Matriksin metalloproteinaasit melanoomasolujen invaasiossa

Tumor microenvironment comprised of extracellular matrix (ECM) and non-malignant cells has profound effects on cancer progression. Membrane-type matrix metalloproteinases (MT-MMPs) are involved in the modulation of tumor microenvironment. MT1-MMP is a prototype of MT-MMP family, which is overexpressed in many types of cancer, where it promotes tumor cell invasion through collagen-rich tissues. The biological functions of another member of the MT-MMP family, MT3-MMP, have remained largely unknown. MT3-MMP is expressed in the adult brain, as well as brain tumors and nodular melanoma. The purpose of this thesis was to elucidate the functions of MT1-MMP and MT3-MMP in melanoma cell invasion. To understand the pericellular growth regulation, we searched for endogenous enzymes which could release latent TGF-β from endothelial cell extracellular matrix. Neovessel formation is a prerequisite for tumor growth. Pericellular modulation of the ECM by MT1-MMP releases matrix-associated growth factors and bioactive peptides, which further affect angiogenesis and tumor cell biology. We found that MT1-MMP modulated subendothelial extracellular matrix, and cleaved latent TGF-β binding protein-1 (LTPB-1), with subsequent release of latent TGF-β complexes from the ECM. Thus, MT1-MMP-mediated LTBP-1 cleavage provides a mechanism for the tightly controlled release of matrix-associated TGF-β at the sites of neovessel formation. To elucidate the functions of MT-MMPs in melanoma cell invasion, we analyzed the expression of MT1-MMP and MT3-MMP from biopsies of normal human skin, benign nevi, and melanoma metastases. MT3-MMP was upregulated in lymph node metastases of human melanoma, while MT1-MMP expression was comparable in all biopsies. By culturing melanoma cells in 3D collagen and fibrin matrices, we found that MT3-MMP was associated with expansive melanoma growth in 3D collagen, but promoted their sprouting growth in 3D fibrin. In in vivo xenograft experiments, MT3-MMP expressing melanoma xenografts grew slowly, while MT3-MMP silencing enhanced tumor growth rate by over twofold. Interestingly, high MT3-MMP expression in murine xenografts and a human melanoma tumor was associated with prominent lymphatic vessel invasion but negligible blood vessel invasion of melanoma cells. Silencing of MT3-MMP reduced lymphatic invasion but facilitated blood vessel invasion of melanoma cells over ~10-fold. MT3-MMP reduced cell surface MT1-MMP in vitro and in vivo, resulting in limited collagen invasion in vitro and collagen accumulation in vivo. This suggested that low collagenolytic ability of MT3-MMP-expressing melanoma cells resulted in decreased blood vascular invasion. These cells invaded instead into more permissive lymphatic vessels. Since lymphatic vessel invasion is associated with metastatic spread in melanoma, MT3-MMP expression may serve as a new prognostic factor in this disease.Kasvaimet koostuvat syöpäsolujen lisäksi soluväliaineesta ja useista muista solutyypeistä, jotka myötävaikuttavat syövän kasvuun. Solukalvoon kiinnittyneet matriksin metalloproteinaasit (MT-MMP:t) pilkkovat solukalvon ja soluväliaineen proteiineja. Näin ollen ne muokkaavat syöpäsolujen proliferaatiota ja invaasiota. MT1-MMP on tämän entsyymiperheen prototyyppi. MT1-MMP:tä ilmennetään monessa syövässä, joissa se lisää syöpäsolujen invaasiota kollageenipitoisten kudosten läpi. MT3-MMP on MT1-MMP:n läheinen homologi, joka ei pysty pilkkomaan tyypin I kollageenia, mutta sen sijaan pilkkoo fibriiniä. Väitöskirjatyöni tarkoituksena on ollut selvittää MT1-MMP:n ja MT3-MMP:n vaikutuksia melanoomasolujen invaasioon. Tähän liittyen selvitimme mitkä endogeeniset entsyymit voivat vapauttaa latenttia transformoivaa kasvutekijä-betaa (TGF-β) endoteelisolujen soluväliaineesta. Soluväliaineen proteiinien pilkkominen voi vapauttaa niihin kiinnittyneitä kasvutekijöitä ja muita bioaktiivisia peptidejä. Tuloksemme osoittavat että MT1-MMP pilkkoo latenttia TGF-β:aa sitovaa proteiinia (LTBP-1) verisuonia verhoavien, eli endoteelisolujen, soluväliaineesta. LTBP-1 sitoo TGF-β:aa ja kiinnittyy soluväliaineen proteiineihin. LTBP-1:n pilkkominen vapauttaa TGF-β -komplekseja soluväliaineesta, mikä myötävaikuttanee tämän latentin kasvutekijän aktivaatioon. Melanooma on yleistymässä oleva ihosyöpätyyppi, ja levinneen melanooman hoitomahdollisuudet ovat rajalliset. MT3-MMP:aa ilmennetään nodulaarisessa melanoomassa, joka on huonoennusteisin melanoomatyyppi. Analysoimme tässä MT3-MMP:n vaikutuksia melanoomasolujen invaasiotapahtumaan käyttäen in vitro invaasiokokeita ja hiirikokeita. MT3-MMP:n ilmentyminen oli lisääntynyt ihmisen melanooman imusolmuke-etäpesäkkeissä. Melanoomasolujen kasvattaminen kolmiulotteisissa geeleissä osoitti, että MT3-MMP esti solujen invasiivista kasvua kollageenissa edistämällä pyöreiden pesäkkeiden kasvua. Fibriinissä MT3-MMP sen sijaan edisti invaasiivista kasvua. Vastaavasti MT3-MMP:tä ilmentävät melanoomaksenograftit kasvoivat hitaasti hiirten subkutaanisissa kudoksissa, kun taas MT3-MMP:n hiljentäminen nopeutti niiden kasvua noin kaksinkertaiseksi. MT3-MMP vähensi solupinnalla olevan MT1-MMP:n tasoja in vitro ja in vivo, mikä johti vähentyneeseen kollageenin pilkkomiseen ja kollageenin määrän nousuun kasvainten sisällä. Havaitsimme MT3-MMP:tä ilmentävissä ksenografteissa ja ihmisen melanoomassa kasvainsolujen voimakkaan invaasion imusuoniin. MT3-MMP:n hiljentäminen puolestaan esti imusuoni-invaasiota ja lisäsi verisuoni-invaasiota noin kymmenkertaiseksi. Koska melanoomasolujen imusuoni-invaasio liittyy etäpesäkkeiden muodostumiseen, MT3-MMP:n ilmentyminen voi osoittautua melanooman huonon ennusteen merkkiaineeksi

