Bortezomib-Induced Apoptosis with Limited Clinical Response Is Accompaniedby InhibitionofCanonicalbutnotAlternativeNuclear Factor-KB Subunits in Head and Neck Cancer

Purpose:Nuclear factor-nB (NF-nB)/REL transcription factors promote cancer cell survival and progression. The canonical (NF-nB1/RELA or cREL) and alternate (NF-nB2/RELB) pathways require the proteasome for cytoplasmic-nuclear translocation, prompting the investigation of bortezomib for cancer therapy. However, limited clinical activity of bortezomibhas beenobserved inmany epithelial malignancies, suggesting this could result from incomplete inhibition of NF-nB/ RELs or other prosurvival signal pathways. Experimental Design:To examine thesepossibilities,matchedbiopsies from24hposttreatment were obtained from accessible tumors of patients who received low-dose bortezomib (0.6 mg/m) before reirradiation in a phase I trial for recurrent head and neck squamous cell carcinoma (HNSCC). Effects of bortezomib on apoptosis and proliferation byTUNEL and Ki67 staining were compared with nuclear staining for all five NF-nB 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-nB subunits in HNSCC cell lines. Results: HNSCC showed increased nuclear staining for all five NF-nB subunits, phosphorylated ERK1/2, andphosphorylated STAT3. Bortezomib treatment significantly enhancedapoptosiswith inhibition of nuclear RELA in three of four tumors, but other NF-nB subunits, ERK1/2, and STAT3 were variably or not affected, and tumor progression was observed within 3 months. In HNSCC cell lines, 10 mol/L bortezomib inhibited cell density while inhibiting tumor necrosis factor-a^ induced and partially inhibiting basal activation of NF-nB1/RELA, but not NF-nB2/RELB. Conclusions: Although low-dose bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-nB or other prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC. Nuclear factor-nB (NF-nB) is an injury, pathogen, growth factor, cytokine, and matrix signal-inducible transcription factor that normally functions to regulate cell survival and proliferation and innate and adaptive immune responses (1). NF-nB transcription factors are heterodimers or homodimers composed from two families of REL proteins. Each member of the REL family contains an NH2 terminal REL homology domain that mediates dimerization and domains mediating nuclear localization and binding to specific nB DNA sequence motifs in target genes (1). The first class, RELA (p65), RELB, and cREL, is synthesized in their mature forms, whereas the second class, consisting of NF-nB1(p105) and NF-nB2(p100), requires proteasome-dependent proteolysis of COOH terminal ankyrin repeats to yield p50 and p52, respectively (2). In the absence of activating stimuli, NF-nB dimers are also bound by a family of ankyrin repeat-containing inhibitor-nB (InB) proteins, sequestering them in the cytoplasm, blocking their nuclear translocation and/or DNA binding (1). The different NF-nB heterodimers and homodimers may be preferentially activated by different ligand-receptor and InB kinase (IKK) signal pathways to undergo proteasome-dependent processing of subunits and InBs, and cytoplasmic-nuclear translocation and activation. The IKK signal pathways and downstream NF-nB components have been broadly classified into canonical and noncanonical (alternative) pathways (3). The canonical pathway involves activation of a trimeric IKK complex, composed of IKKa, IKKh, and IKKg (NEMO), in response to stimuli, such as tumor necrosis factor-a (TNF-a) and interleukin 1. Upon activation, the IKK complex phosphorylates InB, leading to its subsequent ubiquitination and degradation by the proteasome, allowing nuclear translocation and DNA binding of the Cancer Therapy: Clinical Authors’Affiliation: Tumor Biology Section, Head and Neck Surgery Branch, National Institute of Deafness and Other Communication Disorders, NIH, Bethesda, Maryland Received 9/27/07; revised 2/27/08; accepted 3/25/08. Grant support: Intramural Project Z01-DC-00016 (C.V.Waes) and NIH Pfizer Clinical ResearchTraining Program (C. Allen and K. Saigal). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Carter VanWaes, 10 Center Drive CRC, Room 4-2732m Bethesda, MD 20892. Phone: 301-402-4216; Fax: 301-402 1140; E-mail: [email protected]. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-4470 www.aacrjournals.org Clin Cancer Res 2008;14(13) July1, 2008 4175 Research. on May 29, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from NF-nB1/RELA (p50/p65) heterodimer (4). Whereas regulation and function of NF-nB1/cREL is believed to be similar to that of RELA (5), cREL activation is less well studied. The noncanonical (alternate) pathway involves activation of IKKa via NF-nB– inducing kinase, leading to direct phosphorylation, ubiquitination, and degradation of the p100 precursor protein by the proteasome, allowing translocation and DNA binding by p52/ RELB (6). Studies of the consequences of knockout of IKK and NF-nB subunits in mice indicate that the canonical and noncanonical pathways may have some tissue specific developmental functions, as well as potentially redundant roles related to cell survival (3). Consistent with the prevalence of alterations in signal pathways upstream of NF-nB in cancer and its central role in regulating programmed responses, NF-nB has been shown to be aberrantly activated and important in cell proliferation, survival, and malignant potential of a variety of cancers, including head and neck squamous cell carcinomas (HNSCC), in which it has been extensively studied (3, 7, 8). The canonical NF-nB1/RELA (p50/p65) heterodimer was shown to be one of the species aberrantly activated in HNSCC and to regulate expression of cytokines, angiogenesis factors, cell cycle, and antiapoptotic genes (9–11). Increased activation of NF-nB1/ RELA was shown to occur during malignant progression in a murine SCC model (12) and to modulate an altered pattern of gene expression and more aggressive malignant phenotype (13). Consistent with this experimental murine model, increased nuclear activation of RELA has also been shown in squamous dysplasia and HNSCC tumor specimens and correlated with malignant progression and decreased survival (14). Alteration in several upstream cytokine and growth factor pathways contribute to aberrant activation of NF-nB in HNSCC, including autocrine stimulation by interleukin-1, epidermal growth factor receptor, and TNF-a (15–18). Genetic or pharmacologic inhibition of NF-nB was shown to restore altered gene expression and inhibit SCC proliferation, survival, angiogenesis, and resistance to TNF-a and radiotherapy (10, 13, 17–19), providing further evidence for the role of NF-nB as a target for therapy in HNSCC. Based upon the role of the proteasome in canonical and noncanonical NF-nB activation, proteasome inhibitors hold potential for blockade of NF-nB activation and for cancer therapy. Bortezomib (VELCADE) is a small molecule inhibitor that selectively blocks activity of the catalytic site of the 26S proteasome complex (20). Bortezomib has shown anticancer activity in preclinical and clinical studies and been approved for treatment refractory multiple myeloma. Bortezomib has also been tested and shown antitumor activity in preclinical studies for a number of solid tumor types, including HNSCC. Bortezomib blocked NF-nB activation and proliferation, induced apoptosis in HNSCC cell lines, and inhibited growth, radioresistance, and angiogenesis in syngeneic murine SCC and human HNSCC xenograft models (19, 21, 22). In a first-in-human study of bortezomib with reirradiation in patients with HNSCC, we have thus far examined the effects of a relatively low starting dose of proteasome inhibitor bortezomib (0.6 mg/m), with different schedules of drug and reirradiation, to find tolerable regimen(s) to maximize concurrent therapy and to study effects on proteasome and NF-nB–dependent and NF-nB–independent prosurvival signal pathway biomarkers. Although pilot studies provided evidence for inhibition of the proteasome together with phosphorylated RELA and increased apoptosis (23), we have subsequently observed limited clinical antitumor activity. This includes sustained partial responses in 5 of 18 patients and more temporary tumor reductions or stabilization of disease in others. The availability of pretreatment and matched posttreatment tumor biopsies and cell lines from a subset of these patients provide an opportunity to examine if these subtherapeutic responses may relate to differential effects of proteasome inhibition on canonical and alternate NF-nB/REL or other prosurvival pathways. In this study, we examined the nuclear localization of the five NF-nB/REL subunits of the canonical and alternative pathways in tumor and matched nonmalignant mucosa. Using pretreatment and posttreatment tumor biopsies, we examined the effects of bortezomib treatment on their distribution, along with that of the proteasome-independent mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, ERK1/2) and signal transduction and activating transcription factor-3 (STAT3) pathways, two important proliferative and prosurvival pathways activated in HNSCC. The potential linkage between these effects and markers of proliferation and apoptosis were examined. The effect of