Clint T. Allen

Role of activated nuclear factor-κB in the pathogenesis and therapy of squamous cell carcinoma of the head and neck†

Nuclear factor‐κB (NF‐κB), a transcription factor known to modulate expression of factors involved in inflammation, immunity, proliferation, and apoptosis, is constitutively activated and plays a role in pathogenesis and therapeutic resistance in head and neck squamous cell carcinoma (HNSCC). Understanding the molecular alterations leading to aberrant NF‐κB activation in HNSCC may direct investigators to novel therapeutic targets.

Bortezomib-Induced Apoptosis with Limited Clinical Response Is Accompanied by Inhibition of Canonical but not Alternative Nuclear Factor-κB Subunits in Head and Neck Cancer

Purpose: Nuclear factor-κB (NF-κB)/REL transcription factors promote cancer cell survival and progression. The canonical (NF-κB1/RELA or cREL) and alternate (NF-κB2/RELB) pathways require the proteasome for cytoplasmic-nuclear translocation, prompting the investigation of bortezomib for cancer therapy. However, limited clinical activity of bortezomib has been observed in many epithelial malignancies, suggesting this could result from incomplete inhibition of NF-κB/RELs or other prosurvival signal pathways. Experimental Design: To examine these possibilities, matched biopsies from 24 h posttreatment were obtained from accessible tumors of patients who received low-dose bortezomib (0.6 mg/m2) before reirradiation in a phase I trial for recurrent head and neck squamous cell carcinoma (HNSCC). Effects of bortezomib on apoptosis and proliferation by TUNEL and Ki67 staining were compared with nuclear staining for all five NF-κB subunits, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated signal transducers and activators of transcription 3 (STAT3) in tumor biopsies, and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTP) and DNA binding assay for the five NF-κB subunits in HNSCC cell lines. Results: HNSCC showed increased nuclear staining for all five NF-κB subunits, phosphorylated ERK1/2, and phosphorylated STAT3. Bortezomib treatment significantly enhanced apoptosis with inhibition of nuclear RELA in three of four tumors, but other NF-κB subunits, ERK1/2, and STAT3 were variably or not affected, and tumor progression was observed within 3 months. In HNSCC cell lines, 10−8 mol/L bortezomib inhibited cell density while inhibiting tumor necrosis factor-α–induced and partially inhibiting basal activation of NF-κB1/RELA, but not NF-κB2/RELB. Conclusions: Although low-dose bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-κB or other prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC.

Proteomic Signatures of Epidermal Growth Factor Receptor and Survival Signal Pathways Correspond to Gefitinib Sensitivity in Head and Neck Cancer

Purpose: Gefitinib targeting of the epidermal growth factor receptor (EGFR) has shown limited activity in clinical trials of head and neck squamous cell carcinoma (HNSCC). To investigate the underlying molecular mechanism, the proteomic signatures and responses of EGFR and downstream signals have been studied in a panel of HNSCC cell lines and tumor specimens pre- and post-gefitinib treatment. Experimental Design: The IC50 of gefitinib for HNSCC cell lines were determined using 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay. The effects of gefitinib on activation of EGFR and downstream signaling molecules were determined by Western blot, ELISA, and reverse-phase protein microarray (RPMA). The biomarkers involved in the signaling pathways were examined in HNSCC tumor specimens from patients in a phase I gefitinib trial. Results:In vitro, gefitinib inhibited cell proliferation with differing IC50, and suppressed activation of EGFR and downstream signaling molecules protein kinase B (AKT), extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3 (STAT3), and nuclear factor κB. The drug sensitivity was statistically correlated with activation of phosphorylated AKT (p-AKT) and phosphorylated STAT3 (p-STAT3) detected by ELISA, and consistent with results measured by RPMA. In patient samples, a broad suppression of activation of EGFR and downstream signaling molecules was observed in a molecular responder patient, in contrast to a lack of inhibition or increased activation of biomarkers in different pathways in nonresponder patients. Conclusions: Gefitinib sensitivity is correlated with p-AKT and p-STAT3 activation in HNSCC cell lines and tumor specimens. p-AKT and p-STAT3 could serve as potentially useful biomarkers and drug targets for further development of novel therapeutic agents for HNSCC.

