Article | Published:

Cellular and Molecular Biology

Suppression of TGFβ-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition

British Journal of Cancervolume 118pages13591368 (2018) | Download Citation



Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy.


We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo.


From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFβ-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo.


Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.

  • Subscribe to British Journal of Cancer for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Additional information

Note: This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License.


  1. 1.

    Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6, 392–401 (2006).

  2. 2.

    Marusyk, A. et al. Spatial proximity to fibroblasts impacts molecular features and therapeutic sensitivity of breast cancer cells influencing clinical outcomes. Cancer Res. 76, 6495–6506 (2016).

  3. 3.

    Caruana, I. et al. Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes. Nat. Med. 21, 524–529 (2015).

  4. 4.

    Tirosh, I. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Sci. Am. Assoc. Adv. Sci. 352, 189–196 (2016).

  5. 5.

    Hirata, E. et al. Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK signaling. Cancer Cell 27, 574–588 (2015).

  6. 6.

    Chen, D. S. & Mellman, I. Elements of cancer immunity and the cancer?immune set point. Nature 541, 321–330 (2017).

  7. 7.

    Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009).

  8. 8.

    Loeffler, M., Krüger, J. A., Niethammer, A. G. & Reisfeld, R. A. Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake. J. Clin. Invest. 116, 1955–1962 (2006).

  9. 9.

    Chauhan, V. P. et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat. Commun. 4, 2516 (2013).

  10. 10.

    Quante, M. et al. Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth. Cancer Cell 19, 257–272 (2011).

  11. 11.

    Erez, N., Truitt, M., Olson, P., Arron, S. T. & Hanahan, D. Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-kappaB-dependent manner. Cancer Cell 17, 135–147 (2010).

  12. 12.

    Haviv, I., Polyak, K., Qiu, W., Hu, M. & Campbell, I. Origin of carcinoma associated fibroblasts. Cell Cycle 8, 589–595 (2009).

  13. 13.

    Xiao, L. et al. Tumor endothelial cells with distinct patterns of TGFβ-driven endothelial-to-mesenchymal transition. Cancer Res. 75, 1244–1254 (2015).

  14. 14.

    Massagué, J. TGFβ signalling in context. Nat. Rev. Mol. Cell Biol. 13, 616–630 (2012).

  15. 15.

    Dumont, N. et al. Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers. Proc. Natl Acad. Sci. 105, 14867–14872 (2008).

  16. 16.

    Bechtel, W. et al. Methylation determines fibroblast activation and fibrogenesis in the kidney. Nat. Med. 16, 544–550 (2010).

  17. 17.

    Weigel, C., Schmezer, P., Plass, C. & Popanda, O. Epigenetics in radiation-induced fibrosis. Oncogene 34, 2145–2155 (2015).

  18. 18.

    Kojima, Y. et al. Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts. Proc. Natl Acad. Sci. 107, 20009–20014 (2010).

  19. 19.

    Polyak, K. & Weinberg, R. A. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat. Rev. Cancer 9, 265–273 (2009).

  20. 20.

    Bolden, J. E., Peart, M. J. & Johnstone, R. W. Anticancer activities of histone deacetylase inhibitors. Nat. Rev. Drug Discov. 5, 769–784 (2006).

  21. 21.

    Xiao, L., Harrell, J. C., Perou, C. M. & Dudley, A. C. Identification of a stable molecular signature in mammary tumor endothelial cells that persists in vitro. Angiogenesis 17, 511–518 (2014). Springer Netherlands.

  22. 22.

    Xiao, L., McCann, J. V., Dudley, A. C. Isolation and culture expansion of tumor-specific endothelial cells. J. Vis. Exp. 14, e53072–e53072 (2015).

  23. 23.

    Dudley, A. C., Shih, S.-C., Cliffe, A. R., Hida, K. & Klagsbrun, M. Attenuated p53 activation in tumour-associated stromal cells accompanies decreased sensitivity to etoposide and vincristine. Br. J. Cancer 99, 118–125 (2008).

  24. 24.

    Brauer, H. A. et al. Impact of tumor microenvironment and epithelial phenotypes on metabolism in breast cancer. Clin. Cancer Res. 19, 571–585 (2013).

