Defective NF-κB signaling in metastatic head and neck cancer cells leads to enhanced apoptosis by double-stranded RNA

Ligands to several Toll-like receptors (TLR), which mediate innate immune responses and chronic inflammation have been used as adjuvants to immunotherapy to enhance their antitumor activity. In particular, double-stranded RNAs that are cognate ligands of TLR3 have been used to trigger proapoptotic activity in cancer cells. However, a mechanistic understanding of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis has been lacking. In this study, we used paired cell lines and fresh tumor specimens, derived from autologous primary and metastatic head and neck squamous cell carcinoma, to investigate the role of TLR3 signaling in metastatic progression. Compared with primary tumor cells, metastatic tumor cells were highly sensitive to TLR3-mediated apoptosis after double-stranded RNA treatment. Enhanced apoptosis in metastatic cells was dependent on double-stranded RNA and TLR3 and also the TLR3 effector signaling protein TRIF. Downstream responses requiring NF-κB were critical for apoptosis in metastatic cells, the defects in which could be resuscitated by alternative pathways of NF-κB activation. By elucidating how TLR3 ligands trigger apoptosis in metastatic cells, our findings suggest insights into how to improve their clinical use.     

Upregulated interleukin‐6 expression contributes to erlotinib resistance in head and neck squamous cell carcinoma

Despite the role of epidermal growth factor receptor (EGFR) signaling in head and neck squamous cell carcinoma (HNSCC) development and progression, clinical trials involving EGFR tyrosine kinase inhibitors (TKIs) have yielded poor results in HNSCC patients. Mechanisms of acquired resistance to the EGFR TKI erlotinib was investigated by developing erlotinib‐resistant HNSCC cell lines and comparing their gene expression profiles with their parental erlotinib‐sensitive HNSCC cell lines using microarray analyses and subsequent pathway and network analyses. Erlotinib‐resistant HNSCC cells displayed a significant upregulation in immune response and inflammatory pathways compared to parental cells. Interleukin‐6 (IL‐6) was one of thirteen genes that was significantly differentially expressed in all erlotinib‐resistant HNSCC cell lines, which was validated using RT‐PCR and ELISA. Blockade of IL‐6 signaling using the IL‐6 receptor antagonist tocilizumab, was able to overcome erlotinib‐resistance in erlotinib‐resistant SQ20B tumors in vivo. Overall, erlotinib‐resistant HNSCC cells display elevated IL‐6 expression levels compared to erlotinib‐sensitive HNSCC cells and blockade of the IL‐6 signaling pathway may be an effective strategy to overcome resistance to erlotinib and possibly other EGFR TKIs for HNSCC therapy.     

First-line pembrolizumab ± chemotherapy for recurrent/metastatic head and neck cancer: Japanese subgroup of KEYNOTE-048

Background

Here, we report the results of the Japanese subgroup of the phase 3 KEYNOTE-048 study of pembrolizumab alone, pembrolizumab plus platinum and 5-fluorouracil (pembrolizumab–chemotherapy), or cetuximab plus platinum and 5-fluorouracil (EXTREME) in previously untreated recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).

Methods

Primary end points were overall survival (OS) and progression-free survival (PFS). Efficacy was evaluated in patients with PD-L1 combined positive score (CPS) ≥ 20 and ≥ 1 and the total Japanese subgroup (n = 67).

Results

At data cutoff (25 February 2019), pembrolizumab led to longer OS versus EXTREME in the PD-L1 CPS ≥ 20 subgroup (median, 28.2 vs. 13.3 months; HR, 0.29 [95% CI 0.09–0.89]) and to similar OS in the total Japanese (23.4 vs. 13.6 months; HR, 0.51 [95% CI 0.25–1.05]) and CPS ≥ 1 subgroups (22.6 vs. 15.8 months; HR, 0.66 [95% CI 0.31–1.41]). Pembrolizumab–chemotherapy led to similar OS versus EXTREME in the PD-L1 CPS ≥ 20 (median, 18.1 vs. 15.8 months; HR, 0.72 [95% CI 0.23–2.19]), CPS ≥ 1 (12.6 vs. 15.8 months; HR, 1.19 [95% CI 0.55–2.58]), and total Japanese subgroups (12.6 vs. 13.3 months; unadjusted HR, 1.10 [95% CI 0.55–2.22]). Median PFS was similar for pembrolizumab and pembrolizumab–chemotherapy versus EXTREME in all subgroups. Grades 3–5 treatment-related adverse events occurred in 5 (22%), 19 (76%), and 17 (89%) patients receiving pembrolizumab, pembrolizumab–chemotherapy, and EXTREME, respectively. One patient receiving pembrolizumab–chemotherapy died because of treatment-related pneumonitis.

Conclusion

These results support the use of first-line pembrolizumab and pembrolizumab–chemotherapy for Japanese patients with R/M HNSCC.

Clinical trial registry ClinicalTrials.gov, NCT02358031.

