Efekti inhibicije CXCR4 receptora i fokalne adhezione kinaze u supresiji invazije i prevazilaženju rezistencije kod nesitnoćelijskog karcinoma pluća

Abstract

Rak pluća je najčešće dijagnostikovani tip malignih tumora i vodeći uzrok smrti od malignih oboljenja. Na osnovu kliničke prognoze i odgovora na terapiju čak 85% raka pluća se svrstava u tip nesitnoćelijskog karcinoma pluća (NSCLC). Iako manje agresivan od sitnoćelijskog karcinoma pluća (SCLC), NSCLC lošije odgovara na terapiju. Dva najčešća uzroka neuspeha lečenja NSCLC-a klasičnom hemioterapijom su pojave metastaza i rezistencije na citostatike. Ovi procesi su regulisani brojnim signalnim molekulima, među kojima su i CXC hemokinski receptor 4 (CXCR4) i fokalna adheziona kinaza (FAK), čije ciljano inhibiranje predstavlja obećavajući pristup u lečenju NSCLC-a. Cilj ove studije je bio da se ispita uloga CXCR4 i FAK-a u invazivnosti i rezistenciji NSCLC-a, kao i potencijal inhibicije ovih molekula u supresiji invazije i reverziji rezistencije kod ovog tipa karcinoma pluća. Konkretno, za ispitivanje uloge CXCR4 i FAK-a u invazivnosti NSCLC-a uspostavljen je in vitro sistem ćelija sa različitim funkcionalnim statusom p53 i PTEN tumor supresora, kao i in vivo ortotopni metastatski model NSCLC-a sa p53/PTEN deficijentnim tumorima. U ispitivanom in vitro sistemu istovremena inaktivacija p53 i PTEN tumor supresora dovela je do aktivacije CXCR4 i FAK molekula i nishodnih signalnih molekula AKT-a i ERK-a, što je bilo praćeno povećanjem invazivnog i migratornog potencijala ćelija. Primenom specifičnih inhibitora CXCR4 i FAK-a, WZ811 i PF-573228, pokazan je potencijal primene inhibicije ovih molekula u supresiji invazije i migracije p53-/PTEN- NSCLC ćelija, kao i metastatskog širenja agresivnih p53/PTEN deficijentnih tumora u in vivo modelu. U daljem toku studije za ispitivanje uloge CXCR4 i FAK-a u razvoju rezistencije NSCLC-a na hemioterapiju korišćene su rezistentne ćelijske linije, NCIH460/ R i COR-L23, koje se odlikuju povećanom ekspresijom ABCB1, odnosno ABCC1 transportera. Pokazano je da kombinovani tretmani inhibitorima CXCR4 i FAK-a sa doksorubicinom (DOX) uspešno senzitizuju rezistentne ćelije na pomenuti citostatik. Ispitujući mehanizam reverzije rezistencije utvrđeno je da kombinovani tretmani smanjuju fosforilaciju FAK-a i AKT-a i dovode do aktivacije senescence-a. Istovremeno sa senzitizacijom ćelija na DOX, kombinovanim tretmanima je uspešno suprimirana i invazija rezistentnih ćelija. Imajući u vidu da su rezistencija na terapiju i metataziranje dva najvažnija problema u lečenju NSCLC-a, rezultati studije pokazuju da ciljano inhibiranje CXCR4 i FAK-a, kao i njihova kombinacija sa standardnim hemioterapeuticima, može predstavljati dobar terapeutski pristup u lečenju ovog oboljenja.

Lung cancer is the most commonly diagnosed type of malignant tumor and the leading cause of cancer death. Based on clinical prognosis and terapy response, up to 85% of lung cancer is clasified as non-small cell lung carcinoma (NSCLC). Although NSCLC is less agresive than small cell lung carcinoma (SCLC), its response to terapy is worse then SCLC. Two major causes of terapy failure of NSCLC with classic chemotherapeutics are the development of metastasis and chemotherapy resistance. Among other signaling molecules, CXCR4 receptor and focal adhesion kinase (FAK) are known to regulate these processes and their targeted inhibition represents a promising approach in the treatment of NSCLC. The aim of this study was to examine the role of CXCR4 and FAK in the invasiveness and resistance of NSCLC, as well as the potential of their inhibition in the suppression of invasion and the reversal of resistance in this type of lung carcinoma. In order to examine the role of CXCR4 and FAK in the invasiveness of NSCLC, in vitro system of cancer cell lines with different functional status of the p53 and PTEN tumor suppressors was established, as well as in vivo orthoptic metastatic model of NSCLC with p53/PTEN deficient tumors. The simultaneous inactivation of p53 and PTEN tumor suppressors in in vitro system lead to significant increase in invasive and migratory potential associated with activation of CXCR4 and FAK molecules and their downstream signaling molecules, AKT and ERK. The potential for supressing invasion and migration of p53-/PTEN- NSCLC cells, as well as the metastatic spread of aggressive p53/PTEN deficient tumors in in vivo model, has been shown by the application of specific inhibitors of CXCR4 and FAK, PF-573228 and WZ811. Furthermore, resistant cell lines NCI-H460/R and COR-L23 were used to investigate the role of CXCR4 and FAK in the development of NSCLC resistance to chemotherapy. NCI-H460/R and COR-L23 cells are characterized by an increased expression of ABCB1 and ABCC1 transporters, respectively. Our results additionally indicate that inhibition of CXCR4 and FAK in combination with DOX successfully sensitized NSCLC cells to the aforementioned cytostatic. By examining the mechanism of sensitization of NSCLC cells to DOX, it has been discovered that combined treatments reduced phosphorylation of FAK and AKT and lead to activation of senescence. Simultaneously with sensitization of cells to DOX, combined treatment successfully suppressed the invasion of resistant cells. Considering that resistance to therapy and metastasis are the two most important problems in NSCLC treatment, the results of the study show that targeted inhibition of CXCR4 and FAK, as well as their combination with standard chemotherapeutics, could be a promising therapeutic approach in the treatment of this disease.