Liesl Nottingham

Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction

No conventional therapy exists for salivary hypofunction in surviving head and neck cancer patients with Radiation Therapy Oncology Group late grade 2–3 toxicity. We conducted a phase I clinical trial to test the safety and biologic efficacy of serotype 5, adenoviral-mediated aquaporin-1 cDNA transfer to a single previously irradiated parotid gland in 11 subjects using an open label, single-dose, dose-escalation design (AdhAQP1 vector; four dose tiers from 4.8 × 107 to 5.8 × 109 vector particles per gland). Treated subjects were followed at scheduled intervals. Multiple safety parameters were measured and biologic efficacy was evaluated with measurements of parotid salivary flow rate. Symptoms were assessed with a visual analog scale. All subjects tolerated vector delivery and study procedures well over the 42-d study period reported. No deaths, serious adverse events, or dose-limiting toxicities occurred. Generally, few adverse events occurred, and all were considered mild or moderate. No consistent changes were found in any clinical chemistry and hematology parameters measured. Objective responses were seen in six subjects, all at doses <5.8 × 109 vector particles per gland. Five of these six subjects also experienced subjective improvement in xerostomia. AdhAQP1 vector delivery to a single parotid gland was safe and transfer of the hAQP1 cDNA increased parotid flow and relieved symptoms in a subset of subjects.

Nuclear factor-κB p65 small interfering RNA or proteasome inhibitor bortezomib sensitizes head and neck squamous cell carcinomas to classic histone deacetylase inhibitors and novel histone deacetylase inhibitor PXD101

Histone deacetylase inhibitors (HDI) can inhibit proliferation and enhance apoptosis in a wide range of malignancies. However, HDIs show relatively modest activity in head and neck squamous cell carcinomas (HNSCC), in which we have shown the activation of nuclear factor-κB (NF-κB; NF-κB1/RelA or p50/p65), a transcription factor that promotes expression of proliferative and antiapoptotic genes. In this study, we examined if HDIs enhance activation of NF-κB and target genes and if genetic or pharmacologic inhibition of NF-κB can sensitize HNSCC to HDIs. Limited activity of classic HDIs trichostatin A and sodium butyrate was associated with enhanced activation of NF-κB reporter activity in a panel of six HNSCC cell lines. HDIs enhanced NF-κB p50/p65 DNA binding and acetylation of the RelA p65 subunit. Transfection of small interfering RNAs targeting p65 strongly inhibited NF-κB expression and activation, induced cell cycle arrest and cell death, and further sensitized HNSCC cells when combined with HDIs. The p65 small interfering RNA inhibited HDI-enhanced expression of several NF-κB–inducible genes implicated in oncogenesis of HNSCC, such as p21, cyclin D1, and BCL-XL. Bortezomib, an inhibitor of proteasome-dependent NF-κB activation, also increased sensitization to trichostatin A, sodium butyrate, and a novel HDI, PXD101, in vitro, and to the antitumor effects of PXD101 in bortezomib-resistant UMSCC-11A xenografts. However, gastrointestinal toxicity, weight loss, and mortality of the combination were dose limiting and required parenteral fluid administration. We conclude that HDI-enhanced NF-κB activation is one of the major mechanisms of resistance of HNSCC to HDIs. The combination of HDI and proteasome inhibitor produced increased antitumor activity. Low starting dosages for clinical studies combining HDIs with proteasome inhibitors and IV fluid support may be warranted. [Mol Cancer Ther 2007;6(1):37–50]

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.

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.

Deficient TP53 Expression, Function, and Cisplatin Sensitivity Are Restored by Quinacrine in Head and Neck Cancer

Purpose: To determine the nature and potential pharmacologic reversibility of deficient TP53 expression and function in head and neck squamous cell carcinomas (HNSCC) with wild-type TP53, previously associated with decreased sensitivity to cisplatin therapy. Experimental Design:TP53 genotype, mRNA and protein expression, TP53-induced p21 expression, and TP53 DNA–binding and reporter gene function were determined in a panel of nine previously characterized HNSCC cell lines from the University of Michigan squamous cell carcinoma (UM-SCC) series. The genotoxic drug doxorubicin and the anti-inflammatory and antimalarial drug quinacrine, previously identified as inducers of TP53, were used to examine the nature and potential reversibility of deficient TP53 expression and function. The specific role of inducible TP53 on function and cellular proliferation was confirmed using selective TP53 inhibitor pifithrin-α or short hairpin RNA knockdown. The capability of quinacrine to sensitize HNSCC to the cytotoxic effects of cisplatin was assessed. Results: UM-SCC cell lines with wild-type TP53 genotype underexpressed TP53 mRNA and protein when compared with normal human keratinocytes or UM-SCC with mutant TP53. Although doxorubicin failed to induce TP53 expression or functional activity, quinacrine induced TP53 mRNA and protein expression, increased TP53 reporter activity and p21 protein expression, and induced growth inhibition in these wild-type TP53 cell lines. Quinacrine-induced TP53 reporter activity and growth suppression were attenuated by pifithrin-α and TP53 short hairpin RNA knockdown. Furthermore, quinacrine sensitized UM-SCC to cisplatin in vitro. Conclusions: Deficient TP53 mRNA and protein expression underlies decreased function in a subset of HNSCC with wild-type TP53 and can be restored together with cisplatin sensitization by quinacrine.

