PAK4, MCF-7 hücrelerinde E-kaderini baskılayarak PKC-bağımlı invaziv potansiyeli destekler

Yıl 2020, Cilt: 77 Sayı: 1, 107 - 116, 01.03.2020

Suray Pehlivanoğlu Çiğdem Aydın Acar

Öz

Amaç: Normal meme epitel hücrelerinde p21 ile aktive edilen kinaz 4 PAK4 overekspresyonu sağlandığında tek başına tümörigenezis sürecini başlatabilmektedir. Son zamanlarda yapılan araştırmalar, PAK4’ün meme kanserinde önemli bir onkojenik faktör olabileceğini ileri sürmektedir. Bu çalışmada, PAK4 overeksprese eden ve etmeyen MCF-7 meme kanseri hücrelerinde protein kinaz C aktivasyonu ve inhibisyonuna bağlı hücrelerin migrasyon kabiliyeti ve hücre-hücre kontaktını sağlayan E-Kaderin ekspresyon düzeylerinin araştırılması amaçlandı. Yöntem: Çalışmada, meme kanseri modeli olarak MCF7 hücre hattı kullanıldı. MCF7 hücrelerinde PAK4 plasmid kullanılarak yabanıl-tip insan PAK4 geninin ektopik ekspresyonu sağlandı. Kontrol vektör olarak ise p3XFLAGCMV-10 plazmiti kullanıldı. PKC inhibitörü olarak RO318220 ve PKC aktivatörü olarak ise phorbol 12-myristate-13- acetate PMA / TPA kullanıldı. PAK4 plazmid ve kontrol vektör ile transfekte edilen her iki gruptaki hücreler %0,2 FBS, %10 FBS, RO318220 5μM ve TPA 200 nM olacak şekilde 48 saat süre ile kültüre edildi. Meme kanseri hücrelerinde hücre migrasyonu, Oris Hücre migrasyon deneyi ile değerlendirildi. E-kaderin ekspresyonunun değerlendirilmesi için Western blot yöntemi kullanıldı.Bulgular: Yüksek PAK4 ekspresyonu sağlanan MCF7 hücrelerinde mezenkimal-benzer fenotipin meydana geldiği ve podozomal yapıların sayılarının ve uzunluklarının arttığı belirlendi. Ayrıca TPA ile PKC aktivasyonu sağlanan hücrelerde PAK4 overekspresyonuna bağlı hücre migrasyonunda artış görüldü. Fakat PKC ile indüklenen invaziv etkiler PKC inhibitörü olan RO318220 ile muamele edilen hücrelerde bloke edildi. Bunun yanısıra, PAK4 overeksprese eden hücrelerde kontrole göre E-kaderin ekspresyonunda baskılanma meydana geldiği belirlendi. Sonuç: E-kaderin, hücre-hücre kontaktını sağlayan ve hücre göçünü engelleyen temel yapılardan biridir. Birlikte değerlendirildiğinde, bu bulgular PKC ile aktive edilen PAK4 sinyalinin meme kanseri progresyonuna katkıda bulunduğunu göstermektedir. Bu nedenle, sonuçlarımız PKC-PAK4 sinyal yolağının inhibe edilmesinin meme kanseri tedavisi için potansiyel bir terapötik yaklaşım olabileceğini göstermektedir.

Anahtar Kelimeler

Meme kanseri, PAK4, Protein kinaz C

PAK4 promotes invasive potential of MCF-7 cells in PKC-dependent manner through downregulation of E-Cadherin

Öz

Objective: The p21-activated kinase 4 PAK4 overexpression is sufficient to initiate the tumorigenesis process in normal breast epithelial cells. Recent studies suggested that PAK4 could be an important oncogenic factor in breast cancer. The aim of this study was to investigate the migration ability of cells due to protein kinase C activation and inhibition and the expression levels of E-cadherin which provides cell-cell contact in MCF-7 breast cancer cells that PAK4 overexpressing and nonoverexpressing cells.Methods: MCF7 cell line was used as a breast cancer model. Ectopic expression of the wild-type human PAK4 gene was achieved using PAK4 plasmid in MCF7 cells. Plasmid p3XFLAG-CMV-10 was used as control vector. RO318220 was used as PKC inhibitor and phorbol 12-myristate-13-acetate PMA / TPA was used as PKC activator. Cells in both groups transfected with PAK4 plasmid and control vector were cultured for 0.2 h FBS, 10% FBS, RO318220 5μM and TPA 200 nM for 48 hours. Cell migration in breast cancer cells was evaluated by Oris cell migration assay. Western blot method was used to evaluate the expression of E-cadherin.Results: It was determined that mesenchymallike phenotype was formed and the number and length of podosomal structures were increased in these PAK4 overexpressed cells. In addition, PKC activation via TPA treatment increased cell migration due to PAK4 overexpression. However, PKC-induced invasive effects were blocked by the PKC kinase inhibitor RO318220. In addition, PAK4 overexpression leads to downregulation of E-cadherin compared to control. Conclusion: E-cadherin is one of the basic structures that provide cell-cell contacts and prevent cell migration. Taken together, these findings suggest that PKC-activated PAK4 signalling contributes to breast cancer progression. Therefore, our results show that inhibition of the PKC-PAK4 signaling pathway may be a potential therapeutic approach for the treatment of breast cancer

