Genetic modifier HDAC6 was found to control tumor growth and halt metastasis in triple-negative breast cancer in vivo


Genetic modifier HDAC6 was found to control tumor growth and halt metastasis in triple-negative breast cancer in vivo, according to a new study published in the top-tier journal Cancer Research by investigators at the George Washington University (GW) Cancer Center.

Immunotherapy – the use of drugs to stimulate one’s own immune system to recognize and destroy cancer cells – has been wildly successful in melanoma and other cancers.

However, it has been less effective in breast cancer.

“There is an urgent medical need to find new ways to potentiate or increase the efficacy of immunotherapy in breast cancer, especially in aggressive and highly metastatic triple-negative breast cancer,” said Alejandro Villagra, Ph.D., member of the Cancer Biology Program at the GW Cancer Center and assistant professor of biochemistry and molecular medicine at the GW School of Medicine and Health Sciences.

“Our research lays the groundwork for a clinical trial that could lead to new, life-saving treatment options for breast cancer patients that do not respond to conventional immunotherapies.”

Molecularly targeted agents, such as HDAC6 inhibitors, have been widely described in the research literature as cytotoxic – toxic to both cancerous and healthy cells.

Villagra and his research team found new non-canonical regulatory properties of these epigenetic drugs, discovering that the inhibition of HDAC6 has a powerful and strong effect on the immune system unrelated to the previously cytotoxic properties attributed to HDAC inhibitors.

This research demonstrates for the first time that HDAC6 inhibitors can both improve response to immunotherapy and diminish the invasiveness of breast cancer, with minimal cytotoxic effects.

“We are excited about the work because, in addition to the potency of immunotherapy, this drug alone is capable of reducing metastasis,” said Villagra.

“This could have implications beyond breast cancer.”

This research was a multidisciplinary effort, made possible by collaborators across the GW Cancer Center, the GW School of Medicine and Health Sciences and the GW School of Engineering and Applied Sciences.

The project was funded by grants from the GW School of Medicine and Health Sciences, the National Institutes of Health, and the Melanoma Research Foundation.

“HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer” was published in Cancer Research, a journal of the American Association for Cancer Research.

Breast cancer is the most diagnosed cancer and a major cause of death in women worldwide [1]. Triple-negative breast cancer (TNBC) is described by the lack of estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2 [2].

Many reports have shown that TNBC is associated with poor prognosis and is more likely than other breast cancer to recur locally and metastasize to the lung and brain during 3–5 years after diagnosis [2,3].

Radiation therapy (RT) is one of the primary treatments for TNBC. RT reduces the risk of local recurrence and increases overall survival in in situ and infiltrating breast cancer [4].

However, due to the metastatic potential of RT and individual variation in radiosensitivity, many patients experience RT failure, which leads to cancer relapse and metastasis [5].

Therefore, it is necessary to develop novel strategies that can strengthen the effectiveness of radiotherapy.

The autophagy–lysosomal pathway and the ubiquitin–proteasome system (UPS) are two primary self-digestive mechanisms for cellular proteins. The UPS is the selective degradation pathway for the proteolysis of misfolded or short-lived proteins.

In this pathway, misfolded proteins binding with ubiquitin are degraded by proteasome [6]. Previous studies have demonstrated that excessive misfolded or unfolded proteins in the endoplasmic reticulum (ER) cause ER stress and induce unfolded protein response (UPR) pathways [7].

ER stress and UPR pathways in cancer therapies provide very potential for the development of novel anti-cancer strategies [8]. If the stress is too severe, ER stress can also trigger cell death [9,10].

Evidence indicates that ER stress is a trigger of apoptosis and autophagy [8,11,12]. Furthermore, Williams et al. indicated that proteasome inhibitor induced ER stress and cell death in human colon cancer cells [9].

Therefore, the UPS may regulate ER stress. Recent evidence shows that the aggresome is an alternative system to the proteasome for the degradation of polyubiquitinated unfolded/misfolded proteins [13].

Aggresome formation finally causes autophagic clearance, which degrades many substrates (or cargoes) via the lysosomal pathway [14]. Histone deacetylase 6 (HDAC6) plays an important role in aggresomal protein degradation.

HDAC6 binds both dynein and polyubiquitinated proteins for transport to aggresomes [15]. Previously the authors reported that HDAC6 inhibitor panobinostat caused ER stress and autophagy in TNBC cells [16]. These findings suggest that the UPS is closely related to autophagy.

Autophagy is a protein degradation mechanism that recycles damaged organelles and long-lived proteins by transporting them in autophagosomes to lysosomes for degradation [17].

When exposed to adverse environments, such as nutrient starvation or hypoxia, cells induce autophagy to maintain their longevity [18]. In recent years, targeting autophagy to strengthen current treatments in several cancers, including breast cancer, has shown promising results [18,19,20].

Although autophagy plays a critical role in cancer therapy, the role of autophagy in controlling cancer cell death or survival is still being debated. Previous research has shown that ER stress in the breast cancer cells with defective apoptosis mechanism increases irradiation (IR)-induced autophagy and reinforces radiosensitivity.

