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Chronic inflammation plays a fundamental role in tumor promotion, migration, and invasion. With the use of microarray profiling, a profound increase was observed for those transcripts involved in proinflammatory signaling in epidermal growth factor receptor (EGFR) inhibitor–treated head and neck squamous cell carcinoma (HNSCC) cells as compared with their respective controls. As such, it was hypothesized that EGFR inhibitor efficacy is offset by the proinflammatory response that these therapeutics conjure in HNSCC. Systematic evaluation of the clinical EGFR inhibitors—erlotinib, cetuximab, lapatinib, and panitumumab—revealed increased secretion of proinflammatory cytokines such as interleukins (IL-2, IL-4, IL-6, IL-8), granulocyte-macrophage colony-stimulating factor, TNF-α, and IFN-γ. Mechanistic focus on IL-6 revealed that erlotinib induced a time-dependent increase in IL-6 mRNA and protein expression. Importantly, exogenous IL-6 protected HNSCC cells from erlotinib-induced cytotoxicity, whereas tocilizumab, an IL-6 receptor antagonist, sensitized cells to erlotinib in vitro and in vivo. Inhibitors of NF-κB, p38, and JNK suppressed erlotinib-induced IL-6 expression, suggesting critical roles for NF-κB and MAPK in IL-6 regulation. Furthermore, knockdown of NADPH oxidase 4 (NOX4) suppressed erlotinib-induced proinflammatory cytokine expression. Taken together, these results demonstrate that clinical EGFR inhibitors induce the expression of proinflammatory cytokines via NOX4.Implications: The antitumor activity of EGFR inhibitors is reduced by activation of NOX4-mediated proinflammatory pathways in HNSCC. Mol Cancer Res; 11(12); 1574–84. ©2013 AACR.

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PDF file - 170K, EGFR knockout induced IL-6 protein expression in FaDu cells.

PDF file - 69K, Erlotinib induced IL-6 protein expression.

ZVAD protects HNSCC cells from erlotinib-induced cell death.

Human HNSCC tumors display elevated expression of IL-1 pathway components.

SQ20B Network list.

PCR primer description table.

Knockdown of TRIF does not reduce erlotinib-induced IL-6.

Cal-27 Network list.

Transient siRNA Knockdown of IL1R1 reduces erlotinib-induced IL-6 secretion.

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Current therapies for gut inflammation have not reached the desired specificity and are attended by unintended immune suppression. This study aimed to provide evidence for supporting a hypothesis that direct in vivo augmentation of the induction of gut-homing regulatory T (Treg) cells is a strategy of expected specificity for the treatment of chronic intestinal inflammation (e.g., inflammatory bowel disease). We showed that dendritic cells (DCs), engineered to de novo produce high concentrations of both 1,25-dihydroxyvitamin D, the active vitamin D metabolite, and retinoic acid, an active vitamin A metabolite, augmented the induction of T cells that express both the regulatory molecule Foxp3 and the gut-homing receptor CCR9 in vitro and in vivo. In vivo, the newly generated Ag-specific Foxp3+ T cells homed to intestines. Additionally, transfer of such engineered DCs robustly suppressed ongoing experimental colitis. Moreover, CD4+ T cells from spleens of the mice transferred with the engineered DCs suppressed experimental colitis in syngeneic hosts. The data suggest that the engineered DCs enhance regulatory function in CD4+ T cell population in peripheral lymphoid tissues. Finally, we showed that colitis suppression following in vivo transfer of the engineered DCs was significantly reduced when Foxp3+ Treg cells were depleted. The data indicate that maximal colitis suppression mediated by the engineered DCs requires Treg cells. Collectively, our data support that DCs de novo overproducing both 1,25-dihydroxyvitamin D and retinoic acid are a promising novel therapy for chronic intestinal inflammation.

Background Despite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab. Methods Parental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1β, and recombinant IL-1α and IL-1β were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4+ and CD8+ T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS). Results Cetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels. Conclusions Altogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs. Electronic supplementary material The online version of this article (10.1186/s40425-019-0550-z) contains supplementary material, which is available to authorized users.

