c-Jun N terminal kinase modulates NOX-4 derived ROS production and myofibroblasts differentiation in human breast stromal cells

BMC Cancer

Nicolás Tobar²  Marcela Toyos²  Carla Urra²  Nicolás Méndez²  Rodrigo Arancibia¹  Patricio C Smith¹  Jorge Martínez² 

[1] Laboratorio de Fisiología Periodontal, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile

[2] Laboratorio de Biología Celular y Molecular, INTA, Universidad de Chile, El Líbano 5524, Macul, Santiago, Chile

关键词: ROS;    JNK;    NOX-4;    Fibrosis;   

Others  :  1121165
DOI  :  10.1186/1471-2407-14-640

 received in 2013-12-17, accepted in 2014-08-20,  发布年份 2014

【 摘 要 】

Background

Hard consistency, developed under the influence of tumor cell factors, is a characteristic feature of a breast tumor. Activation of resident fibroblasts leading to a myofibroblast phenotype is the principal feature that orchestrates this fibrotic process. The aim of this study was to assess the effects induced by TGF-β1, a growth factor abundantly present in tumor microenvironment, on the molecular mechanisms that mediate myofibroblastic differentiation of normal human mammary fibroblasts.

Methods

We used an immortalized fibroblastic cell line derived from normal mammary tissue (RMF-EG cells) to study the effect of TGF-β1 in the expression of α-SMA and CTGF as markers of myofibroblastic differentiation. The influence of redox status and JNK activity on TGF-β1-induced transcriptional activity was measured by a luciferase reporter assay. We also used a shRNA approach to evaluate the influence of NOX4 in myofibroblastic differentiation.

Results

TGF-β1 stimulates the expression of myofibroblast markers α-SMA and CTGF. Using a NOX inhibitor (DPI) and cells expressing a shRNA for NOX4, we demonstrated that TGF-β1 promotes an oxidative environment that favors myofibroblastic differentiation. We also found that activation of c-Jun N-terminal kinase is required for TGF-β1-dependent expression of CTGF, NOX4 and α-SMA.

Conclusions

Human mammary stromal fibrosis, evaluated by the expression of early and late markers as CTGF and α-SMA, depends on the activation of JNK signaling pathway. Our results show that JNK activation is an early event that precedes the increase in ROS levels leading to myofibroblastic differentiation and tumor fibrosis, suggesting that inhibition of JNK may be used a method to interrupt the development of tumor desmoplasia.