The activation of autophagy through UPR mechanisms may serve as a potential radiosensitization strategy to strengthen the killing efficiency of RT in breast cancer cells [21].

Our previous studies found that a new histone deacetylase inhibitor (HDACi), TMU-35435, enhanced etoposide cytotoxicity by the proteasomal degradation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in TNBC [22].

TMU-35435 had antitumor and enhanced activity of the DNA demethylation reagent against human non-small cell lung cancer [23]. Therefore, we hypothesized that TMU-35435 can sensitize TNBC cells to IR.

We tested this hypothesis by exploring the impacts of TMU-35435 combined with IR on the UPS, ER stress and autophagy in TNBC cell lines. We examined whether TMU-35435 enhanced sensitivity to IR in vitro and in vivo.

Our observations provide novel perceptions into the mechanisms underlying TMU-35435-mediated radiosensitization that may be important for developing strategies to improve the efficiency of TNBC to RT.


Given our data and the existing evidence, we can construct an integrated network of protein aggregation, ER stress and autophagy, as shown in the schematic diagram in Figure 8.

Our results overall suggested that significantly enhanced cytotoxicity was exerted by the combined treatment compared with IR or TMU-35435 alone in TNBC cells.

Moreover, TMU-35435 suppressed the interaction of HDAC6 with dynein. Therefore, significant enhancement of protein aggregation and ER stress was found in the combination treatment.

Combined IR and TMU-35435 treatment increased autophagic flux and autophagic cell death. In the mouse model of orthotopic breast cancer, tumor growth was suppressed by combination treatment with IR and TMU-35435 through the induction of ER stress and autophagy.

Our current study indicated that TMU-35435 could serve as a radiosensitizer against TNBC.

An external file that holds a picture, illustration, etc.
Object name is cancers-11-01703-g008.jpg
Figure 8
TMU-35435 enhances radiation sensitivity through the induction of misfolded protein aggregation and autophagic cell death in triple-negative breast cancer (TNBC). IR causes the aggregation of misfolded protein and ER stress. TMU-35435 suppresses the interaction of HDAC6 with dynein and then causes misfolded protein aggregation. Furthermore, combined treatment-induced ER stress can cause autophagic cell death.


1. Torre L.A., Bray F., Siegel R.L., Ferlay J., Lortet-Tieulent J., Jemal A. Global cancer statistics, 2012. CA Cancer J. Clin. 2015;65:87–108. doi: 10.3322/caac.21262. [PubMed] [CrossRef] [Google Scholar]

2. Voduc K.D., Cheang M.C., Tyldesley S., Gelmon K., Nielsen T.O., Kennecke H. Breast cancer subtypes and the risk of local and regional relapse. J. Clin. Oncol. 2010;28:1684–1691. doi: 10.1200/JCO.2009.24.9284. [PubMed] [CrossRef] [Google Scholar]

3. Chen X., Yu X., Chen J., Zhang Z., Tuan J., Shao Z., Guo X., Feng Y. Analysis in early stage triple-negative breast cancer treated with mastectomy without adjuvant radiotherapy: Patterns of failure and prognostic factors. Cancer. 2013;119:2366–2374. doi: 10.1002/cncr.28085. [PubMed] [CrossRef] [Google Scholar]

4. Early Breast Cancer Trialists’ Collaborative Group Favourable and unfavourable effects on long-term survival of radiotherapy for early breast cancer: An overview of the randomised trials. Lancet. 2000;355:1757–1770. doi: 10.1016/S0140-6736(00)02263-7. [PubMed] [CrossRef] [Google Scholar]

5. Abdulkarim B.S., Cuartero J., Hanson J., Deschenes J., Lesniak D., Sabri S. Increased risk of locoregional recurrence for women with T1-2N0 triple-negative breast cancer treated with modified radical mastectomy without adjuvant radiation therapy compared with breast-conserving therapy. J. Clin. Oncol. 2011;29:2852–2858. doi: 10.1200/JCO.2010.33.4714. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

6. Wojcik S. Crosstalk between autophagy and proteasome protein degradation systems: Possible implications for cancer therapy. Folia Histochem. Cytobiol. 2013;51:249–264. doi: 10.5603/FHC.2013.0036. [PubMed] [CrossRef] [Google Scholar]

7. Walter P., Ron D. The unfolded protein response: From stress pathway to homeostatic regulation. Science. 2011;334:1081–1086. doi: 10.1126/science.1209038. [PubMed] [CrossRef] [Google Scholar]

8. Wang M., Law M.E., Castellano R.K., Law B.K. The unfolded protein response as a target for anticancer therapeutics. Crit. Rev. Oncol. Hematol. 2018;127:66–79. doi: 10.1016/j.critrevonc.2018.05.003. [PubMed] [CrossRef] [Google Scholar]

9. Williams J.A., Hou Y., Ni H.M., Ding W.X. Role of intracellular calcium in proteasome inhibitor-induced endoplasmic reticulum stress, autophagy, and cell death. Pharm. Res. 2013;30:2279–2289. doi: 10.1007/s11095-013-1139-8. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

