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Uticaj in vitro izlaganja STAT3-aktiviranih malignih ćelija dojke višeslojnim nanočesticama obloženim poli-L-glutaminskom kiselinom

dc.contributor.advisorStankov, Karmen
dc.contributor.advisorMikov, Momir
dc.contributor.otherDragojević Simić, Viktorija
dc.contributor.otherKatanić, Jasmina
dc.contributor.otherMikov, Momir
dc.contributor.otherStankov, Karmen
dc.contributor.otherFrank, David
dc.creatorTošić, Isidora
dc.date.accessioned2022-01-17T12:51:53Z
dc.date.available2022-01-17T12:51:53Z
dc.date.issued2021-12-16
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/Disertacija162391395271019.pdf?controlNumber=(BISIS)117882&fileName=162391395271019.pdf&id=17921&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/record.jsf?recordId=117882&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/IzvestajKomisije162391397868332.pdf?controlNumber=(BISIS)117882&fileName=162391397868332.pdf&id=17922&source=NaRDuS&language=srsr
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/18888
dc.description.abstractBreast cancer is the most prevalent malignancy in women worldwide. It is estimated that every eight women in the world will be diagnosed with breast cancer at some moment of life. The modern medical approaches have reshaped treatment and diagnostic strategies in management of patients with breast neoplasms, significantly improving quality of life and therapy outcomes. Despite these advances, breast cancer is currently the second leading cause of all cancer-related mortality. Two of the breast cancer subtypes, hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) positive breast cancers, have specific entities on their surface successfully employed in designing targeted therapies. Such treatments enable distinction between malignant and normal tissue, resulting in selective induction of apoptosis in cancer cells. Therefore, development of targeted therapies strongly advanced the prognosis for patients with these breast cancer subtypes. The third type of breast cancers, triplenegative breast cancer (TNBC) represents its most aggressive form, characterized by substantial metastatic potential and higher recurrence rates. In addition, thus far there are no known cell surface receptors that could differentiate the TNBC from normal cells which could be efficiently employed for targeted therapy desing. Thus, this subset is treated with non-specific approaches alone, such as chemotherapy, radiotherapy and surgical procedures. Therefore, TNBC represents a subset of breast cancers with the greatest need for further elucidation of the pathological mechanisms underlying the malignant behavior and investigation of the novel treatment strategies that could exploit this knowledge. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that regulates the expression of genes involved in essential cellular processes including cell growth, survival, proliferation, differentiation, migration and immune response. Constitutive activation of STAT3 has been reported in a wide spectrum of malignant diseases, including both solid and hematological malignancies. The importance of STAT3 signaling in breast cancer is displayed by its abnormal activation in 70% of all breast cancers, accounting for essentially all triple-negative breast cancers. Therefore, targeting STAT3 holds a great potential in treatment of such malignancies. STAT3 is an intracellular protein that is physiologically activated in response to external stimuli, such as cytokines and growth factors. Upon activation, it shuttles from cytoplasm to nucleus, where it binds to its nine base pair consensus sequence in the promoter of its target genes to regulate their transcription. Since transcription factors like STAT3 have large and relatively flat molecular surfaces to allow protein-DNA and protein-protein interactions, it makes it challenging to directly target these proteins using small molecules. Given the difficulties in direct targeting, we considered whether metabolic changes driven by constitutive activation of STAT3 could provide vulnerability in breast cancer cells that can be exploited therapeutically. Recognizing that malignant transformation is associated with changes in a variety of cellular metabolites, we investigated how STAT3 affects lipid distribution in mammary epithelial cells, in both qualitative and quantitative manner. To evaluate the metabolic architecture resulting from persistent STAT3 activation, we inhibited or activated STAT3 in two complementary mammary epithelial cellular systems and performed mass spectrometry-based lipid profiling. We found that STAT3 prominently modulated several lipid classes, with most profound reduction of N-acyl taurine (NAT) and arachidonic acid (AA). To investigate if