TNF-α Promotes c-REL/ΔNp63α Interaction and TAp73 Dissociation from Key Genes That Mediate Growth Arrest and Apoptosis in Head and Neck Cancer

Inflammation-induced activation of proto-oncogenic NF-κB/REL and dysfunction of tumor suppressor TP53/p63/p73 family transcription factors are key events in cancer progression. How inflammatory signaling coordinates dysregulation of these two transcription factor families during oncogenesis remains incompletely understood. Here, we observed that oncoprotein c-REL and tumor suppressor TAp73 are coexpressed and complex with ΔNp63α in the nucleus of a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with mutant (mt)TP53. TNF-α, a proinflammatory cytokine, promoted c-REL nuclear translocation, c-REL/ΔNp63α interaction, and dissociation of TAp73 from ΔNp63α and the nucleus to the cytoplasm, whereas c-REL siRNA knockdown attenuated this effect. Overexpression of c-REL or a c-REL κB-site DNA-binding mutant enhanced protein interaction with ΔNp63α and TAp73 dissociation, implicating c-REL/ΔNp63α-specific interactions in these effects. We discovered that TNF-α or genetic alteration of c-REL expression inversely modulates ΔNp63α/TAp73 interactions on distinct p63 DNA-binding sites, including those for key growth arrest and apoptotic genes p21WAF1, NOXA, and PUMA. Functionally, c-REL repressed these genes and the antiproliferative effects of TNF-α or TAp73. Conversely, c-REL siRNA depletion enhanced TAp73 promoter interaction and expression of genes mediating growth arrest and apoptosis. Similar to TNF-α-treated HNSCC lines, human HNSCC tumors and hyperplastic squamous epithelia of transgenic mice overexpressing ΔNp63α that exhibit inflammation also show increased nuclear c-REL/ΔNp63α and cytoplasmic TAp73 localization. These findings unveil a novel and reversible dynamic mechanism whereby proinflammatory cytokine TNF-α-induced c-REL/ΔNp63α interactions inactivate tumor suppressor TAp73 function, promoting TNF-α resistance and cell survival in cancers with mtTP53.

Pulsed High-Intensity Focused Ultrasound Enhances Apoptosis and Growth Inhibition of Squamous Cell Carcinoma Xenografts with Proteasome Inhibitor Bortezomib

PURPOSEnTo investigate whether combining pulsed high-intensity focused ultrasound (HIFU) with the chemotherapeutic drug bortezomib could improve antitumor activity against murine squamous cell carcinoma (SCC) tumors.nnnMATERIALS AND METHODSnAll experiments were conducted with animal care and use committee approval. Murine SCC cells were implanted subcutaneously in C3H mice. When tumors reached 100 mm(3), mice were randomized to one of three groups for twice weekly intraperitoneal injections of 1.5 mg of bortezomib per kilogram of body weight, a proteasome inhibitor (n = 10); 1.0 mg/kg bortezomib (n = 11); or a control vehicle (n = 12). Within each group, half of the mice received pulsed HIFU exposure to their tumors immediately prior to each injection. The time for tumors to reach 650 mm(3) was compared among groups. Additional tumors were stained with terminal deoxynucledotidyl transferase-mediated dUTP nick end labeling and CD31 to assess apoptotic index and blood vessel density, respectively.nnnRESULTSnTumors in the control group, pulsed HIFU and control group, and 1.0 mg/kg of bortezomib alone group reached the size end point in 5.2 days +/- 0.8 (standard deviation), 5.3 days +/- 0.8, and 5.6 days +/- 1.1, respectively. However, pulsed HIFU and 1.0 mg/kg bortezomib increased the time to end point to 9.8 days +/- 2.9 (P < .02), not significantly different from the 8.8 days +/- 2.1 in tumors treated with 1.5 mg/kg bortezomib alone (P > .05). Combination therapy was also associated with a significantly higher apoptotic index (P < .05).nnnCONCLUSIONnTreatment of tumors with pulsed HIFU lowered the threshold level for efficacy of bortezomib, resulting in significant tumor cytotoxicity and growth inhibition at lower dose levels.

Aberrant IKKα and IKKβ cooperatively activate NF-κB and induce EGFR/AP1 signaling to promote survival and migration of head and neck cancer

The inhibitor-κB kinase-nuclear factor-κB (IKK-NF-κB) and epidermal growth factor receptor-activator protein-1 (EGFR-AP1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components that enhance co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKβ exhibit increased protein expression, nuclear localization, and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied among different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits. Knock down of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKβ together promoted the expression and activity of transforming growth factor α, EGFR and AP1 transcription factors cJun, JunB and Fra1. Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity and geldanomycins that block IKKα/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKβ cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

Molecular and Clinical Responses in a Pilot Study of Gefitinib With Paclitaxel and Radiation in Locally Advanced Head-and-Neck Cancer