10. Ding W.X., Ni H.M., Gao W., Hou Y.F., Melan M.A., Chen X., Stolz D.B., Shao Z.M., Yin X.M. Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J. Biol. Chem. 2007;282:4702–4710. doi: 10.1074/jbc.M609267200. [PubMed] [CrossRef] [Google Scholar]

11. Li X., Zhu H., Huang H., Jiang R., Zhao W., Liu Y., Zhou J., Guo F.J. Study on the effect of IRE1a on cell growth and apoptosis via modulation PLK1 in ER stress response. Mol. Cell. Biochem. 2012;365:99–108. doi: 10.1007/s11010-012-1248-4. [PubMed] [CrossRef] [Google Scholar]

12. Cheng Y., Yang J.M. Survival and death of endoplasmic-reticulum-stressed cells: Role of autophagy. World J. Biol. Chem. 2011;2:226–231. doi: 10.4331/wjbc.v2.i10.226. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

13. Bennett E.J., Bence N.F., Jayakumar R., Kopito R.R. Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol. Cell. 2005;17:351–365. doi: 10.1016/j.molcel.2004.12.021. [PubMed] [CrossRef] [Google Scholar]

14. Garcia-Mata R., Gao Y.S., Sztul E. Hassles with taking out the garbage: Aggravating aggresomes. Traffic. 2002;3:388–396. doi: 10.1034/j.1600-0854.2002.30602.x. [PubMed] [CrossRef] [Google Scholar]

15. Kawaguchi Y., Kovacs J.J., McLaurin A., Vance J.M., Ito A., Yao T.P. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell. 2003;115:727–738. doi: 10.1016/S0092-8674(03)00939-5. [PubMed] [CrossRef] [Google Scholar]

16. Rao R., Balusu R., Fiskus W., Mudunuru U., Venkannagari S., Chauhan L., Smith J.E., Hembruff S.L., Ha K., Atadja P., et al. Combination of pan-histone deacetylase inhibitor and autophagy inhibitor exerts superior efficacy against triple-negative human breast cancer cells. Mol. Cancer Ther. 2012;11:973–983. doi: 10.1158/1535-7163.MCT-11-0979. [PubMed] [CrossRef] [Google Scholar]

17. Lin N.Y., Beyer C., Giessl A., Kireva T., Scholtysek C., Uderhardt S., Munoz L.E., Dees C., Distler A., Wirtz S., et al. Autophagy regulates TNFalpha-mediated joint destruction in experimental arthritis. Ann. Rheum. Dis. 2013;72:761–768. doi: 10.1136/annrheumdis-2012-201671. [PubMed] [CrossRef] [Google Scholar]

18. Taylor M.A., Das B.C., Ray S.K. Targeting autophagy for combating chemoresistance and radioresistance in glioblastoma. Apoptosis. 2018;23:563–575. doi: 10.1007/s10495-018-1480-9. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

19. Di Fazio P., Matrood S. Targeting autophagy in liver cancer. Transl. Gastroenterol. Hepatol. 2018;3:39. doi: 10.21037/tgh.2018.06.09. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

20. Han Y., Fan S., Qin T., Yang J., Sun Y., Lu Y., Mao J., Li L. Role of autophagy in breast cancer and breast cancer stem cells (Review) Int. J. Oncol. 2018;52:1057–1070. doi: 10.3892/ijo.2018.4270. [PubMed] [CrossRef] [Google Scholar]

21. Kim K.W., Moretti L., Mitchell L.R., Jung D.K., Lu B. Endoplasmic reticulum stress mediates radiation-induced autophagy by perk-eIF2alpha in caspase-3/7-deficient cells. Oncogene. 2010;29:3241–3251. doi: 10.1038/onc.2010.74. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

22. Wu Y.H., Hong C.W., Wang Y.C., Huang W.J., Yeh Y.L., Wang B.J., Wang Y.J., Chiu H.W. A novel histone deacetylase inhibitor TMU-35435 enhances etoposide cytotoxicity through the proteasomal degradation of DNA-PKcs in triple-negative breast cancer. Cancer Lett. 2017;400:79–88. doi: 10.1016/j.canlet.2017.04.023. [PubMed] [CrossRef] [Google Scholar]

23. Shieh J.M., Tang Y.A., Hu F.H., Huang W.J., Wang Y.J., Jen J., Liao S.Y., Lu Y.H., Yeh Y.L., Wang T.W., et al. A histone deacetylase inhibitor enhances expression of genes inhibiting Wnt pathway and augments activity of DNA demethylation reagent against nonsmall-cell lung cancer. Int. J. Cancer. 2017;140:2375–2386. doi: 10.1002/ijc.30664. [PubMed] [CrossRef] [Google Scholar]

More information: Debarati Banik et al, HDAC6 Plays a Noncanonical Role in the Regulation of Antitumor Immune Responses, Dissemination, and Invasiveness of Breast Cancer, Cancer Research (2020). DOI: 10.1158/0008-5472.CAN-19-3738


Please enter your comment!
Please enter your name here

Questo sito usa Akismet per ridurre lo spam. Scopri come i tuoi dati vengono elaborati.