STAT3 activation was accompanied by these metabolic changes in patients, we performed in silico gene set enrichment analyses using publicly available microarray data of 129 breast cancer patients. We confirmed that STAT3 gene expression signature was significantly enriched in patient’s samples with low expression of cysteine dioxygenase (CDO1) and cystathionine gamma lyase (CTH), the two enzymes required for taurine biosynthesis from homocysteine. On the contrary, STAT3 signature was highly enriched in patients’ samples with high expression of AA metalytic enzymes cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX), whereas enzyme that releases AA from plasma membrane, phospholipase A2 (PLA2G4A) did not correlate with STAT3 activity. These findings indicate that STAT3 activation results in reduction of cellular AA and taurine levels by modulating the expression of enzymes involved in their metabolism. As both of taurine and arachidonic acid are involved in plasma membrane remodeling, we examined utilizing these STAT3-driven metabolic alterations in identifying selective nano-size drug carriers that could exploit these lipid properties. We screened a library of 12 layer-by-layer (LbL) nanoparticles (NP) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-L-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to non-transformed cells. This was not a nonspecific consequence of malignant transformation, as nanoparticles coated with other surface chemistries did not display such an effect. Furthermore, the targeting property of PLE-NPs was lost when STAT3 was abrogated using phosphorylation or transcriptional inhibitors, ruxolitinib and pyrimethamine. As STAT3 is constitutively activated in almost all triple-negative breast cancers, we investigated utilizing PLENPs as a STAT3-targeting approach for this subset of breast cancers. The nanoparticles terminally layered with PLE showed potent binding to TNBC cells MDA-MB-231 and SUM159PT, and the heightened NP-cell binding was attenuated when STAT3 expression was diminished using two different small interfering RNAs (siRNAs). As our high-resolution microscopy data indicates that these nanoparticles predominantly bind to cell membrane and might not necessarily be internalized into the cytoplasm, we further examined the tumor penetrating properties of PLE-NPs in three-dimensional breast cell organoids. STAT3-transformed organoids grew at much higher cellular density and formed greater numbers of massive colonies, which could have been expected to impede penetrating ability of nanoparticles through such dense cellular structures. However, PLE-NPs displayed affinity towards STAT3-driven cells even in the three-dimensional breast cellular model and distributed in depth of the STAT3- transformed tumor organoids. Next, we examined the translational potential of these findings. As STAT3- transformed cells show greater resistance to cytotoxic agents, we evaluated if enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven breast cancer cells, including TNBC, at lower concentrations compared to cells lacking activated STAT3. On the contrary, these cells showed significantly greater resistance to apoptosis induced by either un-encapsulated cisplatin or cisplatin-loaded NPs lacking the targeting layer or coated with a non-targeting dextran sulfate (DXS). These findings further emphasize the therapeutic potential of targeting STAT3-driven cells using PLE-coated LbL nanoparticles. In addition, since radiation is commonly used in breast cancer treatment and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using uniquely designed LbL nanoparticles complemented with surface poly-L-glutamic acid. These nanoparticles hold great potential for the delivery of anticancer agents to breast malignant cells, including triple-negative breast cancer cells, with decreased toxicity in normal tissue. The combination treatment with radiotherapy may offer synergy with PLE-nanoparticles and enhance their targeting affinity. Taken together, the results of this dissertation indicate a potential novel treatment approach to triple-negative breast cancers, which currently lack targeted therapeutic options.en
