Analiza Ekspresije Kljuĉnih Molekula PTEN/PI3K/mTOR Signalnog Puta i ABC Transportera Kod Trostruko Negativnih Karcinoma Dojke i Njihova Povezanost Sa Histopatološkim i Kliniĉkim Parametrima

Abstract

Trostruko negativni karcinomi dojke su klinički i genetički visoko heterogena grupa humanih maligniteta a zbog varijabilnog odgovora na terapiju predstavljaju jedan od glavnih izazova za savremeno društvo, istraţivače i kliničare. TNBC ne eksprimiraju molekularne markere, estrogenski receptor-ER, progesteronski receptor-PR i receptor za humani epidermalni faktor rasta 2-HER2. Navedeni molekularni markeri su ključni za izbor prave terapije i u direktnoj su vezi sa kliničkim ponašanjem, rezistencijom na hemioterapeutike i ishodom bolesti.

PTEN/PI3K/AkT/mTOR (PAM) signalni put i prekomerna ekspresija ABC transportera mogu biti odgovorni za rezistenciju na hemoterapeutike. U vezi sa tim, cilj ove studije bio je utvrđivanje bioloških mehanizama koji su u osnovi rezistencije na hemioterapiju.

PAM signalni put je značajan za progresiju humanih tumora a abnormalna aktivacija PAM signalnog puta jedan je od najčešće poremećenih signalnih puteva u karcinomima dojke. Zato je jedan od osnovnih ciljeva ove studije bio ispitivanje imunoekspresionog profila PTEN, PI3K i mTOR protein i ABCB1 (MDR1) membranskog transportera i njihova povezanost i sa kliničkim i histopatološkim parametrima, tokom i ishodom bolesti.

U cilju utvrđivanja molekularnih mehanizama odgovornih za redukciju ili potpuni gubitak ekspresije PTEN proteina, urađena je analiza gubitka heterozigotnosti (LOH), RT-qPCR metodom, kao pretpostavljenog najčešćeg mehanizma inaktivacije PTENtumor supresor gena. Ekspresioni profili PTEN, PI3K, mTORproteina i MDR1 transportera dobijeni su imunohistohemijskom (IHC) analizom.

Pokazali smo da je suprimirana ili odsutna PTEN ekspresija sa visokom ekspresijom PI3K i mTOR proteina u asocijaciji sa lošim ishodom bolesti. Potvrdili smo da su PTEN delecije glavni mehanizam i uzrok smanjene ili odsutne ekspesije PTEN proteina. Takođe, pokazali smo da se agresivno ponašanje i češće javljanje metastaza TNBC tumora mogu pripisati značajno češćem gubitku heterozigotnosti (LOH) PTEN tumor supresor gena. Homozigotne delecije (ne i hemizigotne) bi mogle biti potencijalni marker metastatske bolesti i dobar indikator (pokazatelj) prognoze TNBC tumora. Pored toga, utvrdili smo da bi ABCB1 (MDR1) membranski transporter mogao biti odgovoran za sticanje rezistencije na hemioterapiju TNBC tumora zbog češćeg višeg ekspresionog skora.

Smatramo da bi naši podaci mogli da doprinesu razvoju personalizovane medicine i uspostavljanju novih terapijskih pristupa. Drugim rečima, istovremeno delovanje na ciljne molekule PTEN/PI3K/mTOR signalnog puta i inhibicija MDR1 membranske pumpe, mogla bi biti logična strategija u budućem terapijskom pristupu kod pacijenata obolelih odTNBC.

Alternate abstract:

Triple negative breast cancers are a clinically and genetically highly heterogenous group of human tumours and, due to their variable response to therapy, represent one of the main challenges for modern society, researchers and clinicians. TNBCs do not express molecular markers, estrogen-ER, progesterone-PR, and human growth epidermal factor 2-HER2. These molecular markers are crucial for choosing the right therapy and are directly related to clinical behiavior, resistence to chemotherapeutics and disease outcome. The PTEN/PI3K/Akt/mTOR (PAM) signaling pathway and overexpression of the ABCB1 transporter may be responsible for resistance to chemiotherapeutics. In this regard, the aim of this study was to determine the biological mechanisams underlying chemotherapy resistance. The PAM signaling pathway is one of the most commonly disrupted signaling pathways in breast cancers. Therefore, one of the main objectives of this study was to examine the immunoexpression profile of PTEN, PI3K and mTOR proteins and expression profile of ABCB1 (MDR1) membrane transporter and their association with clinical and histopathological parameters, course of disease and disease outcome. In order to determine the molecular mechanisams responsible for the reduction or complete loss of the PTEN gene allele, we performed LOH analysis as the presumed most common mechanism of PTEN tumour suppressor gene inactivation. Loss of heterozigosity (LOH) of the PTEN tumour suppressor gene was preformed by RT-qPCR method and the expression profile of PTEN, PI3K, mTOR proteins and MDR1 transporter by IHC analysis. . Suppresed or absent PTEN expression with high expression of PI3K and mTOR proteins was found to be associated with poor disease outcome. PTEN deletions are a mayor cause of reduced or absent PTEN protein expression. It has been esthablished that MDR1 could be responsible for multidrug resistance to TNBC tumour chemotherapy due to a more frequent and high expression score. It was found that aggressive behavior and more frequent occurrence of the TNBC tumor metastases can be attributed to significally more frequent loss of heterozigosity (LOH) of the PTENtumour suppressor gene. Homozygous (but not hemyzigotic) could be a potential marker of metastasis formation and a good predictor (indicator) of TNBC outcome. These data could contribute to the development of personalized medicine and the establishment of new therapeutic approaches. In other words, a logical strategy in the therapeutic approach, could be simultaneously targeting molecular markers of PTEN/PI3K/mTOR signaling pathway with MDR1 membrane pump in treatment of TNBC patients.