Human urothelial carcinoma (UC) has a high tendency to recur and progress to life-threatening advanced diseases. Advanced therapeutic regimens are needed to control UC development and recurrence.
We pursued in vitro and in vivo studies to understand the ability of a triple combination of gemcitabine, romidepsin, and cisplatin (Gem+Rom+Cis) to modulate signalling pathways, cell death, drug resistance, and tumour development.
Our studies verified the ability of Gem+Rom+Cis to synergistically induce apoptotic cell death and reduce drug resistance in various UC cells. The ERK pathway and reactive oxygen species (ROS) played essential roles in mediating Gem+Rom+Cis-induced caspase activation, DNA oxidation and damage, glutathione reduction, and unfolded protein response. Gem+Rom+Cis preferentially induced death and reduced drug resistance in oncogenic H-Ras-expressing UC vs. counterpart cells that was associated with transcriptomic profiles related to ROS, cell death, and drug resistance. Our studies also verified the efficacy and safety of the Gem plus Rom+Cis regimen in controlling UC cell-derived xenograft tumour development and resistance.
More than 80% of UCs are associated with aberrant Ras-ERK pathway. Thus the compensatory combination of Rom with Gem and Cis should be seriously considered as an advanced regimen for treating advanced UCs, especially Ras-ERK-activated UCs.
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American Cancer Society. Cancer Facts & Figures 2019. http://www.cancer.org/research/cancerfactsfigures/index (2019).
Kamat, A. M., Hahn, N. M., Efstathiou, J. A., Lerner, S. P., Malmström, P. U., Choi, W. et al. Bladder cancer. Lancet 388, 2796–2810 (2016).
Massari, F., Santoni, M., Ciccarese, C., Brunelli, M., Conti, A., Santini, D. et al. Emerging concepts on drug resistance in bladder cancer: Implications for future strategies. Crit. Rev. Oncol. Hematol. 96, 81–90 (2015).
Teply, B. A. & Kim, J. J. Systemic therapy for bladder cancer - a medical oncologist’s perspective. J. Solid Tumors 4, 25–35 (2014).
von der Maase, H., Sengelov, L., Roberts, J. T., Ricci, S., Dogliotti, L., Oliver, T. et al. Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. J. Clin. Oncol. 23, 4602–4608 (2015).
Cognetti, F., Ruggeri, E. M., Felici, A., Gallucci, M., Muto, G., Pollera, C. F. et al. Adjuvant chemotherapy with cisplatin and gemcitabine versus chemotherapy at relapse in patients with muscle-invasive bladder cancer submitted to radical cystectomy: an Italian, multicenter, randomized phase III trial. Ann. Oncol. 23, 695–700 (2012).
Ramos, P. & Bentires-Alj, M. Mechanism-based cancer therapy: resistance to therapy, therapy for resistance. Oncogene 34, 3617–3626 (2015).
Gifford, J. B., Huang, W., Zeleniak, A. E., Hindoyan, A., Wu, H., Donahue, T. R. et al. Expression of GRP78, master regulator of the unfolded protein response, increases chemoresistance in pancreatic ductal adenocarcinoma. Mol. Cancer Ther. 15, 1043–1052 (2016).
Sau, A., Pellizzari Tregno, F., Valentino, F., Federici, G. & Caccuri, A. M. Glutathione transferases and development of new principles to overcome drug resistance. Arch. Biochem. Biophys. 500, 116–122 (2010).
Bidnur, S., Savdie, R. & Black, P. C. Inhibiting immune checkpoints for the treatment of bladder cancer. Bladder Cancer 2, 15–25 (2016).
Xu, Y., Poggio, M., Jin, H. Y., Shi, Z., Forester, C. M., Wang, Y. et al. Translation control of the immune checkpoint in cancer and its therapeutic targeting. Nat. Med. 25, 301–311 (2019).
