Methylation as a Mechanism of Cisplatin Resistance in Head and Neck Cancer

Tuesday, October 8, 2013: 1:25 PM
Chi T. Viet DDS, PhD, Bluestone Center for Clinical Research, New York University College of Dentistry, New York, NY
Dongmin Dang MD, New York, NY
Yi Ye PhD, New York, NY
Brian L. Schmidt DDS, MD, PhD,FACS, New York University, New York, NY
Cisplatin is the primary chemotherapy for head and neck squamous cell carcinoma (HNSCC).  No equally effective chemotherapeutics are available when cisplatin resistance occurs. We hypothesize that DNA methylation of key genes mediates cisplatin resistance; moreover, pre-treatment with decitabine, a demethylating agent, restores cisplatin sensitivity by mediating expression of genes that are instrumental to cisplatin resistance.

Objectives: 1) Determine whether decitabine treatment of a cisplatin-resistant HNSCC cell line restores the anti-proliferative and apoptotic effects of cisplatin. 2) Evaluate the anti-proliferative effect of decitabine and cisplatin (i.e. combination treatment) on a preclinical HNSCC model. 3) Determine whether combination treatment reduces cancer pain. 4) Create a “gene expression profile of cisplatin resistance” by analyzing cisplatin-sensitive and cisplatin-resistant HNSCC in patients.

Methods: SCC-25, a cisplatin-sensitive HNSCC cell line, and SCC-25/CP, a cisplatin-resistant cell line, were pre-treated with 5µM decitabine and then treated with cisplatin (3-300 µM) for 48 hours. Proliferation was quantified with an MTS assay.  Apoptosis was quantified with a caspase 3/7 assay. A preclinical model was created by inoculating SCC-25/CP cells into the hind-paw of BALB/c mice. Twenty-four mice were placed into one of four treatment groups: control sham, decitabine-only, cisplatin-only, or combination treatment. Decitabine (6 mg/kg) was administered on post-inoculation days (PID) 7 and 9, and cisplatin (6 mg/kg) was administered on PID 12, 15, 18, and 21. Tumor growth was quantified. Mechanical allodynia (i.e. pain) was quantified with a paw withdrawal assay. Formalin-fixed, paraffin-embedded biopsies were obtained from HNSCC patients who underwent chemotherapy with cisplatin. Tumors were classified as either cisplatin-sensitive (RECIST 3 or 4) or cisplatin-resistant (RECIST 1 or 2). Gene expression was quantified in these two sets of samples.

Results: In the in vitro model, pre-treatment with decitabine restored cisplatin sensitivity in the cisplatin-resistant line (SCC-25/CP), and reduced the cisplatin dose required to inhibit proliferation or activate apoptosis to levels comparable to the cisplatin-sensitive line (SCC-25). In the preclinical model, combination treatment of decitabine and cisplatin resulted in significant reduction of tumor growth compared to control, whereas decitabine or cisplatin treatment alone did not. Combination treatment also significantly reduced mechanical allodynia. We quantified gene expression of IGFBP-3, hMLH1, S100, and SAT1, which are genes shown to correlate with cisplatin response in other cancers. We showed that cisplatin-sensitive and cisplatin-resistant patient tumors have distinct expression profiles. Gene expression of IGFBP-3, hMLH1, S100, and SAT1 was higher in cisplatin-sensitive tumors than cisplatin-resistant tumors. Decitabine treatment of cisplatin-resistant HNSCC cells in vitro reversed gene expression toward a cisplatin-sensitive profile.

Conclusion: Decitabine restores cisplatin sensitivity in a preclinical HNSCC model, and has potential use in the HNSCC chemotherapeutic regimen for cisplatin-resistant tumors. The combination of cisplatin and decitabine significantly reduces HNSCC proliferation and HNSCC pain.


Appleton K, Mackay HJ, Judson I, Plumb JA, McCormick C, Strathdee G, Lee C, Barrett S, Reade S, Jadayel D, Tang A, Bellenger K, Mackay L, Setanoians A, Schatzlein A, Twelves C, Kaye SB, Brown R (2007) Phase I and pharmacodynamic trial of the DNA methyltransferase inhibitor decitabine and carboplatin in solid tumors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 25:4603-4609.

Viet CT, Ye Y, Dang D, Lam DK, Achdjian S, Zhang J, Schmidt BL (2011) Re-expression of the methylated EDNRB gene in oral squamous cell carcinoma attenuates cancer-induced pain. Pain 152:2323-2332.