  25. 25.

    Brown, A. C., Fiore, V. F., Sulchek, T. A. & Barker, T. H. Physical and chemical microenvironmental cues orthogonally control the degree and duration of fibrosis-associated epithelial-to-mesenchymal transitions. J. Pathol. 229, 25–35 (2012).

  26. 26.

    Adams, J. C., Bentley, A. A., Kvansakul, M., Hatherley, D. & Hohenester, E. Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region. J. Cell Sci. 121, 784–795 (2008).

  27. 27.

    Magness, S. T., Bataller, R., Yang, L. & Brenner, D. A. A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations. Hepatology 40, 1151–1159 (2004).

  28. 28.

    Ding, S. et al. Mucosal healing and fibrosis after acute or chronic inflammation in wild type FVB-N mice and C57BL6 procollagen α1(I)-promoter-GFP reporter mice. PLoS ONE 7, e42568 (2012).

  29. 29.

    Dunleavey, J. M. et al. Vascular channels formed by subpopulations of PECAM1(+) melanoma cells. Nat. Commun. 5, 5200 (2014).

  30. 30.

    Keen, J. C. et al. A novel histone deacetylase inhibitor, scriptaid, enhances expression of functional estrogen receptor alpha (ER) in ER negative human breast cancer cells in combination with 5-aza 2’-deoxycytidine. Breast Cancer Res. Treat. 81, 177–186 (2003).

  31. 31.

    Hinz, B., Celetta, G., Tomasek, J. J., Gabbiani, G. & Chaponnier, C. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol. Biol. Cell 12, 2730–2741 (2001).

  32. 32.

    Parast, M. M. & Otey, C. A. Characterization of palladin, a novel protein localized to stress fibers and cell adhesions. J. Cell Biol. 150, 643–656 (2000).

  33. 33.

    Labernadie, A. et al. A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nat. Cell Biol. 19, 224–237 (2017).

  34. 34.

    Jenkins, M. H. et al. Multiple murine BRaf V600Emelanoma cell lines with sensitivity to PLX4032. Pigment Cell Melanoma Res. 27, 495–501 (2014).

  35. 35.

    West, A. C. & Johnstone, R. W. New and emerging HDAC inhibitors for cancer treatment. J. Clin. Invest. 124, 30–39 (2014).

  36. 36.

    Nguyen, A. H., Elliott, I. A., Wu, N., Matsumura, C. Histone deacetylase inhibitors provoke a tumor supportive phenotype in pancreatic cancer associated fibroblasts. Oncotarget 8, 19074–19088 (2017).

  37. 37.

    Pazolli, E. et al. Chromatin remodeling underlies the senescence-associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression. Cancer Res. 72, 2251–2261 (2012).

  38. 38.

    Fisher, D. T., Appenheimer, M. M. & Evans, S. S. The two faces of IL-6 in the tumor microenvironment. Semin. Immunol. 26, 38–47 (2014).

  39. 39.

    Zheng, H. et al. HDAC inhibitors enhance T-cell chemokine expression and augment response to PD-1 immunotherapy in lung adenocarcinoma. Clin. Cancer Res. 22, 4119–4132 (2016).

  40. 40.

    Goetz, J. G. et al. Biomechanical remodeling of the microenvironment by stromal Caveolin-1 favors tumor invasion and metastasis. Cell 146, 148–163 (2011).

  41. 41.

    Rönty, M. J. et al. Isoform-specific regulation of the actin-organizing protein palladin during TGF-β1-induced myofibroblast differentiation. J. Invest Dermatol. 126, 2387–2396 (2006).

  42. 42.

    Brentnall, T. A. et al. Arousal of cancer-associated stroma: overexpression of palladin activates fibroblasts to promote tumor invasion. In: D. Gullberg editor. PLoS ONE 7, e30219 (2012).

  43. 43.

    Goicoechea, S. M. et al. Palladin promotes invasion of pancreatic cancer cells by enhancing invadopodia formation in cancer-associated fibroblasts. Oncogene 33, 1265–1273 (2014).

  44. 44.