Choudhary, S., Sood, S. & Wang, H. C. Synergistic induction of cancer cell death and reduction of clonogenic resistance by cisplatin and FK228. Biochem. Biophys. Res. Commun. 436, 325–330 (2013).
Ueda, H., Nakajima, H., Hori, Y., Goto, T. & Okuhara, M. Action of FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum no. 968, on Ha-ras transformed NIH3T3 cells. Biosci. Biotechnol. Biochem. 58, 1579–1583 (1994).
Bertino, E. M. & Otterson, G. A. Romidepsin: a novel histone deacetylase inhibitor for cancer. Expert Opin. Investig. Drugs 20, 1151–1158 (2011).
Tan, J., Cang, S., Ma, Y., Petrillo, R. L. & Liu, D. Novel histone deacetylase inhibitors in clinical trials as anti-cancer agents. J. Hematol. Oncol. 3, 5 (2010).
Choudhary, S. & Wang, H. C. Proapoptotic ability of oncogenic H-Ras to facilitate apoptosis induced by histone deacetylase inhibitors in human cancer cells. Mol. Cancer Ther. 6, 1099–1111 (2007).
Choudhary, S. & Wang, H. C. Role of reactive oxygen species in proapoptotic ability of oncogenic H-Ras to increase human bladder cancer cell susceptibility to histone deacetylase inhibitor for caspase induction. J. Cancer Res. Clin. Oncol. 135, 1601–1613 (2009).
Choudhary, S., Rathore, K. & Wang, H. C. FK228 and oncogenic H-Ras synergistically induce Mek1/2 and Nox-1 to generate reactive oxygen species for differential cell death. Anticancer Drugs 21, 831–840 (2010).
Choudhary, S., Rathore, K. & Wang, H. C. Differential induction of reactive oxygen species through Erk1/2 and Nox-1 by FK228 for preferential apoptosis of oncogenic H-Ras-expressing human urinary bladder cancer J82 cells. J. Cancer Res. Clin. Oncol. 137, 471–480 (2011).
Choudhary, S., Wang, K. K. & Wang, H. C. Oncogenic H-Ras, FK228, and exogenous H2O2 cooperatively activated the ERK pathway in preferential induction of human urinary bladder cancer J82 cell death. Mol. Carcinog. 50, 215–219 (2011).
Pluchino, L. A., Choudhary, S. & Wang, H. C. Reactive oxygen species-mediated synergistic and preferential induction of cell death and reduction of clonogenic resistance in breast cancer cells by combined cisplatin and FK228. Cancer Lett. 381, 124–132 (2016).
Olive, P. L. & Banath, J. P. The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23–29 (2006).
Collins, A. R., Duthie, S. J. & Dobson, V. L. Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733–1735 (1993).
John, B. A., Xu, T., Ripp, S. & Wang, H. C. A real-time non-invasive auto-bioluminescent urinary bladder cancer xenograft model. Mol. Imaging Biol. 19, 10–14 (2016).
Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).
Simes, R. J. An improved Bonferroni procedure for multiple tests of significance. Biometrika 73, 751–754 (1986).
Chou, T. C. & Talalay, P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. 22, 27–55 (1984).
Yin, T., Zhang, Z., Cao, B., Duan, Q., Shi, P., Zhao, H. et al. Bmi1 inhibition enhances the sensitivity of pancreatic cancer cells to gemcitabine. Oncotarget 7, 37192–37204 (2016).
Ju, H. Q., Gocho, T., Aguilar, M., Wu, M., Zhuang, Z. N., Fu, J. et al. Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through the Redox Modulation. Mol. Cancer Ther. 14, 788–798 (2015).
Valdez, B. C., Brammer, J. E., Li, Y., Murray, D., Teo, E. C., Liu, Y. et al. Romidepsin enhances the cytotoxicity of fludarabine, clofarabine and busulfan combination in malignant T-cells. Leuk. Res. 47, 100–108 (2016).