dc.description.abstractKarcinom dojke je na globalnom nivou najučestalije maligno oboljenje kod žena. Prema procenama, svakoj osmoj ženi će u nekom momentu života biti dijagnostikovan karcinom dojke. Savremeni medicinski pristupi su unapredili metode lečenja i dijagnostike u zbrinjavanju pacijenata sa neoplazmom dojke, značajno poboljšavajući kvalitet života i rezultate terapije. Uprkos napretku, karcinom dojke je trenutno drugi vodeći uzrok smrtnosti uzrokovane malignitetom. Dva molekularna podtipa karcinoma dojke, hormon receptor (HR) i humani epidermalni faktor rasta 2 (HER2) pozitivni karcinomi dojke, poseduju specifične strukture na površini maligne ćelije koje su uspešno iskorišćene za dizajniranje ciljanih terapija. Date terapije omogućavaju diferencijaciju između malignog i normalnog tkiva, što rezultira selektivnom indukcijom apoptoze u malignim ćelijama. Stoga je razvoj ciljanih terapija značajno doprineo poboljšanju prognoze za pacijente sa navedenim podtipovima maligniteta dojke. Treći tip karcinoma dojke, trostruko-negativni karcinom dojke (TNBC) predstavlja njegov najagresivniji oblik, koji karakteriše izražen metastatski potencijal i visoka stopa recidiva. Pored toga, trenutno nisu poznati receptori na površini ćelijske membrane koji bi mogli razlikovati TNBC od normalnih ćelija i efikasno bili upotrebljeni za razvoj ciljanih terapija. Stoga se ovaj maligni oblik leči isključivo nespecifičnim pristupima, kao što su hemioterapija, radioterapija i hirurški zahvati. Usled svega navedenog, TNBC predstavlja tip karcinoma dojke sa najvećom potrebom za daljim rasvetljavanjem patoloških mehanizama na kojima zasniva maligno ponašanje kao i istraživanje novih strategija lečenja koje bi mogle iskoristiti dato znanje. Prenosilac signala i aktivator transkripcije 3 (STAT3) je onkogeni transkripcioni faktor koji reguliše ekspresiju gena uključenih u esencijalne ćelijske procese, uključujući ćelijski rast, preživljavanje, proliferaciju, diferencijaciju, migraciju i imunološki odgovor. Konstitutivna aktivacija STAT3 je zabeležena u širokom spektru malignih bolesti, uključujući solidne i hematološke malignitete. Značaj STAT3 signalizacije u karcinomu dojke se odlikuje njegovom patološkom aktivacijom u 70% svih karcinoma dojke, i praktično svim trostruko-negativnim karcinomima dojke. Stoga, terapije selektivno usmerene na STAT3 imaju veliki potencijal u lečenju takvih malignih oboljenja. STAT3 je intracelularni protein koji se fiziološki aktivira kao odgovor na spoljašnje stimuluse, poput citokina i faktora rasta. Kada je aktiviran, STAT3 se translocira iz citoplazme u jedro, gde se vezuje za svoju konsenzus sekvencu od devet baznih parova u promoteru ciljnih gena i reguliše njihovu transkripciju. Budući da faktori transkripcije poput STAT3 imaju velike i relativno ravne molekularne površine kako bi omogućili interakcije sa drugim proteinima i DNK, istovremeno takva molekulska struktura je poprilično problematična za direktno vezivanje malih molekula. S obzirom na poteškoće u direktnom ciljanom delovanju na STAT3, u okviru ove disertacije smo razmotrili mogućnost da metaboličke promene uzrokovane konstitutivnom aktivacijom STAT3 mogu predstavljati vulnerabilnost ćelija karcinoma dojke koja se može terapijski iskoristiti. Imajući u vidu da je maligna transformacija povezana sa promenama različitih ćelijskih metabolita, istražili smo kako STAT3 utiče na kvantitativnu i kvalitativnu distribuciju lipida u epitelnim ćelijama karcinoma dojke. Kako bismo ispitali efekte konstitutivne STAT3 aktivacije na metabolički profil ćelija karcinoma dojke, inhibirali smo ili aktivirali STAT3 u dva komplementarna ćelijska sistema i potom analizirali lipidni profil korišćenjem masene spektrometrije. Otkrili smo da STAT3 vrši modulaciju nekoliko klasa lipida, sa najizraženijom redukcijom ćelijskog nivoa N-acil taurina (NAT) i arahidonske kiseline (AA). Da bismo ispitali da li je aktivacija STAT3 praćena datim metaboličkim promenama kod pacijenata, izvršili smo in silico analize obogaćivanja gena pomoću javno dostupnih podataka RNK mikromatrica od 129 pacijenata obolelih od karcinoma dojke. Potvrdili smo da je STAT3 genska signatura značajno obogaćena u uzorcima pacijenta sa niskom ekspresijom cistein dioksigenaze (CDO1) i cistationin gama liaze (CTH), dva enzima potrebna za biosintezu taurina iz homocisteina. Suprotno tome, STAT3 genska signatura je značajno obogaćena u uzorcima pacijenata sa visokom ekspresijom metaboličkih enzima AA ciklooksigenaze 2 (COX-2) i 5-lipoksigenaze (5-LOX), dok ekspresija enzima koji oslobađa AA iz plazma membrane, fosfolipaze A2 (PLA2G4A), nije u korelaciji sa STAT3 aktivnošću. Ovi nalazi ukazuju na to da aktivacija STAT3 rezultira smanjenjem ćelijskog nivoa AA i taurina modulacijom ekspresije enzima koji su uključeni u njihov metabolizam. Kako su i taurin i arahidonska kiselina uključeni u modeliranje plazme membrane, ispitali smo korišćenje datih STAT3-uzrokovanih metaboličkih promena u identifikovanju selektivnih nanonosača lekova koji bi mogli da iskoriste date lipidne karakteristke. Analizirali smo ćelijsko vezivanje biblioteke od 12 višeslojnih (Layer-byLayer, LbL) nanočestica (NP) koje se razlikuju u površinskom sloju, s obzirom da omotač modulira njihovu interaktivnost sa ćelijskom membranom. Otkrili smo da se NP obložene poli-L-glutaminskom kiselinom (PLE) vezuju za ćelije karcinoma dojke