    Maleszewska, M., Gjaltema, R. A. F., Krenning, G., Harmsen, M. C. Enhancer of Zeste homolog-2 (EZH2) methyltransferase regulates transgelin/smooth muscle-22α expression in endothelial cells in response to interleukin-1β and transforming growth factor-β2. Cell. Signal. 27, 1–28 (2015).

  45. 45.

    Guo, W., Shan, B., Klingsberg, R. C., Qin, X. & Lasky, J. A. Abrogation of TGF- 1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition. AJP: Lung Cel. Mol. Physiol. 297, L864–L870 (2009).

  46. 46.

    Hu, M. et al. Distinct epigenetic changes in the stromal cells of breast cancers. Nat. Genet. 37, 899–905 (2005).

  47. 47.

    Salmon, M., Gomez, D., Greene, E., Shankman, L. & Owens, G. K. Cooperative binding of KLF4, pELK-1, and HDAC2 to a G/C repressor element in the SM22α promoter mediates transcriptional silencing during SMC phenotypic switching in vivo. Circ. Res. 111, 685–696 (2012).

  48. 48.

    Glénisson, W., Castronovo, V. & Waltregny, D. Histone deacetylase 4 is required for TGFbeta1-induced myofibroblastic differentiation. Biochim. Biophys. Acta 1773, 1572–1582 (2007).

  49. 49.

    Waltregny, D. et al. Histone deacetylase HDAC8 associates with smooth muscle alpha-actin and is essential for smooth muscle cell contractility. FASEB J. 19, 966–968 (2005).

  50. 50.

    Barter, M. J. et al. HDAC-mediated control of ERK- and PI3K-dependent TGF-β-induced extracellular matrix-regulating genes. Matrix Biol. 29, 602–612 (2010).

  51. 51.

    Albrengues, J. et al. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat. Commun. 6, 1–15 (2015).

  52. 52.

    Kroesen, M. et al. HDAC inhibitors and immunotherapy; a double edged sword?. Oncotarget 5, 6558–6572 (2014).

Download references

Author information


  1. Department of Microbiology, Immunology, and Cancer Biology, The University of Virginia, Charlottesville, VA, 22908, USA

    • Dae Joong Kim
    •  & Andrew C. Dudley
  2. National Cancer Institute, Tumor Angiogenesis Unit, Center for Cancer Research, Frederick, MD, 21702, USA

    • James M. Dunleavey
  3. Children’s Cancer Institute, Kensington, NSW 2750, Australia

    • Lin Xiao
  4. Department of Surgery, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    • David W. Ollila
  5. Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    • Melissa A. Troester
  6. Department of Cell Biology and Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    • Carol A. Otey
  7. Department of Biomedical Engineering, The University of Virginia, Charlottesville, VA, 22908, USA

    • Wei Li
    •  & Thomas H. Barker
  8. Emily Couric Cancer Center, The University of Virginia, Charlottesville, VA, 22908, USA

    • Andrew C. Dudley


  1. Search for Dae Joong Kim in:

  2. Search for James M. Dunleavey in:

  3. Search for Lin Xiao in:

  4. Search for David W. Ollila in:

  5. Search for Melissa A. Troester in:

  6. Search for Carol A. Otey in:

  7. Search for Wei Li in:

  8. Search for Thomas H. Barker in:

  9. Search for Andrew C. Dudley in:


DJ.K designed and carried out the experiments and wrote the manuscript. J.M.D. assisted with microarray analysis, L.X. isolated tumour associated endothelial cells, D.W.O. provided human melanoma biopsies, M.A.T. provided human breast tissue CAFs, C.A.O. provided palladin antibodies and advice on the experimental design, W.L. carried out atomic force microscopy and analysis, T.H.B. provided the atomic force microscope, reagents, materials, and advice for the experimental design. A.C.D. designed the experiments and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Ethics approval and consent

No human subjects were used. All animal experiments were performed in accordance with the University of North Carolina at Chapel Hill and the University of Virginia guidelines for animal handling and care.

Availability of materials

All materials and datasets will be made available by sending an email request to the corresponding author. Raw data from the array will also be deposited here (

Corresponding author

Correspondence to Andrew C. Dudley.

Electronic supplementary material

About this article

Publication history






Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.