Miyajima, A., Nakashima, J., Tachibana, M., Nakamura, K., Hayakawa, M. & Murai, M. N-acetylcysteine modifies cis-dichlorodiammineplatinum-induced effects in bladder cancer cells. Jpn. J. Cancer Res. 90, 565–570 (1999).
Kim, H. J., Lee, J. H., Kim, S. J., Oh, G. S., Moon, H. D., Kwon, K. B. et al. Roles of NADPH oxidases in cisplatin-induced reactive oxygen species generation and ototoxicity. J. Neurosci. 30, 3933–3946 (2010).
Hecht, F., Pessoa, C. F., Gentile, L. B., Rosenthal, D., Carvalho, D. P. & Fortunato, R. S. The role of oxidative stress on breast cancer development and therapy. Tumor Biol. 37, 4281–4291 (2016).
Khongkow, P., Middleton, A. W., Wong, J. P., Kandola, N. K., Kongsema, M., de Moraes, G. N. et al. In vitro methods for studying the mechanisms of resistance to DNA-damaging therapeutic drugs. Methods Mol. Biol. 1395, 39–53 (2016).
Siddik, Z. H. Cisplatin: mode of action and molecular basis of resistance. Oncogene 22, 7265–7279 (2003).
Wang, H. C. & Choudhary, S. Reactive oxygen species-mediated therapeutic control of bladder cancer. Nat. Rev. Urol. 8, 608–616 (2001).
Chuang, J. I., Chang, T. Y. & Liu, H. S. Glutathione depletion-induced apoptosis of H-Ras-transformed NIH3T3 cells can be prevented by melatonin. Oncogene 22, 1349–1357 (2003).
Wang, Y. Y., Chen, W. H., Xiao, P. P., Xie, W., Luo, Q., Bork, P. et al. GEAR: a database of genomic elements associated with drug resistance. Sci. Rep. 7, 44085 (2017).
Yan, M. M., Ni, J. D., Song, D., Ding, M. & Huang, J. Interplay between unfolded protein response and autophagy promotes tumor drug resistance. Oncol. Lett. 10, 1959–1969 (2015).
Casas, C. GRP78 at the centre of the stage in cancer and neuroprotection. Front. Neurosci. 11, 177 (2017).
Piekarz, R. L., Frye, R., Prince, H. M., Kirschbaum, M. H., Zain, J., Allen, S. L. et al. Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma. Blood 117, 5827–5834 (2011).
DTP/DCTD/NCI/NIH/DHHS. Equivalent surface area dosage conversion factors. http://dtp.nci.nih.gov (2007).
Faustino-Rocha, A., Oliveira, P. A., Pinho-Oliveira, J., Teixeira-Guedes, C., Soares-Maia, R., da Costa, R. G. et al. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab. Anim. 42, 217–224 (2013).
The Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507, 315–322 (2014).
He, F., Melamed, J., Tang, M. S., Huang, C. & Wu, X. R. Oncogenic HRAS activates epithelial-to-mesenchymal transition and confers stemness to p53-deficient urothelial cells to drive muscle invasion of basal subtype carcinomas. Cancer Res. 75, 2017–2028 (2015).
Sarkisian, S. & Davar, D. MEK inhibitors for the treatment of NRAS mutant melanoma. Drug Des. Dev. Ther. 12, 2553–2565 (2018).
Heinzerling, L., Eigentler, T. K., Fluck, M., Hassel, J. C., Heller-Schenck, D., Leipe, J. et al. Tolerability of BRAF/MEK inhibitor combinations: adverse event evaluation and management. ESMO Open 4, e000491 (2019).
Sun, J., Zager, J. S. & Eroglu, Z. Encorafenib/binimetinib for the treatment of BRAF-mutant advanced, unresectable, or metastatic melanoma: design, development, and potential place in therapy. Onco Targets Ther. 11, 9081–9089 (2018).