Anahtar Kelimeler

Breast cancer, PAK4, Protein kinase C

Kaynakça

• 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin, 2019; 69(1):7-34.
• 2. Mahjoubin-Tehran M, Rezaei S, Jalili A, AghaeeBakhtiari SH, Orafai HM, Jamialahmadi T, Sahebkar A. Peptide decoys: a new technology offering therapeutic opportunities for breast cancer. Drug Discov Today, 2020; pii: S1359-6446(20)30036-2.
• 3. Dillekas H, Rogers MS, Straume O. Are 90% of deaths from cancer caused by metastases? Cancer Med, 2019; 8(12):5574-6.
• 4. Bokoch GM. Biology of the p21-activated kinases. Annu Rev Biochem, 2003; 72:743–781.
• 5. Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer. Nat Rev Cancer, 2014; 14:13–25.
• 6. Abo A, Qu J, Cammarano MS, Dan C, Fritsch A, Baud V, et al. PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia. EMBO J, 1998; 17:6527–40.
• 7. Dan C, Kelly A, Bernard O, Minden A. Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin. J Biol Chem, 2001 ;276:32115–21.
• 8. Qu J, Li X, Novitch BG, Zheng Y, Kohn M, Xie JM, et al. PAK4 kinase is essential for embryonic viability and for proper neuronal development. Mol Cel Biol, 2003; 23:7122–33.
• 9. Arias-Romero LE, Chernoff J. A tale of two Paks. Biologie Cellulaire, 2008; 100:97–108.
• 10. Won SY, Park JJ, Shin EY, Kim EG. PAK4 signaling in health and disease: defining the PAK4-CREB axis. Exp Mol Med, 2019; 12;51(2):11.
• 11. Minden A. The pak4 protein kinase in breast cancer. ISRN Oncol, 2012; 2012:694201.
• 12. Yang JX, Han YJ, Zheng H, Luo RC. Expression of PAK4 in breast cancer and benign breast pathological changes. Nan Fang Yi Ke Da Xue Xue Bao, 2010; 30:981–3.
• 13. Li D, Zhang Y, Li Z, Wang X, Qu X, Liu Y. Activated Pak4 expression correlates with poor prognosis in human gastric cancer patients. Tumour Biol, 2015; 36:9431–6.
• 14. Xue J, Chen LZ, Li ZZ, Hu YY, Yan SP, Liu LY. MicroRNA-433 inhibits cell proliferation in hepatocellular carcinoma by targeting p21 activated kinase (PAK4). Mol Cell Biochem, 2015; 399:77–86.
• 15. Shu XR, Wu J, Sun H, Chi LQ, Wang JH. PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway. Diagn Pathol, 2015; 10:177.
• 16. Tyagi N, Marimuthu S, Bhardwaj A, Deshmukh SK, Srivastava SK, Singh AP et al. p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling. Cancer Lett, 2016; 370:260–7.
• 17. Nekrasova T, Minden A. PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression. J Cell Biochem, 2011; 112:1795–806.
• 18. Kumar R, Gururaj AE, Barnes CJ. p21-activated kinases in cancer. Nat Rev Cancer, 2006; 6(6):459–71.
• 19. King H, Thillai K, Whale A, Arumugam P, Eldaly H, Kocher HM, et al. PAK4 interacts with p85 alpha: implications for pancreatic cancer cell migration. Sci Rep, 2017;7:42575.
• 20. Liu Y, Chen N, Cui X, Zheng X, Deng L, Price S et al. Karantza V, Minden . The protein kinase Pak4 disrupts mammary acinar architecture and promotes mammary tumorigenesis. Oncogene, 2010; 29:5883–94.
• 21. Fu X, Feng J, Zeng D, Ding Y, Yu C, Yang B. PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways. Biosci Rep, 2014; (2)1;34.
• 22. Wang F, Gao Y, Tang L, Ning K, Geng N, Zhang H, et al. A novel PAK4-CEBPB-CLDN4 axis involving in breast cancer cell migration and invasion. Biochem Biophys Res Commun, 2019; 2;511(2):404-8.
• 23. Arias-Romero LE, Villamar-Cruz O, Pacheco A, Kosoff R, Huang M, Muthuswamy SK, et al. A rac-pak signaling pathway is essential for ErbB2-mediated transformation of human breast epithelial cancer cells. Oncogene, 2010; 29:5839–49.
• 24. Arias-Romero LE, Chernoff J. p21-activated kinases in Erbb2-positive breast cancer: A new therapeutic target? Small GTPases, 2010; 1:124–8.
• 25. Callow MG, Clairvoyant F, Zhu S, Schryver B, Whyte DB, Bischoff JR, et al. Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines. J Biol Chem, 2002; 277: 550–8.