Differential bortezomib sensitivity in head and neck cancer lines corresponds to proteasome, nuclear factor-κB and activator protein-1 related mechanisms

Head and neck squamous cell carcinomas (HNSCC) exhibit constitutive activation of transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which are modulated by the proteasome and promote resistance to cell death. HNSCC show variable sensitivity to the proteasome inhibitor bortezomib in vitro as well as in murine xenografts and patient tumors in vivo, and the mechanisms are not well understood. To address this question, the sensitivities of nine HNSCC cell lines to bortezomib were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and the potential relationship between the sensitivity and bortezomib effects on biological processes was examined in HNSCC lines of differential bortezomib sensitivity. The most sensitive cell line (UM-SCC-11B) underwent cell death at 10−9 mol/L in vitro and tumor regression at a maximally tolerated dose of bortezomib in a murine xenograft model. The differential sensitivity between UM-SCC-11A and UM-SCC-11B cells corresponded to differences in the extent of suppression of proteasome activity, ubiquitinated protein degradation, and NF-κB and AP-1 activation. Lower concentrations of bortezomib transiently increased NF-κB and sustained AP-1 activation in UM-SCC-11A cells. AP-1 reporter activity and cell density of UM-SCC-11A were suppressed when bortezomib was combined with c-Jun NH2-terminal kinase and p38 kinase pathways inhibitors. Thus, the differential sensitivities to bortezomib corresponded to dissimilar effects on the proteasome, NF-κB and AP-1 activities. Inhibition of c-Jun NH2-terminal kinase and p38 pathways blocked AP-1 activity and enhanced the antitumor effects. These findings revealed molecular mechanisms of bortezomib sensitivity and resistance, which are under development as biomarkers for clinical trials in patients with HNSCC. [Mol Cancer Ther 2008;7(7):1949–60]

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

PURPOSE Epidermal 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). METHODS AND MATERIALS This 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. RESULTS Ten 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. CONCLUSIONS Inhibition 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.

Inhibitor-κB Kinase in Tumor Promotion and Suppression During Progression of Squamous Cell Carcinoma

Previous studies have provided evidence that activation of inhibitor-κB kinases (IKK) leads to nuclear translocation and activation of nuclear factor-κB (NF-κB), a transcription factor that can promote cell survival and malignant phenotypic changes important in development and progression of

Abstract 5005: Combined targeting of IKKα and β effectively suppresses NF-κB activation, cell survival and migration in head and neck squamous cell cancers

Activation of the transcription factor NF-kappaB (NF-kB) has been found to be a key promoter of tumorigenesis and a contributor of chemotherapy resistance in many cancers, including head and neck squamous cell carcinomas (HNSCC). The NK-kB pathway is regulated through both canonical and noncanonical pathways involving the IkB kinases (IKK) α and β which together, have been implicated in cancer pathogenesis. However, the individual roles of the IKK subunits have not been previously elucidated and potential therapeutic targets still largely remain to be characterized. Endogenous IKKα and β expression and phosphorylation were analyzed by IHC staining and western blot in HNSCC tumor specimens and cell lines (UM-SCC 11A and 11B). Individual and dual overexpression and knockdown of IKKα and β were assessed for their effects on NF-kB activation by a luciferase reporter assay, and on tumor cell proliferation and migration in vitro via MTT and wound healing assays, respectively. HNSCC tissue and cell lines exhibited aberrant expression with increased nuclear localization and phosphorylation of IKKα and β when compared to normal controls. In a panel of 9 HNSCC cell lines, endogenous IKKα was more abundantly expressed than IKKβ. While individual overexpression of wt or constitutively activated IKKβ was more potent than IKKα, dual overexpression of IKKα and β exhibited the strongest induction of NF-kB activation. siRNA knockdown of either kinase resulted in decreased cell proliferation and migration and NF-kB activation, and a combinatorial effect was seen with double IKKα and β knockdown. Additionally, chemical inhibitors targeting IKKα and β were used to evaluate their potential for suppressing NF-kB activation and targeting the HNSCC malignant phenotype in vitro. Four small molecule inhibitors were studied that preferentially targeted IKKα>β (17-DMAG), IKKβ (MLN120b and SC514), or both IKKα and β (wedelolactone). Compared to the IKKβ specific inhibitor, MLN120b, 17-DMAG more potently inhibited NF-kB DNA binding in electromobility shift assays (EMSA) and NF-kB reporter gene activation. MTT proliferation assays and flow cytometric DNA cell cycle analysis demonstrated more potent effects of 17-DMAG and wedelolactone in inhibiting cell proliferation. We conclude that overexpression and signaling mediated by both IKKα and β contribute to NF-kB activation, and pathogenesis of proliferation and migration of HNSCC cells in vitro. Furthermore, dual siRNA silencing or chemical inhibition of IKKα and β kinases resulted in combinatorial inhibitory effects on NF-kB activation and the malignant phenotype. In the face of intrinsic and acquired chemoresistance limiting treatment options, IKKα/β represent promising novel therapeutic targets for fighting HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5005. doi:10.1158/1538-7445.AM2011-5005