EGFR is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). However, many patients with HNSCC respond poorly to the EGFR inhibitors (EGFRI) cetuximab and erlotinib, despite tumor expression of EGFR. Gene expression analysis of erlotinib-treated HNSCC cells revealed an upregulation of genes involved in MyD88-dependent signaling compared with their respective vehicle-treated cell lines. We therefore investigated whether MyD88-dependent signaling may reduce the antitumor efficacy of EGFRIs in HNSCC. Erlotinib significantly upregulated IL6 secretion in HNSCC cell lines, which our laboratory previously reported to result in reduced drug efficacy. Suppression of MyD88 expression blocked erlotinib-induced IL6 secretion in vitro and increased the antitumor activity of erlotinib in vivo. There was little evidence of Toll-like receptor or IL18 receptor involvement in erlotinib-induced IL6 secretion. However, suppression of IL1R signaling significantly reduced erlotinib-induced IL6 production. A time-dependent increase of IL1α but not IL1β was observed in response to erlotinib treatment, and IL1α blockade significantly increased the antitumor activity of erlotinib and cetuximab in vivo. A pan-caspase inhibitor reduced erlotinib-induced IL1α secretion, suggesting that IL1α was released because of cell death. Human HNSCC tumors showed higher IL1α mRNA levels compared with matched normal tissue, and IL1α was found to be negatively correlated with survival in patients with HNSCC. Overall, the IL1α/IL1R/MYD88/IL6 pathway may be responsible for the reduced antitumor efficacy of erlotinib and other EGFRIs, and blockade of IL1 signaling may improve the efficacy of EGFRIs in the treatment of HNSCC. Cancer Res; 75(8); 1657–67. ©2015 AACR.

Chronic inflammation plays a fundamental role in tumor promotion, migration, and invasion. With the use of microarray profiling, a profound increase was observed for those transcripts involved in proinflammatory signaling in epidermal growth factor receptor (EGFR) inhibitor–treated head and neck squamous cell carcinoma (HNSCC) cells as compared with their respective controls. As such, it was hypothesized that EGFR inhibitor efficacy is offset by the proinflammatory response that these therapeutics conjure in HNSCC. Systematic evaluation of the clinical EGFR inhibitors—erlotinib, cetuximab, lapatinib, and panitumumab—revealed increased secretion of proinflammatory cytokines such as interleukins (IL-2, IL-4, IL-6, IL-8), granulocyte-macrophage colony-stimulating factor, TNF-α, and IFN-γ. Mechanistic focus on IL-6 revealed that erlotinib induced a time-dependent increase in IL-6 mRNA and protein expression. Importantly, exogenous IL-6 protected HNSCC cells from erlotinib-induced cytotoxicity, whereas tocilizumab, an IL-6 receptor antagonist, sensitized cells to erlotinib in vitro and in vivo. Inhibitors of NF-κB, p38, and JNK suppressed erlotinib-induced IL-6 expression, suggesting critical roles for NF-κB and MAPK in IL-6 regulation. Furthermore, knockdown of NADPH oxidase 4 (NOX4) suppressed erlotinib-induced proinflammatory cytokine expression. Taken together, these results demonstrate that clinical EGFR inhibitors induce the expression of proinflammatory cytokines via NOX4. Implications: The antitumor activity of EGFR inhibitors is reduced by activation of NOX4-mediated proinflammatory pathways in HNSCC. Mol Cancer Res; 11(12); 1574–84. ©2013 AACR.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) including erlotinib have demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date compared to the EGFR monoclonal antibody cetuximab. Therefore we seek to identify molecular mechanisms involved in tumor response to erlotinib and ultimately improve the anti-tumor efficacy of erlotinib for HNSCC patients. We have shown that erlotinib-treated HNSCC cell lines induced the expression and secretion of a wide variety of genes involved in inflammation and immune response including interleukin-1 (IL-1) and interleukin-6 (IL-6) compared to control-treated cells. The secretion of IL-6 from HNSCC cells in response to erlotinib was found to be triggered by MyD88-dependent IL-1 signaling and suppression of IL-1 signaling (using a neutralizing IL-1α antibody) significantly enhanced the anti-tumor efficacy of erlotinib in vivo. In erlotinib-resistant HNSCC cell lines, IL-1 signaling was significantly upregulated compared to their respective parental cell lines. Blockade of IL-1 signaling using a neutralizing IL-1α antibody or an IL-1 receptor (IL-1R) antagonist was able to significantly suppress the growth of erlotinib-resistant HNSCC xenografts as a single agent and in combination with erlotinib which was associated with reduced immune cell recruitment and angiogenesis. Altogether, blockade of the IL-1 pathway may represent a novel strategy to improve tumor response to erlotinib and other EGFR TKIs in HNSCC patients. Citation Format: Andrean Simons, Aditya Stanam, Adam Koch, Madelyn Espinosa-Cotton, Katherine Gibson-Corley. Targeting IL-1 signaling to improve tumor response to erlotinib in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 48.

