PAK4, MCF-7 hücrelerinde E-kaderini baskılayarak PKC-bağımlı invaziv potansiyeli destekler
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.
PAK4 promotes invasive potential of MCF-7 cells in PKC-dependent manner through downregulation of E-Cadherin
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
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- 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.