Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M value). From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection (P < 0.05; ROC area under the curve = 0.96, 95% CI: 0.91–1.0) similar to the subset of four CDKN2A-specific CpG loci (0.90, 95% CI: 0.82–0.99) with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products.
Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares – Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9μM (range 6.6μM – 13.9μM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7μM (range, 8.8μM to 12.7μM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.
Objectives: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. Methods: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. Results: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. Conclusion: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals.
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