Membrane-Type-3 Matrix Metalloproteinase (MT3-MMP) Functions as a Matrix Composition-Dependent Effector of Melanoma Cell Invasion

Peer reviewe

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and beta 1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and beta 1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident beta 1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype.Peer reviewe

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and beta 1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and beta 1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident beta 1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype

Membrane-type matrix metalloproteases as diverse effectors of cancer progression

Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.Peer reviewe

Vaginal microbiota and its relationship with human papilloma virus infection

Objetivos: Determinar la prevalencia de infección por el virus del papiloma humano de alto riesgo (VPH-ar) en distintos grupos etarios y según grado de lesión. Establecer la prevalencia de vaginosis bacteriana, candidiasis y trichomoniasis en los grupos estudiados y caracterizar las especies de lactobacilos según el tipo de lesión. Evaluar la disfunción vaginal mediante los estados vaginales básicos por balance del contenido de la vagina en pacientes infectadas por el virus del papiloma humano, según tipo de lesión, en comparación con pacientes control no infectadas. Materiales y Métodos: Se llevó a cabo un estudio transversal. Se realizó un examen clínico y se tomó una muestra para estudio de estados vaginales básicos y cultivo. La identificación de especies se realizó por MALDI TOF-MS. Se determinaron los tipos de VPH-ar con qPCR multiplex. Se utilizó test de chi cuadrado y Fisher y se consideró significativo p50 años) n = 147. Además, las pacientes fueron subdivididas en VPH negativas, VPH positivas sin lesión, con lesión intraepitelial escamosa de bajo grado (L-SIL), con lesión intraepitelial escamosa de alto grado / carcinoma de cuello uterino (H-SIL/CC). El VPH16 fue el tipo de VPH-ar más asociado con H-SIL/CC (grupo 1: 63,6 %, grupo 2: 43,8 % y grupo 3: 100%). La prevalencia de estados vaginales básicos de desbalance en H-SIL/CC fue para el grupo 1 de 72,7 % (p = 0,03), para el grupo 2 de 53,1 % (p = 0,05) y no se detectaron casos en el grupo 3. La patología más prevalente fue la vaginosis bacteriana en los tres grupos (p50 years) n=147. They were also divided into HPV negative patients, HPV positive without lesion, Low Grade Squamous Intraepithelial Lesion (L-SIL), and High-Grade Squamous Intraepithelial Lesion/ Cervical Cancer (H-SIL/CC). HPV16 was the hrHPV type most associated with H-SIL/CC (Group 1: 63.6%, Group 2: 43.8% and Group 3 100%). The prevalence of basic vaginal states of unbalance in H-SIL/CC was: Group 1: 72.7% (p=0.03), Group 2: 53.1% (p=0.05) and Group 3: no cases. In the three groups, the most prevalent infection was bacterial vaginosis (p<0.001). In Group 1, 54.2% of the HPV16 patients had associated bacterial vaginosis (p=0.054). Patients with H-SIL/ CC had a prevalence of 21.4% of Lactobacillus crispatus, 42.9% of L. jensenii, and 14.3% of L. iners. Conclusions: HPV16 was the most prevalent type in Groups 1 and 2. Results also showed a higher unbalanced microbiota in H-SIL/CC patients in Groups 1 and 2 and a low prevalence of L. crispatus and an increase in L. jensenii and Liners.Fil: Perazzi, Beatriz Elizabeth. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Payalef, Sandra Noemi. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Fleider, Laura. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Reyes, Ana Paula. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Maldonado, Veronica. Atila BioSystems; ArgentinaFil: Losada, Mirta Olga. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Chen, Xin. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Díaz, Lilí Beatriz. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Wang, Youxiang. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Gomez Cherey, Facundo. Atila Biosystems; Estados UnidosFil: Tatti, Silvio Alejandro. Atila Biosystems; Estados Unido