【 授权许可】

   
2014 Tobar et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Mueller MM, Fusenig NE: Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 2004, 4(11):839-849.
• [2]Ronnov-Jessen L, Bissell MJ: Breast cancer by proxy: can the microenvironment be both the cause and consequence? Trends Mol Med 2009, 15(1):5-13.
• [3]Egeblad M, Littlepage LE, Werb Z: The fibroblastic coconspirator in cancer progression. Cold Spring Harb Symp Quant Biol 2005, 70:383-388.
• [4]Beacham DA, Cukierman E: Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol 2005, 15(5):329-341.
• [5]Elenbaas B, Weinberg RA: Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res 2001, 264(1):169-184.
• [6]Bissell MJ, Radisky DC, Rizki A, Weaver VM, Petersen OW: The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation 2002, 70(9–10):537-546.
• [7]Kuperwasser C, Chavarria T, Wu M, Magrane G, Gray JW, Carey L, Richardson A, Weinberg RA: Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci U S A 2004, 101(14):4966-4971.
• [8]Fiaschi T, Chiarugi P: Oxidative stress, tumor microenvironment, and metabolic reprogramming: a diabolic liaison. Int J Cell Biol 2012, 2012:8.
• [9]Storz P: Reactive oxygen species in tumor progression. Front Biosci 2005, 10:1881-1896.
• [10]Dröge W: Free radicals in the physiological control of cell function. Physiol Rev 2002, 82(1):47-95.
• [11]Tobar N, Guerrero J, Smith PC, Martinez J: NOX4-dependent ROS production by stromal mammary cells modulates epithelial MCF-7 cell migration. Br J Cancer 2010, 103(7):1040-1047.
• [12]De Wever O, Demetter P, Mareel M, Bracke M: Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer 2008, 123(10):2229-2238.
• [13]Radisky DC, Przybylo JA: Matrix Metalloproteinase–induced Fibrosis and Malignancy in Breast and Lung. Proc Am Thorac Soc 2008, 5(3):316-322.
• [14]Shimoda M, Mellody KT, Orimo A: Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression. Semin Cell Dev Biol 2010, 21(1):19-25.
• [15]Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA: Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 2002, 3(5):349-363.
• [16]Moustakas A, Heldin C-H: Non-Smad TGF-β signals. J Cell Sci 2005, 118(16):3573-3584.
• [17]Samarakoon R, Overstreet JM, Higgins PJ: TGF-β signaling in tissue fibrosis: redox controls, target genes and therapeutic opportunities. Cell Signal 2013, 25(1):264-268.
• [18]Wrana JL, Attisano L, Cárcamo J, Zentella A, Doody J, Laiho M, Wang X-F, Massague J: TGFβ signals through a heteromeric protein kinase receptor complex. Cell 1992, 71(6):1003-1014.
• [19]Benhar M, Dalyot I, Engelberg D, Levitzki A: Enhanced ROS production in oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress. Mol Cell Biol 2001, 21(20):6913-6926.
• [20]Arancibia R, Caceres M, Martinez J, Smith PC: Triclosan inhibits tumor necrosis factor-alpha-stimulated urokinase production in human gingival fibroblasts. J Periodontal Res 2009, 44(6):726-735.
• [21]Ushio-Fukai M, Nakamura Y: Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett 2008, 266(1):37-52.
• [22]Engel ME, McDonnell MA, Law BK, Moses HL: Interdependent SMAD and JNK signaling in transforming growth factor-β-mediated transcription. J Biol Chem 1999, 274(52):37413-37420.
• [23]Curtin JF, Cotter TG: JNK Regulates HIPK3 Expression and promotes resistance to Fas-mediated apoptosis in DU 145 prostate carcinoma cells. 17100 2004, 279(17):17090-17100.
• [24]Hasebe T, Tsuda H, Hirohashi S, Shimosato Y, Tsubono Y, Yamamoto H, Mukai K: Fibrotic focus in infiltrating ductal carcinoma of the breast: a significant histopathological prognostic parameter for predicting the long-term survival of the patients. Breast Cancer Res Treat 1998, 49(3):195-208.
• [25]Sampson N, Koziel R, Zenzmaier C, Bubendorf L, Plas E, Jansen-Dürr P, Berger P: ROS Signaling by NOX4 drives fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Mol Endocrinol 2011, 25(3):503-515.
• [26]Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, Sorescu D: NAD(P)H oxidase 4 mediates transforming growth factor-β1–induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res 2005, 97(9):900-907.
• [27]Jain M, Rivera S, Monclus EA, Synenki L, Zirk A, Eisenbart J, Feghali-Bostwick C, Mutlu GM, Budinger GR, Chandel NS: Mitochondrial reactive oxygen species regulate transforming growth factor-beta signaling. J Biol Chem 2013, 288(2):770-777.
• [28]Li Y, Trush MA: Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem Biophys Res Commun 1998, 253(2):295-299.
• [29]Liu RM, Choi J, Wu JH, Gaston Pravia KA, Lewis KM, Brand JD, Mochel NS, Krzywanski DM, Lambeth JD, Hagood JS, Forman HJ, Thannickal VJ, Postlethwait EM: Oxidative modification of nuclear mitogen-activated protein kinase phosphatase 1 is involved in transforming growth factor beta1-induced expression of plasminogen activator inhibitor 1 in fibroblasts. J Biol Chem 2010, 285:16239-16247.
• [30]Massagué J, Chen Y-G: Controlling TGF-β signaling. Genes Dev 2000, 14(6):627-644.
• [31]Mu Y, Gudey S, Landström M: Non-Smad signaling pathways. Cell Tissue Res 2012, 347(1):11-20.
• [32]Ginnan R, Jourd'heuil FL, Guikema B, Simons M, Singer HA, Jourd'heuil D: NADPH oxidase 4 is required for interleukin-1beta-mediated activation of protein kinase Cdelta and downstream activation of c-jun N-terminal kinase signaling in smooth muscle. Free Radic Biol Med 2013, 54:125-134.
• [33]Shi L, Chang Y, Yang Y, Zhang Y, Yu FS, Wu X: Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing. PLoS One 2012, 7(2):e32128.
• [34]Reich N, Tomcik M, Zerr P, Lang V, Dees C, Avouac J, Palumbo K, Horn A, Akhmetshina A, Beyer C, Xie W, Bennett BL, Distler O, Schett G, Distler JH: Jun N-terminal kinase as a potential molecular target for prevention and treatment of dermal fibrosis. Ann Rheum Dis 2012, 71(5):737-745.