Kshitij Parag-Sharma1, Jason Tasoulas2, Adele M Musicant3, Carlos H Viesi do Nascimento-Filho4, Zhichuan Zhu5, Chloe Twomey6, Pengda Liu5, Rogerio M Castilho4, Antonio L Amelio7. 1. Graduate Curriculum in Cell Biology and Physiology, Biological and Biomedical Sciences Program, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 2. Lineberger Comprehensive Cancer Center, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Division of Oral and Craniofacial Health Sciences, UNC Adams School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 3. Graduate Curriculum in Genetics and Molecular Biology, Biological and Biomedical Sciences, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 4. Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA. 5. Lineberger Comprehensive Cancer Center, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Biochemistry and Biophysics, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 6. Carolina Research Scholar, Undergraduate Curriculum in Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 7. Division of Oral and Craniofacial Health Sciences, UNC Adams School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Cell Biology and Physiology, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Cancer Cell Biology Program, Lineberger Comprehensive Cancer Center, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Biomedical Research Imaging Center, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: antonio_amelio@unc.edu.
Abstract
OBJECTIVES: Mucoepidermoid carcinoma (MEC) is the most common type of salivary gland malignancy. Advanced or high-grade MECs are refractory to chemotherapy, often leading to tumor recurrence/metastasis and abysmal ~35% 5-year survival. Causal links have been established between Epithelial Growth Factor Receptor (EGFR) activation and poor outcome. Herein we investigated the therapeutic efficacy of EGFR inhibition against MEC using in vitro pre-clinical models. MATERIALS AND METHODS: Five human MEC cell lines were used in cell viability, cytotoxicity, apoptosis, cell cycle, 2D-clonogenicity, and 3D-spheroid formation assays following treatment with Erlotinib (EGFR inhibitor), SAHA (Histone Deacetylase inhibitor; HDAC) and CUDC-101 (dual EGFR-HDAC inhibitor). Effects on MEC cancer stem cells were evaluated using flow cytometry. Gene expression and pathway regulation were evaluated via qPCR and Western blot, respectively. RESULTS: MEC cells enter a quiescent, non-proliferative yet rapidly reversible drug tolerant state upon EGFR inhibition. Despite robust suppression of MEC cell proliferation, no discernable apoptosis is detected. Combination of EGFR and HDAC inhibitors exhibits synergistic effects, exerting ~5-fold more potent cell cytotoxicity compared to HDAC or EGFR monotherapy. CUDC-101, a single molecule with dual EGFR-HDAC inhibitor moieties, exerts irreversible and potent cytotoxic activity against MEC cells and blunts MEC cancer stem-cell tumorigenicity. CONCLUSION: MEC cells are intrinsically tolerant to EGFR inhibition. Combining EGFR and HDAC inhibitors exerts synergistic and potent cytotoxic effects, suggesting that EGFR inhibitors still hold significant promise against MEC. Future studies are needed to assess the applicability and efficacy of dual EGFR-HDAC inhibitors for the clinical management of MEC.
OBJECTIVES: Mucoepidermoid carcinoma (MEC) is the most common type of salivary gland malignancy. Advanced or high-grade MECs are refractory to chemotherapy, often leading to tumor recurrence/metastasis and abysmal ~35% 5-year survival. Causal links have been established between Epithelial Growth Factor Receptor (EGFR) activation and poor outcome. Herein we investigated the therapeutic efficacy of EGFR inhibition against MEC using in vitro pre-clinical models. MATERIALS AND METHODS: Five human MEC cell lines were used in cell viability, cytotoxicity, apoptosis, cell cycle, 2D-clonogenicity, and 3D-spheroid formation assays following treatment with Erlotinib (EGFR inhibitor), SAHA (Histone Deacetylase inhibitor; HDAC) and CUDC-101 (dual EGFR-HDAC inhibitor). Effects on MEC cancer stem cells were evaluated using flow cytometry. Gene expression and pathway regulation were evaluated via qPCR and Western blot, respectively. RESULTS: MEC cells enter a quiescent, non-proliferative yet rapidly reversible drug tolerant state upon EGFR inhibition. Despite robust suppression of MEC cell proliferation, no discernable apoptosis is detected. Combination of EGFR and HDAC inhibitors exhibits synergistic effects, exerting ~5-fold more potent cell cytotoxicity compared to HDAC or EGFR monotherapy. CUDC-101, a single molecule with dual EGFR-HDAC inhibitor moieties, exerts irreversible and potent cytotoxic activity against MEC cells and blunts MEC cancer stem-cell tumorigenicity. CONCLUSION: MEC cells are intrinsically tolerant to EGFR inhibition. Combining EGFR and HDAC inhibitors exerts synergistic and potent cytotoxic effects, suggesting that EGFR inhibitors still hold significant promise against MEC. Future studies are needed to assess the applicability and efficacy of dual EGFR-HDAC inhibitors for the clinical management of MEC.
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