Kanserde P21-Aktive Kinaz 4 (PAK4)'ün Hipoksi Sinyal Mekanizmasındaki Rolünün Araştırılması
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Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Kanserde hücresel sinyal mekanizmalarının aydınlatılması terapötik hedeflerin belirlenmesi
açısından oldukça önemlidir. Bu yolaklardaki defektler kanser progresyonunu tetikleyebilmektedir. p-21
aktive kinaz 4 (PAK4) bu sinyal mekanizlamarında rol alan önemli onkogenlerden biridir. Literatürde
endojen PAK4 ifadesinin artışı tümör gelişimini desteklediği bilinmektedir. Fonksiyonel olarak PAK4,
Ras, NFκB, AKT, gibi pek çok kanser sinyal yolaklarında rol oynayarak kanser hücrelerinin
sağkalımını, çoğalmasını ve metastazını teşvik eder. Tümör büyümesi ile beraber doku içerisinde
hipoksi gelişir. Hipoksik ortamda, kanser sürecinin ilerlemesini destekleyen hipoksi ile indüklenebilir
faktör 1α (HIF1α) aktive olur. Normoksik koşullarda hücre içinde HIF1α hızlı bir şekilde yıkılır. Ancak,
normoksik şartlarda dahi HIF1α’nın stabilite kazanabildiği ve bu sayede kanser progresyonunu
destekleyebildiğini kanıtlayan çalışmalar mevcuttur.
Tez projemizde, p21-aktive kinaz 4 (PAK4) enzimini endojen olarak yüksek düzeyde ifade eden
metastatik MDA-MB-231 meme kanseri hücrelerinde normoksik şartlarda PAK4 aktivasyonuna bağlı
olarak PAK4 ile HIF1α arasındaki fiziksel ilişkiyi, aktivasyon durumunu ve buna bağlı olarak kanser
gelişimini destekleyip desteklemediğini in vitro olarak araştırmayı amaçladık. Bunun için, MDA-MB-231
hücreleri PDGF-BB ile uyararak PDGFRβ aracılı PAK4 aktivasyonu ve PF-3758309 ajanı ile muamele
edilerek PAK4 inhibisyonu sağlandı. PDGFRβ aracılı PAK4 aktivasyon zamanı belirlendikten sonra,
PDGF-BB, PF-3758309 ve PDGF-BB ile PF-3758309 beraber muamele edilen hücrelerde kontrole
kıyasla proliferasyon ve migrasyon oranları belirlendi. Ayrıca elde ettiğimiz hücre lizatları ile PDGFRβ PAK4 ve HIF1α-PAK4 interaksiyonları immünpresipitasyon deneyi ile doğrulandı. Western blot deneyi
ile HIF1α stabilizasyonu, p21 ifade düzeyi, bazı epitelyal-mezenkimal transisyon (EMT), apoptotik ve
immünmodülatör belirteçlerin düzeyleri muamele edilen hücrelerde incelendi. Buna ilave olarak, HIF1α
hedefi olduğu düşünülen Rantes geninin mRNA seviyesinde ifade değişimleri de araştırıldı.
Çalışma sonucunda; PAK4’ün PDGFRβ fosforilasyonu ile birlikte aktive olarak HIF1α’ya
fiziksel olarak bağlandığı ve normoksik şartlarda HIF1α stabilizasyonunun desteklendiği gösterildi.
Ayrıca PDGFRβ-PAK4-HIF1α aktivasyonuna bağlı olarak MDA-MB-231 meme kanseri hücrelerinde
HIF1α miktar artışı ile birlikte proliferasyon ve migrasyon düzeylerinin arttığı, EMT sürecinin
desteklendiği ve HIF1α hedefi olarak öngördüğümüz Rantes ifadesinin arttığı saptandı. Ayrıca, PAK4
inhibisyonunun apoptoza direci düşürdüğü ve immünmodülasyonu desteklediği belirlendi. Sonuç olarak,
PDGFRβ-PAK4-HIF1α aksisinin meme kanseri gelişimini desteklediği ve tedavi açısından önemli bir
hedef olabileceği ileri sürülebilir.