     

IKK phosphorylation of NF-κB at serine 536 contributes to acquired cisplatin resistance in head and neck squamous cell cancer

Current treatment methods for advanced head and neck squamous cell carcinoma (HNSCC) include surgery, radiation therapy and chemotherapy. For recurrent and metastatic HNSCC, cisplatin is the most common treatment option, but most of patients will eventually develop cisplatin resistance. Therefore, it is imperative to define the mechanisms involved in cisplatin resistance and find novel therapeutic strategies to overcome this deadly disease. In order to determine the role of nuclear factor-kappa B (NF-κB) in contributing to acquired cisplatin resistance in HNSCC, the expression and activity of NF-κB and its upstream kinases, IKKα and IKKβ, were evaluated and compared in three pairs of cisplatin sensitive and resistant HNSCC cell lines, including a pair of patient derived HNSCC cell line. The experiments revealed that NF-κB p65 activity was elevated in cisplatin resistant HNSCC cells compared to that in their parent cells. Importantly, the phosphorylation of NF-κB p65 at serine 536 and the phosphorylation of IKKα and IKKβ at their activation loops were dramatically elevated in the resistant cell lines. Furthermore, knockdown of NF-κB or overexpression of p65-S536 alanine (p65-S536A) mutant sensitizes resistant cells to cisplatin. Additionally, the novel IKKβ inhibitor CmpdA has been shown to consistently block the phosphorylation of NF-κB at serine 536 while also dramatically improving the efficacy of cisplatin in inhibition of cell proliferation and induction of apoptosis in the cisplatin resistant cancer cells. These results indicated that IKK/NF-κB plays a pivotal role in controlling acquired cisplatin resistance and that targeting the IKK/NF-κB signaling pathway may provide a possible therapeutic method to overcome the acquired resistance to cisplatin in HNSCC.     

HPV DNA,E6I-mRNA expression and p16 immunohistochemistry in head and neck cancer – How valid is p16 as surrogate marker?

It has been proposed that p16(INK4A) qualifies as a surrogate marker for viral oncogene activity in head and neck cancer (HNSCC). By analyzing 78 HNSCC we sought to validate the accuracy of p16(INK4A) as a reliable marker of active HPV infections in HNSCC. To this end we determined HPV DNA (HPVD) and E6*I mRNA (HPVR) expression status and correlated these results with p16(INK4A) staining. In tonsillar SCC 12/20 were HPVD+ and 12/12 of these showed active HPV infections whereas in non-tonsillar SCC 10/58 were HPVD+ and 5/10 showed active HPV infections. Thus, we prove about 8% of non-tonsillar SCC to be also correlated with HPV-associated carcinogenesis. Strikingly, 3/14 (21.4%) of tonsillar and non-tonsillar HPVD+/HPVR+ cases did not show p16(INK4A) overexpression and these cases would have been missed when applying initial p16(INK4A) staining only. However, in 13 cases negative for HPV, DNA p16(INK4A) was overexpressed. In conclusion, our data confirm tonsillar SCC to be predominantly but not only associated with active HPV infections. Furthermore, our data show that p16(INK4A) overexpression is not evident in a subgroup of HNSCC with active HPV infection. Definitive HPV data should therefore be utilized in diagnostics and treatment modalities of HPV positive and HPV negative HNSCC patients, resulting in a paradigm shift regarding these obviously different tumor entities.     

NF-κB participates in chemokine receptor 7-mediated cell survival in metastatic squamous cell carcinoma of the head and neck

Metastatic squamous cell carcinoma of the head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7), which activates phosphoinositide-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signal pathway to promote the invasion and survival of SCCHN cells. Since nuclear factor-kappa B (NF-κB) is shown to be the downstream signal molecule of PI3K/Akt in many tumors, we investigated whether it also exists in the CCR7 pathway in SCCHN, and the relationship between NF-κB and PI3K/Akt/mTOR, and the role it plays in SCCHN. We assayed the phosphorylation of the inhibitor of NF-κB (IκBα), the NF-κB DNA-binding capacity and location. The results showed that the interaction between CCR7 and the ligand for CCR7, CCL19, induces phosphorylation of IκBα, causes NF-κB to translocate to the nucleus and raises the DNA-binding capacity of NF-κB. The phosphorylation and DNA-binding capacity were abolished by the inhibition of CCR7, PI3K, Akt and mTOR. Further research demonstrated that inhibitors of NF-κB and CCR7-PI3K attenuate the survival of CCR7-mediated cells, causing decreased viability, increased apoptosis and increased cell cycle arrest in SCCHN cells. In clinical samples from 78 patients, immunohistochemical assay also showed that CCR7 and NF-κB are not only highly expressed in SCCHN, but also correlated with each other, and related to lymph node metastasis and clinical stage. Together, our data indicate that NF-κB is activated by CCR7 via PI3K/Akt/mTOR, and this signal pathway plays an important role in regulating the cell survival and prognosis of SCCHN.     