Treatment for head and neck squamous cell carcinoma (HNSCC) patients includes the epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab. However, response to cetuximab is low and resistance remains a critical issue. Therefore, it is imperative to develop strategies to enhance tumor response to this drug. Inflammation plays an important role in tumor progression by increasing the release of cytokines that promote tumor cell survival. Our prior work has shown that a variety of pro-inflammatory cytokines involved in tumor survival are upregulated upon treatment with EGFR inhibitors in HNSCC cells, suggesting that poor tumor response to EGFR inhibitors may be due to increased tumor-associated inflammation. Our lab has shown that the increased proinflammatory tumor response due to EGFR inhibition may be activated by interleukin-1 (IL-1) signaling, suggesting that the IL-1 pathway may be an important target to enhance the efficacy of anti-EGFR therapy. Here we investigated the effect of IL-1 blockade on HNSCC tumor response to cetuximab treatment. We found that cetuximab induced IL-6 secretion from SQ20B HNSCC cells and knockdown of the adapter protein MyD88, which is essential for IL-1 signaling, suppressed IL-6 secretion at baseline and in response to cetuximab treatment. Similarly, knock down of the IL-1 receptor (IL-1R) decreased baseline and cetuximab-induced IL-6 secretion, although IL-1R knockdown did not improve response to cetuximab in SQ20B xenografts in vivo. We additionally blocked IL-1 signaling using a recombinant IL-1R antagonist (IL-1RA). IL-1RA pre-treatment reduced baseline and cetuximab-induced IL-6 secretion in vitro. Although IL-1RA did not significantly enhance SQ20B tumor response to cetuximab, IL-1RA as a single agent significantly suppressed tumor growth in female mice but not in male mice. Finally, treatment with a human-specific neutralizing IL-1 alpha (IL-1α) antibody (but not IL-1 beta (IL-1β)) reduced baseline and cetuximab-induced IL-6 secretion in vitro, and significantly increased SQ20B tumor response to cetuximab in vivo. Altogether we propose that targeting IL-1α specifically may be a viable strategy to improve HNSCC tumor response to cetuximab. Citation Format: Madelyn Espinosa-Cotton, Ayana J. McLaren, Adam T. Koch, Andrean L, Simons. Targeting interleukin-1 to increase cetuximab efficacy in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3705. doi:10.1158/1538-7445.AM2017-3705

Epidermal growth factor receptor (EGFR) is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). However many HNSCC patients respond poorly to EGFR inhibitors (EGFRIs) despite tumor expression of EGFR. Gene expression analysis of erlotinib-treated HNSCC cell lines revealed an upregulation of genes involved in MyD88-dependent interleukin-6 (IL-6) expression compared to their respective vehicle treated cell lines. We therefore proposed that MyD88-dependent signaling may reduce the anti-tumor efficacy of the EGFR inhibitor erlotinib in HNSCC. Erlotinib significantly upregulated IL-6 secretion which we have previously reported to result in reduced drug efficacy and drug resistance (Fletcher et al, 2013). Suppression of MyD88 expression significantly blocked erlotinib-induced IL-6 secretion in vitro and increased the anti-tumor activity of erlotinib in vivo. There was little to no evidence of toll-like receptor (TLR) or interleukin-18 receptor (IL-18R) involvement in erlotinib-induced IL-6 secretion. However, suppression of the interleukin-1 receptor (IL-1R) signaling through pharmacologic and genetic methods significantly reduced erlotinib-induced IL-6 production and increased HNSCC cell sensitivity to erlotinib in vitro. A time-dependent increase of IL-1 alpha (IL-1α) but not IL-1 beta (IL-1β) was observed in response to erlotinib treatment and IL-1α blockade significantly increased the anti-tumor activity of erlotinib and the EGFR antibody inhibitor cetuximab in vivo. Pre-treatment with a pan-caspase inhibitor but not a caspase-1 inhibitor reduced erlotinib-induced IL-1α secretion suggesting that IL-1α was released due to cell death. Human HNSCC tumors showed higher IL-1α mRNA levels compared to matched normal tissue, and IL-1α was found to be negatively correlated with survival in HNSCC patients. Overall, the IL-1α/IL-1R/MYD88/IL-6 pathway may be responsible for the reduced anti-tumor efficacy of erlotinib and other EGFRIs; and blockade of the MyD88-dependent signaling may improve the efficacy of EGFRIs in the treatment of HNSCC. Citation Format: Andrean L. Simons-Burnett, Adam T. Koch, Laurie Love-Homan, Aditya Stanam. MyD88-dependent signaling decreases the anti-tumor efficacy of epidermal growth factor receptor inhibition in head and neck cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2015-5505