sa aktiviranim STAT3 sa 50% većom efikasnošću nego za netransformisane ćelije. Navedena pojava ne predstavlja nespecifičnu posledicu maligne transformacije, jer nanočestice presvučene drugim strukturama nisu pokazale dati efekat. Pored toga, svojstvo ciljanog delovanja PLE-NP nestaje kada se inhibira fosforilacija ili transkripciona aktivnost STAT3 korišćenjem ruksolitiniba i pirimetamina. S obzirom da je STAT3 konstitutivno aktiviran u gotovo svim trostruko-negativnim karcinomima dojke, ispitali smo korišćenje PLE-NP kao seletivnog pristupa ovom tipu karcinoma  dojke zasnovanog na ciljanom delovanju na STAT3. Nanočestice obložene sa PLE pokazale su povećano vezanje za TNBC ćelijske linije MDA-MB-231 i SUM159PT, koje se značajno smanjuje inhibicijom STAT3 ekspresije korišćenjem dve različite kratke interferirajuće RNK (siRNK). Kako naši mikroskopski podaci visoke rezolucije pokazuju da se date nanočestice pretežno vezuju za ćelijsku membranu i ne moraju nužno biti internalizovane u citoplazmu, dodatno smo ispitali svojstva penetracije PLENP u trodimenzionalne strukture organoida ćelija dojke. Rast STAT3-transformisanih organoida pokazuje izraženo veću ćelijsku gustinu uz formiranje većeg broja masivnih kolonija, te se mogla očekivati otežana penetracija nanočestica kroz tako guste ćelijske strukture. Međutim, PLE-NP su pokazale afinitet prema ćelijama sa aktiviranim STAT3 čak i u trodimenzionalnom modelu karcinoma dojke i distribuciju u dubini STAT3 transformisanih tumorskih organoida. Nakon datih otkrića, evaluirali smo translacioni potencijal ovih nalaza. Uzimajući u obzir da STAT3-transformisane ćelije pokazuju veću otpornost na citotoksične agense, ispitali smo da li bi poboljšana ciljana isporuka lekova putem PLE-NP-a pružila terapijsku prednost. Otkrili smo da PLE-NP ispunjene sa cisplatinom indukuju apoptozu ćelija karcinoma dojke sa aktiviranim STAT3, uključujući TNBC, u nižim koncentracijama u poređenju sa ćelijama kojima nedostaje STAT3 aktivnost, uključujući nemaligne ćelije dojke. Suprotno tome, STAT3- aktivirane ćelije su pokazale značajno veću apoptotsku otpornost na slobodan cisplatin kao i na nanočestice ispunjene cisplatinom koje nemaju ciljni sloj ili su obložene neciljajućim dekstran sulfatom (DXS). Ovi nalazi dodatno ističu terapijski potencijal selektivnog delovanja na maligne ćelije sa aktiviranim STAT3, korišćenjem PLE obloženih LbL nanočestica. Konačno, s obzirom da se terapija gama zraćenjem često koristi u lečenju maligniteta dojke i može delovati na ćelijsku distribuciju lipida, analizirali smo njen uticaj na ćelijsko vezivanje PLE-NP. Ozračivanje ćelija poboljšalo je selektivna svojstva PLE-NP ka STAT3-transformisanim ćelijama na dozno zavisan načina, sugerišući potencijalnu sinergiju između datih terapijskih modaliteta. Rezultati ove disertacije ukazuju da ćelijske lipidne promene uzrokovane aktiviranim STAT3 mogu biti terapijski iskorišćene LbL nanočesticama obloženim površinskom poli-L-glutaminskom kiselinom. Navedene nanočestice imaju veliki potencijal za isporuku lekova malignim ćelijama dojke, uključujući trostrukonegativne ćelije karcinoma dojke, uz smanjenu toksičnost ka normalnom tkivu. Kombinovani tretman sa radioterapijom može ponuditi sinergiju sa PLEnanočesticama i povećati njihov afinitet ciljanog delovanja. U zaključku, rezultati ove doktorske disertacije ukazuju na potencijalni novi pristup lečenju trostruko-negativnog karcinoma dojke, kome trenutno nedostaju ciljani terapijski modaliteti.sr
dc.languageen
dc.publisherУниверзитет у Новом Саду, Медицински факултетsr
dc.rightsopenAccessen
dc.sourceУниверзитет у Новом Садуsr
dc.subjectSTAT3 Transcription Factor; Transcription Factors; Breast Neoplasms; Triple Negative Breast Neoplasms; Nanoparticles; Molecular Targeted Therapy; Lipidomics; Polyglutamic Aciden
dc.subjectSTAT3 transkripcioni faktor; transkripcioni faktori; neoplazme dojke; trostruko negativni karcinom dojke; nanočestice; molekularna ciljana terapija; lipidomika; poliglutaminska kiselinasr
dc.titleTargeting STAT3-driven breast cancer cells using poly-L-glutamic acid-coated Layer-by-Layer nanoparticlesen
dc.title.alternativeUticaj in vitro izlaganja STAT3-aktiviranih malignih ćelija dojke višeslojnim nanočesticama obloženim poli-L-glutaminskom kiselinomsr
dc.typedoctoralThesissr
dc.rights.licenseAttribution-NonCommercial-ShareAlike
dcterms.abstractСтанков, Кармен; Миков, Момир; Драгојевић Симић, Викторија; Франк, Давид; Катанић, Јасмина; Миков, Момир; Станков, Кармен; Тошић, Исидора;
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/141868/Izvestaj_komisije_12030.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/141867/Disertacija_12030.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_18888


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