Lymphatic Vessels in Tumor Dissemination versus Immunotherapy

During the growth of various cancers, primary tumors can escape antitumor immune responses of their host and eventually disseminate into distant organs. Peritumoral lymphatic vessels connect the primary tumor to lymph nodes, facilitating tumor entry into lymph nodes, systemic circulation, and metastasis. Lymph node metastases that occur frequently provide sites of tumor cell spread, whereas tumor antigen transfer into and presentation in tumor-draining lymph nodes induce activation of tumor-specific T-lymphocyte responses that can result in cytolytic targeting of the tumor. Here, we discuss the recently emerged controversial role of the lymphatic vessels in tumor dissemination and cancer immunotherapy.Non peer reviewe

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.Syöpägeenejä aktivoivat ja kasvurajoitegeenien toimintaa estävät DNA- mutaatiot aiheuttavat solujen hallitsematonta jakautumisesta ja lopulta syöpäkasvaimen kehittymisen. Mutaatioiden vuoksi syöpäsolu ei enää reagoi kudoksen tai solun itsensä solukasvua rajoittaviin signaaleihin, vaan jakautuu olosuhteissa, joissa normaalisolu ei jakautuisi. LKB1 on eräs merkittävimmistä kasvurajoitegeeneistä. Sporadisia LKB1-geenin inaktivoivia mutaatioita on jopa 30%:ssa keuhkojen ei-pienisoluisista adenokarsinoomista ja 20%:ssa kohdunkaulasyövistä. Perinnölliset LKB1-mutaatiot puolestaan aiheuttavat Peutz-Jeghers-oireyhtymän, jolle on tunnusomaista polyyppien kehittyminen mahavatsakanavaan. Polyypit koostuvat liiallisesti jakautuvasta epiteelisolukosta ja stroomasta, joka muodostuu mm. sileälihassolulinjan soluista. Tämän väitöskirjatyön tarkoituksena oli selvittää, mitkä signalointireitit ovat tärkeitä LKB1-kasvurajoitetehtävälle. Tietämys LKB1-signalointireiteistä voi jatkossa mahdollistaa lääkityksen kehittämisen potilaille, jotka kärsivät syöpäkasvaimista. LKB1-geenin ilmentäminen syöpäsolulinjoissa estää solujakautumista. Tässä väitöskirjatyössä saatiin selville, että G361-melanoomasoluissa LKB1-kinaasin aktiivisuus solulimassa aktivoi kasvurajoittaja p53-proteiinin, mikä puolestaan johti solujakautumisen hidastumiseen. Selvittääksemme LKB1:n tehtävää mahavatsakanavan polypoosissa estimme LKB1-geenin ilmenemisen hiiren sileälihassoluissa. Estämisen seurauksena hiirille kehittyi polypoosi, joka oli samankaltainen kuin Peutz-Jeghers-syndrooman potilailla. Tutkimuksemme primaarisilla sileälihassolulinjan soluilla paljasti, että LKB1 estää polypoosia mahdollisesti vahvistamalla TGFβ-kasvutekijäsignalointia, aktiinitukirangan stressisäikeitä ja sileälihassolujen erilaistumista. Jatkotutkimukset HeLa-solulinjalla paljastivat, että NUAK2, yksi LKB1-kinaasin kohdeproteiineista, säätelee positiivisesti aktiinitukirangan stressisäikeitä. Koska NUAK2-kinaasin tasot ovat toisaalta riippuvaisia aktiinitukirangan stressisäikeistä, NUAK2 ja aktiinistressisäikeet muodostavat positiivisen takaisinkytkennän. Tämän väitöskirjatyön tulosten perusteella ehdotan, että LKB1-kasvurajoittaja estää syövän kehittymistä kahden keskeisen kasvurajoitereitin (p53 ja TGFβ) kautta. On mahdollista, että NUAK2 on oleellinen LKB1-kohdeproteiini LKB1-kinaasin kasvurajoitetehtävässä

SUMOylation of AMPK alpha 1 by PIAS4 specifically regulates mTORC1 signalling

AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of Z attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPK alpha 1 SUMOylation and the inhibition of AMPK alpha 1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPK alpha 1a mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKa-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPK alpha 1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.Peer reviewe

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Publisher Copyright: © 2022 American Society for Clinical Investigation. All rights reserved.Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with smallmolecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.Peer reviewe

Lymphatic exosomes promote dendritic cell migration along guidance cues

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.Peer reviewe

Cardiac lymphatics in health and disease

The lymphatic vasculature, which accompanies the blood vasculature in most organs, is indispensable in the maintenance of tissue fluid homeostasis, immune cell trafficking, and nutritional lipid uptake and transport, as well as in reverse cholesterol transport. In this Review, we discuss the physiological role of the lymphatic system in the heart in the maintenance of cardiac health and describe alterations in lymphatic structure and function that occur in cardiovascular pathology, including atherosclerosis and myocardial infarction. We also briefly discuss the role that immune cells might have in the regulation of lymphatic growth (lymphangiogenesis) and function. Finally, we provide examples of how the cardiac lymphatics can be targeted therapeutically to restore lymphatic drainage in the heart to limit myocardial oedema and chronic inflammation.Peer reviewe

Shape and Function of Interstitial Chemokine CCL21 Gradients Are Independent of Heparan Sulfates Produced by Lymphatic Endothelium

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.Peer reviewe

Lymphangiogenesis guidance by paracrine and pericellular factors

Lymphatic vessels are important for tissue fluid homeostasis, lipid absorption, and immune cell trafficking and are involved in the pathogenesis of several human diseases. The mechanisms by which the lymphatic vasculature network is formed, remodeled, and adapted to physiological and pathological challenges are controlled by an intricate balance of growth factor and biomechanical cues. These transduce signals for the readjustment of gene expression and lymphatic endothelial migration, proliferation, and differentiation. In this review, we describe several of these cues and how they are integrated for the generation of functional lymphatic vessel networks