NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression
Abstract
:1. Introduction
2. Results
2.1. NAB2 Is Expressed in Cancer-Associated Fibroblasts (CAFs) of Head and Neck Squamous Cell Carcinoma (HNSCC) Patient Tumor Tissues
2.2. CAF Marker and Matrix Metalloproteinase (MMP) Expression Is Upregulated in CAFs of HNSCC Patients
2.3. NAB2 and CAF Marker Expression Levels in Fibroblasts Are Correlated
2.4. NAB2 Expressed by CAFs Enhances FaDu Cell Invasion
2.5. NAB2 Expressed by CAFs Increases the Tumorigenicity of FaDu Spheroids In Vivo
3. Discussion
4. Materials and Methods
4.1. Immunohistochemical Analysis of Clinical Specimens
4.2. Chemicals and Reagents
4.3. Primary Fibroblast Cultures and Preparation of Conditioned Medium (CM)
4.4. Real-Time Quantitative Polymerase Chain Reaction (qPCR)
4.5. Western Blot Analysis
4.6. Transfection of Small Interfering (si)RNA or Overexpression Vector
4.7. Matrigel Invasion Assay
4.8. Mouse Xenograft Model
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
α-SMA | alpha smooth muscle actin |
CAF | cancer-associated fibroblast |
CM | conditioned medium |
ECM | extracellular matrix |
FAP | fibroblast activation protein |
H&E | hematoxylin and eosin |
MMP | matrix metalloproteinase |
NAB2 | NGFI-A-binding protein 2 |
NTF | non-tumor fibroblast |
HNSCC | head and neck squamous cell carcinoma |
siRNA | small interfering RNA |
TGF-β | transforming growth factor beta |
Appendix A
Patients | TNM Stage | Cell Differentiation |
---|---|---|
P1 | T1N0M0 | well-differentiated |
P2 | T4N0M0 | well-differentiated |
P3 | T2N0M0 | poorly-differentiated |
References
- Eck, S.M.; Cote, A.L.; Winkelman, W.D.; Brinckerhoff, C.E. CXCR4 and matrix metalloproteinase-1 are elevated in breast carcinoma-associated fibroblasts and in normal mammary fibroblasts exposed to factors secreted by breast cancer cells. Mol. Cancer Res. 2009, 7, 1033–1044. [Google Scholar] [CrossRef] [PubMed]
- Gonda, T.A.; Varro, A.; Wang, T.C.; Tycko, B. Molecular biology of cancer-associated fibroblasts: Can these cells be targeted in anti-cancer therapy? Semin. Cell Dev. Biol. 2010, 21, 2–10. [Google Scholar] [CrossRef] [PubMed]
- Ayala, G.; Tuxhorn, J.A.; Wheeler, T.M.; Frolov, A.; Scardino, P.T.; Ohori, M.; Wheeler, M.; Spitler, J.; Rowley, D.R. Reactive stroma as a predictor of biochemical-free recurrence in prostate cancer. Clin. Cancer Res. 2003, 9, 4792–4801. [Google Scholar] [PubMed]
- Bhowmick, N.A.; Neilson, E.G.; Moses, H.L. Stromal fibroblasts in cancer initiation and progression. Nature 2004, 432, 332–337. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Suetsugu, A.; Osawa, Y.; Nagaki, M.; Saji, S.; Moriwaki, H.; Bouvet, M.; Hoffman, R.M. Imaging the recruitment of cancer-associated fibroblasts by liver-metastatic colon cancer. J. Cell. Biochem. 2011, 112, 949–953. [Google Scholar] [CrossRef] [PubMed]
- Olumi, A.F.; Grossfeld, G.D.; Hayward, S.W.; Carroll, P.R.; Tlsty, T.D.; Cunha, G.R. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 1999, 59, 5002–5011. [Google Scholar] [PubMed]
- Noel, A.; De Pauw-Gillet, M.C.; Purnell, G.; Nusgens, B.; Lapiere, C.M.; Foidart, J.M. Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts. Br. J. Cancer 1993, 68, 909–915. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- De Wever, O.; Demetter, P.; Mareel, M.; Bracke, M. Stromal myofibroblasts are drivers of invasive cancer growth. Int. J. Cancer 2008, 123, 2229–2238. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sappino, A.P.; Skalli, O.; Jackson, B.; Schurch, W.; Gabbiani, G. Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. Int. J. Cancer 1988, 41, 707–712. [Google Scholar] [CrossRef] [PubMed]
- Lazard, D.; Sastre, X.; Frid, M.G.; Glukhova, M.A.; Thiery, J.P.; Koteliansky, V.E. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc. Natl. Acad. Sci. USA 1993, 90, 999–1003. [Google Scholar] [CrossRef]
- Radisky, D.C.; Kenny, P.A.; Bissell, M.J. Fibrosis and cancer: Do myofibroblasts come also from epithelial cells via EMT? J. Cell. Biochem. 2007, 101, 830–839. [Google Scholar] [CrossRef]
- Elmusrati, A.A.; Pilborough, A.E.; Khurram, S.A.; Lambert, D.W. Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma. Br. J. Cancer 2017, 117, 867–875. [Google Scholar] [CrossRef]