The evaluation of the cellular signaling mechanisms in cancer is very important in terms of determining specific therapeutic targets. Defects in these pathways can trigger cancer progression. p-21 activated kinase 4 (PAK4) is one of the important oncogenes involved in these signaling mechanisms. It is known in the literature that increased endogenous PAK4 expression promotes tumor development. As function, PAK4 promotes the survival, proliferation and metastasis abilities of cancer cells by playing important roles in many cancer signaling pathways such as Ras, NFκB, AKT. Parallel to tumor growth, hypoxia develops in the tissue. In the hypoxic environment, hypoxia-inducible factor 1α (HIF1α) is become activated, which promotes the cancer progression. Its well-known that HIF1α is rapidly degraded in normoxic conditions. However, there are many studies showing that HIF1α could gains stability and triggers cancer progression even under normoxic conditions. In our thesis project, we aimed to investigate the physical relationship between PAK4 and HIF1α dependence on the activation of PAK4 under normoxic conditions in metastatic MDA-MB-231 breast cancer cells, which endogenously express the p21-activated kinase 4 (PAK4) at high level. Also, whether PAK4 supports the activation status of HIF1α and accordingly cancer development. Here, we showed PDGFRβ-mediated activation of PAK4 by treating MDA-MB-231 cells with PDGF-BB and PAK4 inhibition by PF3758309. After time-course study of PDGFRβ-mediated PAK4 activation, we dinvestigated proliferation and migration rates of the cells treated with PDGF-BB, PF-3758309 and PDGF-BB+PF3758309 compared to control. In addition, we confirmed the interaction of PDGFRβ-PAK4 and HIF1α-PAK4 by immunoprecipitation assay. Then, we investigated the HIF1α stabilization, p21 level, levels of some epithelial-mesenchymal transition (EMT), apoptotic and immunomodulatory markers by Western blot assay. In addition, we showed the mRNA expression changes of the Rantes gene, which was considered as a potential HIF1α target. As a result of the study, we showed that PAK4 could activated by PDGFRβ activation, physically could in interaction with HIF1α and promoting HIF1α stabilization under normoxic conditions. In addition, due to the activation of PDGFRβ-PAK4-HIF1α axis, the proliferation and migration levels elevated of MDA-MB-231 breast cancer cells. Also, the intracellular HIF1α amounts increased, the EMT process was supported, and the Rantes expression was enhanced. Consequently, we showed that PAK4 inhibition reduces resistance to apoptosis and promotes immunomodulation. As a result, we suggest that the PDGFRβ-PAK4-HIF1α axis could triggers breast cancer progression and be considered as an important target for treatment.
The evaluation of the cellular signaling mechanisms in cancer is very important in terms of determining specific therapeutic targets. Defects in these pathways can trigger cancer progression. p-21 activated kinase 4 (PAK4) is one of the important oncogenes involved in these signaling mechanisms. It is known in the literature that increased endogenous PAK4 expression promotes tumor development. As function, PAK4 promotes the survival, proliferation and metastasis abilities of cancer cells by playing important roles in many cancer signaling pathways such as Ras, NFκB, AKT. Parallel to tumor growth, hypoxia develops in the tissue. In the hypoxic environment, hypoxia-inducible factor 1α (HIF1α) is become activated, which promotes the cancer progression. Its well-known that HIF1α is rapidly degraded in normoxic conditions. However, there are many studies showing that HIF1α could gains stability and triggers cancer progression even under normoxic conditions. In our thesis project, we aimed to investigate the physical relationship between PAK4 and HIF1α dependence on the activation of PAK4 under normoxic conditions in metastatic MDA-MB-231 breast cancer cells, which endogenously express the p21-activated kinase 4 (PAK4) at high level. Also, whether PAK4 supports the activation status of HIF1α and accordingly cancer development. Here, we showed PDGFRβ-mediated activation of PAK4 by treating MDA-MB-231 cells with PDGF-BB and PAK4 inhibition by PF3758309. After time-course study of PDGFRβ-mediated PAK4 activation, we dinvestigated proliferation and migration rates of the cells treated with PDGF-BB, PF-3758309 and PDGF-BB+PF3758309 compared to control. In addition, we confirmed the interaction of PDGFRβ-PAK4 and HIF1α-PAK4 by immunoprecipitation assay. Then, we investigated the HIF1α stabilization, p21 level, levels of some epithelial-mesenchymal transition (EMT), apoptotic and immunomodulatory markers by Western blot assay. In addition, we showed the mRNA expression changes of the Rantes gene, which was considered as a potential HIF1α target. As a result of the study, we showed that PAK4 could activated by PDGFRβ activation, physically could in interaction with HIF1α and promoting HIF1α stabilization under normoxic conditions. In addition, due to the activation of PDGFRβ-PAK4-HIF1α axis, the proliferation and migration levels elevated of MDA-MB-231 breast cancer cells. Also, the intracellular HIF1α amounts increased, the EMT process was supported, and the Rantes expression was enhanced. Consequently, we showed that PAK4 inhibition reduces resistance to apoptosis and promotes immunomodulation. As a result, we suggest that the PDGFRβ-PAK4-HIF1α axis could triggers breast cancer progression and be considered as an important target for treatment.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
HIF1α, PAK4, PDGF-BB, Kanser, Cancer
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Kostak, F. (2022). Kanserde p21-aktive kinaz 4 (PAK4)'ün hipoksi sinyal mekanizmasındaki rolünün araştırılması. (Yayımlanmamış yüksek lisans tezi). Necmettin Erbakan Üniversitesi, Fen Bilimleri Enstitüsü Moleküler Biyoloji ve Genetik Anabilim Dalı, Konya.