PURPOSEnEpidermal growth factor receptor (EGFR) overexpression in head-and-neck squamous cell carcinoma (HNSCC) stimulates tumor cell proliferation, inhibits apoptosis, and increases chemotherapy and radiation resistance. We examined the toxicity, safety and the effects on EGFR signaling in tumor biopsy samples from patients with locally advanced HNSCC treated with the EGFR signaling inhibitor gefitinib (GEF) combined with weekly intravenous paclitaxel (PAC) and radiation therapy (RT).nnnMETHODS AND MATERIALSnThis was a pilot Phase I dose-escalation study. Eligibility included Stage III to IVB HNSCC, age >or=18 years, no prior RT or chemotherapy, adequate organ function, and informed consent. Endpoints included determination of maximum tolerated dose (MTD) and analysis of treatment effect on EGFR signaling, tumor cell proliferation, and apoptosis in biopsy samples.nnnRESULTSnTen patients were treated. The MTD of this combination was GEF 250 mg/d with PAC 36 mg/m(2) intravenously weekly x 6 with concurrent RT. Grade 3/4 toxicities included prolonged (>8 weeks) stomatitis (7 patients), infection (2 patients), and interstitial pneumonitis (1 patient). There were five complete responses (CR) and two partial responses (PR). Of 7 patients undergoing serial biopsies, only 1 patient demonstrated a reduction in phosphorylated EGFR, decreased downstream signaling, and reduced cellular proliferation after initiating GEF.nnnCONCLUSIONSnInhibition of EGFR by GEF was observed in only one of seven tumors studied. The addition of GEF to PAC and RT did not appear to improve the response of locally advanced HNSCC compared with our prior experience with PAC and RT alone. This treatment appeared to delay recovery from stomatitis.

The p53 Homologue ΔNp63α Interacts with the Nuclear Factor-κB Pathway to Modulate Epithelial Cell Growth

The p53 homologue DeltaNp63alpha is overexpressed and inhibits apoptosis in a subset of human squamous cell carcinomas (SCC). Here, we report that in normal keratinocytes overexpressing DeltaNp63alpha and in human squamous carcinoma cells, DeltaNp63alpha physically associates with phosphorylated, transcriptionally active nuclear c-Rel, a nuclear factor-kappaB family member, resulting in increased c-Rel nuclear accumulation. This accumulation and the associated enhanced proliferation driven by elevated DeltaNp63alpha are attenuated by c-Rel small interfering RNA or overexpression of mutant IkappaBalphaM, indicating that c-Rel-containing complex formation is critical to the ability of elevated DeltaNp63alpha to maintain proliferation in the presence of growth arresting signals. Consistent with a role in growth regulation, DeltaNp63alpha-c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human squamous carcinoma cells and normal keratinocytes overexpressing DeltaNp63alpha. The relationship between DeltaNp63alpha and activated c-Rel is reflected in their strong nuclear staining in the proliferating compartment of primary head and neck SCC. This is the first report indicating that high levels of DeltaNp63alpha interact with activated c-Rel in keratinocytes and SCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers.

Syngeneic Mouse Models of Oral Cancer Are Effectively Targeted by Anti–CD44-Based NIR-PIT

Oral cavity squamous cell carcinoma (OSCC) is considered one of the most aggressive subtypes of cancer. Anti-CD44 monoclonal antibodies (mAb) are a potential therapy against CD44 expressing OSCC; however, to date the therapeutic effects have been disappointing. Here, a new cancer treatment is described, near-infrared photoimmunotherapy (NIR-PIT), that uses anti-CD44 mAbs conjugated to the photoabsorber IR700DX. This conjugate is injected into mice harboring one of three CD44 expressing syngeneic murine oral cancer cell (MOC) lines, MOC1 (immunogenic), MOC2 mKate2 (moderately immunogenic), and MOC2-luc (poorly immunogenic). Binding of the anti-CD44–IR700 conjugate was shown to be specific and cell-specific cytotoxicity was observed after exposure of the cells to NIR light in vitro. The anti-CD44–IR700 conjugate, when assessed in vivo, demonstrated deposition within the tumor with a high tumor-to-background ratio. Tumor-bearing mice were separated into four cohorts: no treatment; 100 μg of anti-CD44–IR700 i.v. only; NIR light exposure only; and 100 μg of anti-CD44–IR700 i.v. with NIR light exposure. NIR-PIT therapy, compared with the other groups, significantly inhibited tumor growth and prolonged survival in all three cell model systems. In conclusion, these data reveal that anti-CD44 antibodies are suitable as mAb–photoabsorber conjugates for NIR-PIT in MOC cells. Implications: This study using syngeneic mouse models, which better model the disease in humans than conventional xenografts, suggests that NIR-PIT with anti-CD44–IR700 is a potential candidate for the treatment of OSCC. Mol Cancer Res; 15(12); 1667–77. ©2017 AACR.

CCR 20th Anniversary Commentary: Preclinical Study of Proteasome Inhibitor Bortezomib in Head and Neck Cancer

In a study published in the May 1, 2001, issue of Clinical Cancer Research, Sunwoo and colleagues provided evidence for proteasome inhibition of NF-κB and tumorigenesis, supporting early-phase clinical trials in solid malignancies of the upper aerodigestive tract. Subsequent clinical studies uncovered a dichotomy of responses in patients with hematopoietic and solid malignancies, and the mechanisms of resistance. Clin Cancer Res; 21(5); 942–3. ©2015 AACR. See related article by Sunwoo et al., Clin Cancer Res 2001;7(5) May 2001;1419–28