• 26. He LF, Xu HW, Chen M, Xian ZR, Wen XF, Chen MN et al. Activated-PAK4 predicts worse prognosis in breast cancer and promotes tumorigenesis through activation of PI3K/AKT signaling. Oncotarget, 2017; 8(11): 17573-85.
• 27. Wong LE, Chen N, Karantza V, Minden A. The Pak4 protein kinase is required for oncogenic transformation of MDA-MB-231 breast cancer cells. Oncogenesis, 2013; 2: e50.
• 28. Zhang H, Li Z, Viklund EK, Stromblad S. P21- activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration. J Cell Biol, 2002; 158: 1287–97.
• 29. Van Roy F, Berx G. The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci, 2008; 65(23): 3756- 88.
• 30. Platet N, Prevostel C, Derocq D, Joubert D, Rochefort H, Garcia M. Breast cancer cell invasiveness: correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells. Int J Cancer, 1998; 2;75(5):750-6.
• 31. Brenner W, Beitz S, Schneider E, Benzing F, Unger RE, Roos FC, et al. Adhesion of renal carcinoma cells to endothelial cells depends on PKCmu. BMC Cancer, 2010; 6;10:183.
• 32. Goyal P, Pandey D, Behring A, Siess W. Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser283. J Biol Chem, 2005;280(30):27569-77.
• 33. Lau MT, So WK, Leung PC. Fibroblast growth factor 2 induces E-cadherin down-regulation via PI3K/ Akt/mTOR and MAPK/ERK signaling in ovarian cancer cells. PLoS One, 2013;8(3):e59083.
• 34. Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res, 2008; 68(10): 3645-54.
• 35. Ramos-Alvarez I, Jensen RT. P21-activated kinase 4 in pancreatic acinar cells is activated by numerous gastrointestinal hormones/neurotransmitters and growth factors by novel signaling, and its activation stimulates secretory/growth cascades. Am J Physiol Gastrointest Liver Physiol, 2018; 315(2): 302–17.
• 36. Cordover E, Wei J, Patel C, Shan NL, Gionco J, Sargsyan D, et al. KPT-9274, an Inhibitor of PAK4 and NAMPT, Leads to downregulation of mTORC2 in triple negative breast cancer cells. Chem Res Toxicol, 2020; 9.
• 37. Arowosegbe MA, Amusan OT, Adeola SA, Adu OB, Akinola IA, Ogungbe BF, et al. Kaempferol as a potential PAK4 inhibitor in triple negative breast cancer: extra precision glide docking and free energy calculation. Curr Drug Discov Technol, 2019; 23.
• 38. Rabieifar P, Zhuang T, Costa TDF, Zhao M, Strömblad S. Normal mammary gland development after MMTV-Cre mediated conditional PAK4 gene depletion. Sci Rep, 2019; 8;9(1):14436.
• 39. Santiago-Gomez A, Kedward T, Simoes BM, Dragoni I, NicAmhlaoibh R, Trivier E, et al. PAK4 regulates stemness and progression in endocrine resistant ER-positive metastatic breast cancer. Cancer Lett, 2019; 28;458:66-75.
• 40. Li Y, Zhang H, Zhao Y, Wang C, Cheng Z, Tang L, et al. A mandatory role of nuclear PAK4-LIFR axis in breast-to-bone metastasis of ERα-positive breast cancer cells. Oncogene, 2019;38(6):808-21.
• 41. Costa TDF, Zhuang T, Lorent J, Turco E, Olofsson H, Masia-Balague M, et al. PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer. Nat Commun, 2019; 9;10(1):3589.
• 42. Li SQ, Wang ZH, Mi XG, Liu L, Tan Y. MiR-199a/b-3p suppresses migration and invasion of breast cancer cells by downregulating PAK4/MEK/ERK signaling pathway. IUBMB Life, 2015;67(10):768-77
• 43. Kumar R, Sanawar R, Li X, Li F. Structure, biochemistry, and biology of PAK kinases. Gene, 2017; 605: 20–31.
• 44. Shao YG, Ning K, Li F. Group II p21-activated kinases as therapeutic targets in gastrointestinal cancer. World J Gastroenterol, 2016; 22: 1224–35.
• 45. Ye DZ, Field J. PAK signaling in cancer. Cell Logist, 2012; 2: 105–16.
• 46. Tse JC, Kalluri R. Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment. J Cell Biochem, 2007; 101(4): 816-29.
• 47. Koh W, Mahan RD, Davis GE. Cdc42- and Rac1- mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling. J Cell Sci, 2008; 1;121(Pt 7):989-1001.
• 48. Gavert N, Ben-Ze’ev A. beta-Catenin signaling in biological control and cancer. J Cell Biochem, 2007; 102(4): 820-8.