This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches.

Transforming growth factor-beta (TGF-β) has a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-β signaling enhances de novo tumor development, whereas TGF-β overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-β-induced growth arrest response is attenuated, in association with aberrant activation of nuclear factor-κB (NF-κB), a transcription factor, which promotes malignant progression in HNSCC. However, what role cross-talk between components of the TGF-β and NF-κB pathways plays in altered activation of these pathways has not been established. Here, we show TGF-β receptor II and TGF-β-activated kinase 1 (TAK1) are predominantly expressed in a subset of HNSCC tumors with nuclear activation of NF-κB family member RELA (p65). Further, TGF-β1 treatment induced sequential phosphorylation of TAK1, IKK, IκBα and RELA in human HNSCC lines. TAK1 enhances TGF-β-induced NF-κB activation, as TAK1 siRNA knockdown decreased TGF-β1-induced phosphorylation of IKK, IκB and RELA, degradation of IκBα, RELA nuclear translocation and DNA binding, and NF-κB-induced reporter and target gene transcription. Functionally, TAK1 siRNA inhibited cell proliferation, migration and invasion. Celastrol, a TAK1 inhibitor and anti-inflammatory compound used in traditional Chinese medicine, also decreased TGF-β1-induced phosphorylation of TAK1 and RELA, and suppressed basal, TGF-β1- and tumor necrosis factor-alpha (TNF-α)-induced NF-κB reporter gene activity. Celastrol also inhibited cell proliferation, while increasing sub-G0 DNA fragmentation and Annexin V markers of apoptosis. Furthermore, TGF-β and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-β-induced SMAD and NF-κB reporters when compared with constitutive or TNF-α-induced NF-κB reporter gene activation. Thus, cross-talk by TGF-β via TAK1 and NF-κB promotes the malignant phenotype of HNSCC. Moreover, NF-κB may contribute to the downstream attenuation of canonical TGF-β signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-β-NF-κB signal pathway.

Nuclear factor-κB (NF-κB) is a signal transcription factor that has emerged as an important modulator of altered gene programs and malignant phenotype in development of cancer. Major carcinogens and oncogenic viruses induce NF-κB activation, and a variety of subsequent oncogenic events contribute to a progressive increase in constitutive NF-κB activation as an important common pathway in most forms of cancer. NF-κB target genes promote tumor cell proliferation, survival, migration, inflammation, and angiogenesis. Inhibition of NF-κB has been found to be an important mechanism of action of steroids, nonsteroidal anti-inflammatory drugs, and natural and synthetic compounds that show therapeutic and preventive activity. Newer agents targeting the proteasome, inhibitor-κB kinase, and other upstream kinases involved in NF-κB activation have shown anticancer activity in clinical or preclinical studies.

Recruitment of myeloid-derived suppressor cells (MDSCs) into tumors induces local immunosuppression in carcinomas. Here, we assessed whether SX-682, an orally bioavailable small-molecule inhibitor of CXCR1 and CXCR2, could block tumor MDSC recruitment and enhance T cell activation and antitumor immunity following multiple forms of immunotherapy. CXCR2 ⁺ neutrophilic MDSCs (PMN-MDSCs) were the most abundant myeloid cell subset within oral and lung syngeneic carcinomas. PMN-MDSCs demonstrated greater suppression of tumor-infiltrating lymphocyte killing of targets compared with macrophages. SX-682 significantly inhibited trafficking of PMN-MDSCs without altering CXCR2 ligand expression. Trafficking of CXCR1 ⁺ macrophages was unaltered, possibly due to coexpression of CSF1R. Reduced PMN-MDSC tumor infiltration correlated with enhanced accumulation of endogenous or adoptively transferred T cells. Accordingly, tumor growth inhibition or the rate of established tumor rejection following programed death–axis (PD-axis) immune checkpoint blockade or adoptive cell transfer of engineered T cells was enhanced in combination with SX-682. Despite CXCR1/2 expression on tumor cells, SX-682 appeared to have little direct antitumor effect on these carcinoma models. These data suggest that tumor-infiltrating CXCR2 ⁺ PMN-MDSCs may prevent optimal responses following both PD-axis immune checkpoint blockade and adoptive T cell transfer therapy. Abrogation of PMN-MDSC trafficking with SX-682 enhances T cell–based immunotherapeutic efficacy and may be of benefit to patients with MDSC-infiltrated cancers.

We previously reported that expression of angiogenesis factors interleukin‐8 (IL‐8) and vascular endothelial growth factor (VEGF) is promoted by coactivation of transcription factors nuclear factor‐kappaB (NF‐κB) and activator protein‐1 (AP‐1) by interleukin‐1α in human head and neck squamous cell carcinomas (HNSCC). However, expression of IL‐1 receptor antagonist incompletely blocked reporter gene activity and cytokine expression, suggesting that other upstream signals may contribute to activation. Overexpression and autocrine activation of epidermal growth factor receptor (EGFR) is detected in 90% of HNSCC, and EGFR inhibitors have been reported to inhibit IL‐8 and VEGF expression, but the intermediary signal pathways and transcription factors by which EGFR modulates proangiogenic factors is unknown. EGFR can activate the phosphotidylinositol‐3 kinase (PI3K) and mitogen‐activated/extracellular signal‐regulated kinase (MEK) pathways, which can potentially modulate activation of NF‐κB and AP‐1, respectively. In our study, we examined the effect of EGF and antagonists of EGFR, PI3K and MEK on NF‐κB and AP‐1 activation and IL‐8 and VEGF expression in HNSCC cell lines UM‐SCC‐9 and 11B in which EGFR is overexpressed and activated. Recombinant EGF induced EGFR phosphorylation, activation of NF‐κB and AP‐1 reporter genes and IL‐8 and VEGF expression, indicating that EGFR can mediate coactivation of both transcription factors and cytokine genes in HNSCC. EGFR antagonist PD153035 and anti‐EGFR antibody C225 completely inhibited EGF‐induced reporter activity and cytokine expression, but only partially inhibited constitutive activity. MEK inhibitor U0126 preferentially blocked AP‐1 activity and expression of both IL‐8 and VEGF, while PI3K inhibitor LY‐294002 or a dominant negative inhibitor‐κB preferentially blocked NF‐κB activation and expression of IL‐8 but not VEGF. EGFR, PI3K and MEK antagonists inhibited growth of HNSCC. We conclude that antagonists of EGFR, PI3K and MEK signal pathways have inhibitory activity against EGFR‐induced NF‐κB and AP‐1 activation, IL‐8 and VEGF expression and growth by HNSCC. Published 2002 Wiley‐Liss, Inc.

Importance of the field: Recent advances in the understanding of the oncogenesis of head and neck squamous cell carcinomas (HNSCC) have revealed multiple dysregulated signaling pathways. One freque...