Choudhary, S. & Wang, H. R. Pro-apoptotic activity of oncogenic H-Ras for histone deacetylase inhibitor to induce apoptosis of human cancer HT29 cells. J. Cancer Res. Clin. Oncol. 133, 725–739 (2007).
Choi, Y. M., Kim, H. K., Shim, W., Anwar, M. A., Kwon, J. W., Kwon, H. K. et al. Mechanism of cisplatin-induced cytotoxicity is correlated to impaired metabolism due to mitochondrial ROS generation. PLoS ONE 10, e0135083 (2015).
Jones, R. M., Kotsantis, P., Stewart, G. S., Groth, P. & Petermann, E. BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine. Mol. Cancer Ther. 13, 2412–2421 (2014).
Rudin, C. M., Yang, Z., Schumaker, L. M., VanderWeele, D. J., Newkirk, K., Egorin, M. J. et al. Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res. 63, 312–318 (2003).
Godwin, A. K., Meister, A., O’Dwyer, P., Huang, C. S., Hamilton, T. C. & Anderson, M. E. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc. Natl Acad. Sci. USA 89, 3070–3074 (1992).
Gifford, J. B. & Hill, R. GRP78 influences chemoresistance and prognosis in cancer. Curr. Drug Targets 19, 701–708 (2018).
Chern, Y. J., Wong, J. C. T., Cheng, G. S. W., Yu, A., Yin, Y., Schaeffer, D. F. et al. The interaction between SPARC and GRP78 interferes with ER stress signaling and potentiates apoptosis via PERK/eIF2α and IRE1α/XBP-1 in colorectal cancer. Cell Death Dis. 10, 504 (2019).
Burikhanov, R., Zhao, Y., Goswami, A., Qiu, S., Schwarze, S. R. & Rangnekar, V. M. The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis. Cell 138, 377–388 (2009).
Wang, J., Li, Y., Ma, F., Zhou, H., Ding, R., Lu, B. et al. Inhibitory effect of Par-4 combined with cisplatin on human Wilms’ tumor cells. Tumor Biol. 39, 1010428317716689 (2017).
Qiu, S. G., Krishnan, S., el-Guendy, N. & Rangnekar, V. M. Negative regulation of Par-4 by oncogenic Ras is essential for cellular transformation. Oncogene 18, 7115–7123 (1999).
Mabe, N. W., Fox, D. B., Lupo, R., Decker, A. E., Phelps, S. N., Thompson, J. W. et al. Epigenetic silencing of tumor suppressor Par-4 promotes chemoresistance in recurrent breast cancer. J. Clin. Invest. 128, 4413–4428 (2018).
von der Masse, H. Gemcitabine and cisplatin in locally advanced and/or metastatic bladder cancer. Eur. J. Cancer 36, 13–16 (2000).
Roberts, J. T., von der Maase, H., Sengeløv, L., Conte, P. F., Dogliotti, L., Oliver, T. et al. Long-term survival results of a randomized trial comparing gemcitabine/cisplatin and methotrexate/vinblastine/doxorubicin/cisplatin in patients with locally advanced and metastatic bladder cancer. Ann. Oncol. 17, 118–122 (2006).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
We are grateful to Dr. L. Pluchino and Ms. D.J. Trent for technical supports, Dr. A. Odoi for statistical consultation, and Ms. A. Hand for textual editing of the manuscript.
Ethics approval and consent to participate
All animal procedures were approved by the University of Tennessee Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
All the data related to this study are included in this article and its supplementary file.
The authors declare no competing interests.
This study was supported by the National Institutes of Health [CA177834 to H.-C.R.W.] and the University of Tennessee, Center of Excellence in Livestock Diseases and Human Health [to H.-C.R.W.].
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Pattarawat, P., Hong, T., Wallace, S. et al. Compensatory combination of romidepsin with gemcitabine and cisplatin to effectively and safely control urothelial carcinoma. Br J Cancer 123, 226–239 (2020). https://doi.org/10.1038/s41416-020-0877-8