Epidermal growth factor receptor (EGFR) is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). Unfortunately, the incorporation of EGFR inhibitors (EGFRIs) into the management of HNSCC has not improved long-term survival rates in patients with advanced stage HNSCC tumors. Therefore, the identification and understanding of strategies that will improve the efficacy of EGFRIs may potentially improve patient treatment and survival. Using microarray analysis, our laboratory has observed a profound increase in genes involved in MYD88-dependent pro-inflammatory pathways in EGFRI-treated HNSCC cell lines compared to their respective vehicle-treated cell lines. Additionally, we have previously shown that EGFRIs induce production of the inflammatory cytokine interleukin-6 (IL-6), leading to the reduced efficacy and acquired resistance to EGFRIs in HNSCC cells. Since MYD88-dependent signaling may lead to IL-6 production and secretion, the purpose of these studies is to determine if MYD88-dependent pathways such as Toll-like receptor (TLR), interleukin-1 receptor (IL-1R) and interleukin-18 receptor (IL-18R) pathways are responsible for the IL-6 production induced by EGFRIs. We found that knockdown of MYD88 (using siRNA and shRNA) significantly inhibited IL-6 secretion induced by the EGFRI erlotinib in Cal-27 and SQ20B HNSCC cells in vitro, increased the anti-tumor activity of erlotinib in vitro and enhanced the anti-tumor efficacy of erlotinib in a HNSCC xenograft mouse model. These results suggested that MYD88-dependent signaling was involved in IL-6 production and in the reduced efficacy of erlotinib. In order to elucidate which MYD88-dependent pathway was involved in erlotinib-induced IL-6 production, we investigated the involvement of TLR, IL-1R or IL-18R-mediated signaling. Although TLRs and the IL-18R were expressed and active on our HNSCC cells, we found little to no evidence of their involvement in erlotinib-induced IL-6 production. However, inhibition of the IL-1R through pharmacologic (anakinra) or genetic (siRNA and shRNA transfection) methods significantly reduced erlotinib-induced IL-6 production and increased HNSCC cell sensitivity to erlotinib in vitro. These observations point to the IL-1R/MYD88 pathway being involved in IL-6 production and in the reduced efficacy of erlotinib. We observed a time-dependent increase of IL-1 alpha (IL-1α) but not IL-1 beta (IL-1β) in response to erlotinib treatment implicating IL-1α as the damage associated molecular pattern (DAMP) responsible for activating the IL-1R. Furthermore, suppression of cell death with a pan-caspase inhibitor reduced erlotinib-induced IL-1α secretion. Finally, human HNSCC tumors were found to have higher IL-1α mRNA levels than matched normal tissue, and comparison of high and low IL-1α expressing HNSCC tumors (from the TCGA PANCAN databank) showed IL-1α to be negatively correlated with survival. Altogether, erlotinib-induced cell death may result in the release of IL-1α which activates IL-1R on adjacent surviving cells resulting in MYD88-mediated IL-6 secretion and the development of acquired resistance to erlotinib. In summary, the IL-1α/IL-1R/MYD88/IL-6 pathway may be responsible for the reduced anti-tumor efficacy of erlotinib and other EGFRIs; and blockade of the IL-1 pathway may improve the efficacy of EGFRIs in the treatment of HNSCC. This work was supported by the grants NIH K01CA134941 and ACS IRG7700434. Citation Format: Andrean L. Simons, Adam T. Koch, Laurie Love-Homan, Aditya Stanam. The role of MYD88-dependent signaling in the anti-tumor efficacy of the EGFR inhibitor erlotinib in head and neck cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B29.