- Malanchi, I.; Santamaria-Martinez, A.; Susanto, E.; Peng, H.; Lehr, H.A.; Delaloye, J.F.; Huelsken, J. Interactions between cancer stem cells and their niche govern metastatic colonization. Nature 2011, 481, 85–89. [Google Scholar] [CrossRef] [Green Version]
- Kinugasa, Y.; Matsui, T.; Takakura, N. CD44 expressed on cancer-associated fibroblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cells in the tumor microenvironment. Stem. Cells 2014, 32, 145–156. [Google Scholar] [CrossRef]
- Russo, M.W.; Sevetson, B.R.; Milbrandt, J. Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription. Proc. Natl. Acad. Sci. USA 1995, 92, 6873–6877. [Google Scholar] [CrossRef]
- Svaren, J.; Sevetson, B.R.; Apel, E.D.; Zimonjic, D.B.; Popescu, N.C.; Milbrandt, J. NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli. Mol. Cell Biol. 1996, 16, 3545–3553. [Google Scholar] [CrossRef]
- Swirnoff, A.H.; Apel, E.D.; Svaren, J.; Sevetson, B.R.; Zimonjic, D.B.; Popescu, N.C.; Milbrandt, J. Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol. Cell Biol. 1998, 18, 512–524. [Google Scholar] [CrossRef]
- Madar, S.; Goldstein, I.; Rotter, V. ‘Cancer associated fibroblasts’—More than meets the eye. Trends Mol. Med. 2013, 19, 447–453. [Google Scholar] [CrossRef]
- Kunz-Schughart, L.A.; Knuechel, R. Tumor-associated fibroblasts (part I): Active stromal participants in tumor development and progression? Histol. Histopathol. 2002, 17, 599–621. [Google Scholar] [CrossRef]
- Elenbaas, B.; Weinberg, R.A. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp. Cell Res. 2001, 264, 169–184. [Google Scholar] [CrossRef]
- Tuxhorn, J.A.; Ayala, G.E.; Smith, M.J.; Smith, V.C.; Dang, T.D.; Rowley, D.R. Reactive stroma in human prostate cancer: Induction of myofibroblast phenotype and extracellular matrix remodeling. Clin. Cancer Res. 2002, 8, 2912–2923. [Google Scholar]
- Valentini, V.; de Paoli, A.; Gambacorta, M.A.; Mantini, G.; Ratto, C.; Vecchio, F.M.; Barbaro, B.; Innocente, R.; Rossi, C.; Boz, G.; et al. Infusional 5-fluorouracil and ZD1839 (Gefitinib-Iressa) in combination with preoperative radiotherapy in patients with locally advanced rectal cancer: A phase I and II Trial (1839IL/0092). Int. J. Radiat. Oncol. Biol. Phys. 2008, 72, 644–649. [Google Scholar] [CrossRef]
- Aref, A.R.; Huang, R.Y.; Yu, W.; Chua, K.N.; Sun, W.; Tu, T.Y.; Bai, J.; Sim, W.J.; Zervantonakis, I.K.; Thiery, J.P.; et al. Screening therapeutic EMT blocking agents in a three-dimensional microenvironment. Integr. Biol. 2013, 5, 381–389. [Google Scholar] [CrossRef] [Green Version]
- Naber, H.P.; Wiercinska, E.; Ten Dijke, P.; van Laar, T. Spheroid assay to measure TGF-beta-induced invasion. J. Vis. Exp. 2011, 57. [Google Scholar] [CrossRef]
- Bhattacharyya, S.; Wei, J.; Melichian, D.S.; Milbrandt, J.; Takehara, K.; Varga, J. The transcriptional cofactor nab2 is induced by tgf-Beta and suppresses fibroblast activation: Physiological roles and impaired expression in scleroderma. PLoS ONE 2009, 4, e7620. [Google Scholar] [CrossRef]
- Fang, M.; Yuan, J.; Peng, C.; Li, Y. Collagen as a double-edged sword in tumor progression. Tumour Biol. 2014, 35, 2871–2882. [Google Scholar] [CrossRef]
- Kim, J.; Kang, H.S.; Lee, Y.J.; Lee, H.J.; Yun, J.; Shin, J.H.; Lee, C.W.; Kwon, B.M.; Hong, S.H. EGR1-dependent PTEN upregulation by 2-benzoyloxycinnamaldehyde attenuates cell invasion and EMT in colon cancer. Cancer Lett. 2014, 349, 35–44. [Google Scholar] [CrossRef]
- Vinci, M.; Gowan, S.; Boxall, F.; Patterson, L.; Zimmermann, M.; Court, W.; Lomas, C.; Mendiola, M.; Hardisson, D.; Eccles, S.A. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol. 2012, 10, 29. [Google Scholar] [CrossRef]
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Choi, S.-Y.; Oh, S.Y.; Kang, S.H.; Kang, S.-M.; Kim, J.; Lee, H.-J.; Kwon, T.-G.; Kim, J.-W.; Hong, S.-H. NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression. Cancers 2019, 11, 388. https://doi.org/10.3390/cancers11030388
Choi S-Y, Oh SY, Kang SH, Kang S-M, Kim J, Lee H-J, Kwon T-G, Kim J-W, Hong S-H. NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression. Cancers. 2019; 11(3):388. https://doi.org/10.3390/cancers11030388
Chicago/Turabian StyleChoi, So-Young, Su Young Oh, Soo Hyun Kang, Sung-Min Kang, Jinkyung Kim, Heon-Jin Lee, Tae-Geon Kwon, Jin-Wook Kim, and Su-Hyung Hong. 2019. "NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression" Cancers 11, no. 3: 388. https://doi.org/10.3390/cancers11030388