LRIG1 modulates cancer cell sensitivity to Smac mimetics by regulating TNFα expression and receptor tyrosine kinase signaling

Smac mimetics block inhibitor of apoptosis proteins to trigger TNFα-dependent apoptosis in cancer cells. However, only a small subset of cancer cells seem to be sensitive to Smac mimetics and even sensitive cells can develop resistance. Herein, we elucidated mechanisms underlying the intrinsic and acquired resistance of cancer cells to Smac mimetics. In vitro and in vivo investigations revealed that the expression of the cell surface protein LRIG1, a negative regulator of receptor tyrosine kinases (RTK), is downregulated in resistant derivatives of breast cancer cells sensitive to Smac mimetics. RNA interference-mediated downregulation of LRIG1 markedly attenuated the growth inhibitory activity of the Smac mimetic SM-164 in drug-sensitive breast and ovarian cancer cells. Furthermore, LRIG1 downregulation attenuated TNFα gene expression induced by Smac mimetics and increased the activity of multiple RTKs, including c-Met and Ron. The multitargeted tyrosine kinase inhibitors Crizotinib and GSK1363089 greatly enhanced the anticancer activity of SM-164 in all resistant cell derivatives, with the combination of SM-164 and GSK1363089 also completely inhibiting the outgrowth of resistant tumors in vivo. Together, our findings show that both upregulation of RTK signaling and attenuated TNFα expression caused by LRIG1 downregulation confers resistance to Smac mimetics, with implications for a rational combination strategy.     

PD-1 blockade attenuates immunosuppressive myeloid cells due to inhibition of CD47/SIRPα axis in HPV negative head and neck squamous cell carcinoma

Myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) play key roles in the tumor immune suppressive network and tumor progression. However, precise roles of programmed death-1 (PD-1) in immunological functions of MDSCs and TAMs in head and neck squamous cell carcinoma (HNSCC) have not been clearly elucidated. In the present study, we show that PD-1 and PD-L1 levels were significantly higher in human HNSCC specimen than in normal oral mucosa. MDSCs and TAMs were characterized in mice and human HNSCC specimen, correlated well with PD-1 and PD-L1 expression. αPD-1 treatment was well tolerated and significantly reduced tumor growth in the HNSCC mouse model along with significant reduction in MDSCs and TAMs in immune organs and tumors. Molecular analysis suggests a reduction in the CD47/SIRPα pathway by PD-1 blockade, which regulates MDSCs, TAMs, dendritic cell as well as effector T cells. Hence, these data identify that PD-1/PD-L1 axis is significantly increased in human and mouse HNSCC. Adoptive αPD-1 immunotherapy may provide a novel therapeutic approach to modulate the micro- and macro- environment in HNSCC.     

Synergistic effects of imatinib and carboplatin on VEGF, PDGF and PDGF-Rα/ß expression in squamous cell carcinoma of the head and neck in vitro

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. The development of new treatment modalities in order to improve long-term survival of patients with HNSCC is imperative. Numerous studies have demonstrated that carcinogenesis and tumor cell dissemination is influenced by the tumor microenvironment. The protein-kinase-receptors (PTKs) are essential elements of the intracellular signal transduction pathway and regulate cell growth, development and apoptosis. Cell proliferation, migration, induction of tumor vascularization and carcinogenesis, invasion is regulated by a variety of angiogenic factors, such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor) and their respective tyrosine kinase receptors (PDGF-R and VEGF-R). They present promising targets for anti-cancer therapy through abrogation of impaired signaling pathways. Indeed, imatinib, a small molecule drug targeting these protein kinases, has antiproliferative effects in several cancer types. The purpose of this study was to investigate the potential synergism of imatinib and carboplatin on the expression of PDGF, PDGF-R α/? and VEGF in different HNSCC cell lines. Several tumor cell lines were subjected to increasing concentrations of carboplatin (3 and 7.5 μmol/l) and imatinib (18 and 30 μmol/l) and ELISA, immunohistochemical methods and RQ-PRC after 48, 72, 120 and 240 h were used to assess their expression levels. While PDGF-Rα/? expression was unimpaired at lower imatinib concentrations (18 μmol/l), PDGF-Rα/? expression was suppressed at 30 μmol/l, and suppression was enhanced by the presence of carboplatin. By RQ-PCR, a significant reduction of PDGF-Rα/? expression was detected (p<0.5). We observed explicit significant reduction in VEGF levels with increasing concentrations of imatinib and with the combination of the two chemotherapeutic drugs (p<0.5). We report for the first time evidence of synergism of imatinib and carboplatin in suppressing VEGF, PDGF and PDGF-Rα/? expression in HNSCC.     

Increased topoisomerase IIα expression in colorectal cancer is associated with advanced disease and chemotherapeutic resistance via inhibition of apoptosis

Topoisomerase IIα is a nuclear enzyme that regulates the tertiary structure of DNA. The influence of topoisomerase IIα gene (TOP2A) or protein alterations on disease progression and treatment response in colorectal cancer (CRC) is unknown. The study investigated the clinical relevance of topoisomerase IIα in CRC using in vivo and in vitro models.     

The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy ± chemotherapy

Background  

Several parameters of the tumor microenvironment, such as hypoxia, inflammation and angiogenesis, play a critical role in tumor aggressiveness and treatment response. A major question remains if these markers can be used to stratify patients to certain treatment protocols.