bortezomib on basal and inducible activation of NF-nB/REL subunits by TNF-a, one of the factors implicated in autocrine and paracrine activation of the canonical pathway in HNSCC (17, 18), was examined. Our findings suggest that incomplete inhibition of nuclear activation of basal canonical and lack of inhibition of alternate NF-nB/REL pathway subunits, as well as other prosurvival pathways, may contribute to the limited responsiveness observed with lower dosages of bortezomib and reirradiation. Materials andMethods Patient treatment and tumor specimens. Patients with histologically proved recurrent HNSCC provided informed consent before being enrolled into protocol 01-C-0104, a phase I study of bortezomib and reirradiation for recurrent HNSCC, approved by Institutional Review Board and Cancer Treatment Evaluation Program of the National Cancer Institute. Full eligibility criteria and initial study design and results are included in a previous report (23). Bortezomib produced by Millennium Pharmaceuticals was supplied through the National Cancer Institute Cancer Treatment Evaluation Program. Bortezomib was given as an i.v. bolus for 3 to 5 s on days 1 and 4 of weeks when drug was given. Radiation, delivered with a high-energy linear accelerator using 6-MV photons and electrons, was given in standard fractions of 1.8 to 2.0 Gy daily (Monday to Friday), to a total dose of 60 to 70 Gy. Among 17 patients enrolled to investigate tolerability of different schedules of bortezomib 0.6 mg/m twice weekly with 6 to 7 wk of reirradiation, two of seven among the initial cohort progressed early and died (22), leaving 15 patients who completed treatment and were fully evaluable for toxicities. Of those who received the initial schedule of bortezomib every week with reirradiation, two of five had dose-limiting toxicities, prompting study of scheduled treatment breaks. Of those treated with a midcourse break of 2 wk from both modalities, one of six had a doselimiting toxicity, and among those treated with a midcourse break of 2 wk from bortezomib while receiving continuous reirradiation, zero of four had dose-limiting toxicities. Overall, 8 of 17 showed reductions in tumor size of 30% or more, and 5 of 17 qualified as having durable partial responses of at least 3 mo. Six of 17 patients consented to optional pretreatment tumor biopsies, and four of these with tumors of the oral cavity, oropharynx, or neck were safely accessible for both pretreatment Cancer Therapy: Clinical www.aacrjournals.org Clin Cancer Res 2008;14(13) July1, 2008 4176 Research. on May 29, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from and posttreatment biopsy in the outpatient clinic. The timing of tumor sampling 24 h posttreatment from protocol patients was based upon prior pharmacodynamic data indicating proteasome inhibition by bortezomib peak to trough between 1 and 72 h, and antiproliferative and apoptotic effects observed at 24 h posttreatment in HNSCC cells and tumors in clinical and preclinical studies (19, 20, 22). Posttreatment tumor biopsies were taken 24 h after the initial bortezomib treatment, before the initiation of radiotherapy to evaluate effects of drug alone. Specimens were immediately embedded in optimum cutting temperature media and frozen at -80jC. As standard controls, tumor blocks of two sets of matched carcinoma and noncancerous epithelium from deidentified subjects from the cooperative human tissue network of National Cancer Institute were used for analysis. Frozen tissues were sectioned at a thickness of 10 Am at the largest area and placed on silanated glass slides (Histoserv) for immunostaining. Immunoassays and scoring. Immunohistochemistry for NF-nB/REL, ERK, STAT3, apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labeling, TUNEL) and proliferation (Ki-67) was done using standard methods (23). Scoring of nuclear phosphorylated RELA, RELB, c-REL, p105/p50, p100/p52, phosphorylated ERK1/2, and phosphorylated STAT3 was done using a modified scoring method previously reported by Nenutil et al. (24), as described in supplementary methods. Cell culture and reagents. University of Michigan HNSCC (UMSCC) cell lines 9, 11A, and 11B were a generous gift from T.E. Carey (University of Michigan), and their characteristics and culture conditions are described in supplementary methods. Recombinant TNF-a was purchased from R&D Systems. MTT assays. Cellular cytotoxicity was measured with an MTT calorimetric cell proliferation kit (Roche), as described by others (25) and in supplementary methods. Growth effects at different drug concentrations were analyzed for statistical significance using the