sen toiminta on estynyt yli 50 %:ssa syövistä. p53-geenin tuottaman proteiinin syövältä suojaava vaikutus perustuu sen kykyyn aktivoitua monissa solun stressitilanteissa ja avustaa virheiden korjauksessa. p53 voi pysäyttää solusyklin etenemisen ja antaa korjausmekanismeille aikaa poistaa perimäaineksen vauriot, jotteivat ne siirry myöhemmin solun jakautuessa tytärsoluihin. p53 itse myös osallistuu näihin korjaustapahtumiin. Jos solussa olevat vauriot ovat liian suuret korjattavaksi, voi p53 aiheuttaa ohjelmoidun solukuoleman, apoptoosin, haitallisten solujen poistamiseksi. p53:n aktiivisuutta soluissa säätelee sen hajotukseen osallistuva proteiini, Mdm2. Vaikka p53:n aktivoitumista solun stressitilanteessa on tutkittu jo vuosia, ei kaikkia siihen osallistuvia tekijöitä ja yksityiskohtia tunneta vielä tarkoin. Useimmat nykyisin käytössä olevat syövän kemoterapeuttiset hoidot johtavat toiminnalliseen p53:een ja hoitojen vaste on usein myös riippuvaista p53:n toimintakyvystä. Koska p53:n merkitys syöpähoitojen ja syövän hoidon vasteen kannalta on siis huomattava, on tärkeää tuntea sen aktivoitumiseen johtavat tapahtumat yksityiskohtaisesti. Tämän tutkimuksen tavoitteena on ollut selvittää tapahtumia, jotka saavat aikaan p53 vasteen UV-säteilytyksen seurauksena. UV-säteilytyksellä tiedetään olevan samankaltaisia vaikutuksia solujen toimintoihin kuin monilla kemoterapeuttisilla aineilla. Tavoitteena on ollut löytää p53:n aktiivisuuteen vaikuttavia proteiineja DNA-vaurion seuraksena. Tutkimuksessa on löydetty kaksi uutta p53:een ja sen negatiiviseen säätelijään, Mdm2:een sitoutuvaa proteiinia. Näiden proteiinien, Promyelosyyttisen leukemia proteiinin (PML) ja nukleofosmiinin (NPM) havaittiin sitoutuvan sekä toisiinsa että p53:een ja Mdm2:een nopeasti UV-säteilytetyissä soluissa. Nämä havaitut proteiinikompleksit todennäköisesti osallistuvat p53:n välittämään stressivasteeseen solujen vaurioiden poistamiseksi. Lisäksi havaittiin interaktioiden liittyvän solun sisäisiin lokalisaatioiden muutoksiin, joilla voi myös olla olennainen merkitys p53:n stressivasteen kannalta. Tutkimuksessa tarkasteltiin myös samojen proteiinien toimintaa eräässä leukemiatyypin solulinjassa. Kyseisessä solulinjassa PML geeni on yhdistynyt solujen erilaistumiseen vaikuttavaan RARα geeniin muodostaen fuusioproteiinin, joka estää PML:n ja RARα:n geenituotteiden normaalin toiminnan ja aiheuttaa leukemian kehittymisen. Tämän fuusioproteiinin havaittiin muuttavan NPM-proteiinin sijaintia solun tumajyväsistä tuman nukleoplasmaan, häiriten todennäköisesti sen normaaleja toimintoja solussa. Lisäksi näiden solujen käsittely terapeuttisesti merkittävällä aineella, retinoihapolla (RA), joka johtaa leukemiasolujen fenotyypin normalisoitumiseen, sai aikaan vastaavan proteiinikompleksin muodostumisen kuin UV-vauriosta palautuvissa soluissa. RA-käsittely johti myös p53:n aktivoitumiseen. Tutkimus antaa uuttaa tietoja kasvunrajoiteproteiini p53:n toimintaan vaikuttavista tekijöistä solujen UV-vaurion jälkeen. Lisäksi tutkimus antaa mekanistista tietoa retinoihapon vaikutuksesta tutkitun leukemiatyypin soluissa

TRPV Family Ion Channels in the Mammary Epithelium: Role in Normal Tissue Homeostasis and along Breast Cancer Progression

Calcium homeostasis directs various intracellular cascades and therefore strict spatio-temporal control of calcium influx is also crucial for diverse physiological processes. In the mammary gland, calcium is important for the specialized tasks of this organ during lactation, but it also guides other structural and functional features of the mammary epithelium and in this way the maintenance of the whole tissue. Transient receptor potential, TRP, family ion channels are cationic channels, permeable to both monovalent and divalent cations and play a role in the influx of calcium mainly through the plasma membrane. These channels also represent vital calcium entry routes in the mammary epithelium and may thus act as central players in the preservation of calcium balance within this tissue. Moreover, TRP family channel proteins are abnormally expressed in breast cancers and may promote cancer progression through deregulation of intracellular signaling, consequently triggering several hallmarks of cancer. This chapter concentrates on the role of transient receptor potential vanilloid, TRPV, a subfamily of proteins in the calcium-dependent functions of normal mammary epithelium and the evident role of these channel-forming proteins along breast cancer progression

Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines

Peer reviewe

CaMKK2 Regulates Mechanosensitive Assembly of Contractile Actin Stress Fibers

Stress fibers are contractile actomyosin bundles that guide cell adhesion, migration, and morphogenesis. Their assembly and alignment are under precise mechanosensitive control. Thus, stress fiber networks undergo rapid modification in response to changes in biophysical properties of the cell's surroundings. Stress fiber maturation requires mechanosensitive activation of 5 0 AMP-activated protein kinase (AMPK), which phosphorylates vasodilator- stimulated phosphoprotein (VASP) to inhibit actin polymerization at focal adhesions. Here, we identify Ca2+-calmodulin-dependent kinase kinase 2 (CaMKK2) as a critical upstream factor controlling mechanosensitive AMPK activation. CaMKK2 and Ca2+ influxes were enriched around focal adhesions at the ends of contractile stress fibers. Inhibition of either CaMKK2 or mechanosensitive Ca2+ channels led to defects in phosphorylation of AMPK and VASP, resulting in a loss of contractile bundles and a decrease in cell-exerted forces. These data provide evidence that Ca2+, CaMKK2, AMPK, and VASP form a mechanosensitive signaling cascade at focal adhesions that is critical for stress fiber assembly.Peer reviewe

TRPV6 calcium channel directs homeostasis of the mammary epithelial sheets and controls epithelial mesenchymal transition

Epithelial integrity is lost upon cancer progression as cancer cells detach from the primary tumor site and start to invade to the surrounding tissues. Invasive cancers of epithelial origin often express altered levels of TRP-family cation channels. Upregulation of TRPV6 Ca2+-channel has been associated with a number of human malignancies and its high expression in breast cancer has been linked to both proliferation and invasive disease. The mechanisms behind the potential of TRPV6 to induce invasive progression have, however, not been well elucidated. Here we show that TRPV6 is connected to both E-cadherin-based adherens junctions and intracellular cytoskeletal structures. Loss of TRPV6 from normal mammary epithelial cells led to disruption of epithelial integrity and abnormal 3D-mammo sphere morphology. Furthermore, expression level of TRPV6 was tightly linked to the levels of common EMT markers, suggesting that TRPV6 may have a role in the mesenchymal invasion of breast cancer cells. Thus, either too low or too high TRPV6 levels compromise homeostasis of the mammary epithelial sheets and may promote the progression of pathophysiological conditions.Peer reviewe

Assembly of Peripheral Actomyosin Bundles in Epithelial Cells Is Dependent on the CaMKK2/AMPK Pathway

Summary Defects in the maintenance of intercellular junctions are associated with loss of epithelial barrier function and consequent pathological conditions, including invasive cancers. Epithelial integrity is dependent on actomyosin bundles at adherens junctions, but the origin of these junctional bundles is incompletely understood. Here we show that peripheral actomyosin bundles can be generated from a specific actin stress fiber subtype, transverse arcs, through their lateral fusion at cell-cell contacts. Importantly, we find that assembly and maintenance of peripheral actomyosin bundles are dependent on the mechanosensitive CaMKK2/AMPK signaling pathway and that inhibition of this route leads to disruption of tension-maintaining actomyosin bundles and re-growth of stress fiber precursors. This results in redistribution of cellular forces, defects in monolayer integrity, and loss of epithelial identity. These data provide evidence that the mechanosensitive CaMKK2/AMPK pathway is critical for the maintenance of peripheral actomyosin bundles and thus dictates cell-cell junctions through cellular force distribution.Peer reviewe

A beta2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation

beta2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of beta2-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the b2-integrin (TTT/AAA-b2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-b2-integrin KI dendritic cells, which leads to a failure ofMRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of b2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of b2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.Peer reviewe

Tropomodulins Control the Balance between Protrusive and Contractile Structures by Stabilizing Actin-Tropomyosin Filaments

Eukaryotic cells have diverse protrusive and contractile actin filament structures, which compete with one another for a limited pool of actin monomers. Numerous actin-binding proteins regulate the dynamics of actin structures, including tropomodulins (Tmods), which cap the pointed end of actin filaments. In striated muscles, Tmods prevent actin filaments from overgrowing, whereas in non-muscle cells, their function has remained elusive. Here, we identify two Tmod isoforms, Tmod1 and Tmod3, as key components of contractile stress fibers in non-muscle cells. Individually, Tmodl and Tmod3 can compensate for one another, but their simultaneous depletion results in disassembly of actin-tropomyosin filaments, loss of force-generating stress fibers, and severe defects in cell morphology. Knockout-rescue experiments reveal that Tmod's interaction with tropomyosin is essential for its role in the stabilization of actin-tropo-myosin filaments in cells. Thus, in contrast to their role in muscle myofibrils, in non-muscle cells, Tmods bind actin-tropomyosin filaments to protect them from depolymerizing, not elongating. Furthermore, loss of Tmods shifts the balance from linear actin-tropomyosin filaments to Arp2/3 complex-nucleated branched networks, and this phenotype can be partially rescued by inhibiting the Arp2/3 complex. Collectively, the data reveal that Tmods are essential for the maintenance of contractile actomyosin bundles and that Tmod-dependent capping of actin-tropomyosin filaments is critical for the regulation of actin homeostasis in non-muscle cells.Peer reviewe