Clinical investigation
Head and neck
Inhibition of nuclear factor-κB and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma

https://doi.org/10.1016/j.ijrobp.2005.05.007Get rights and content

Purpose: To examine the effects the proteasome inhibitor bortezomib (VELCADE) on transcription factor nuclear factor-κB (NF-κB) and target genes and the feasibility of combination therapy with reirradiation in patients with recurrent head-and-neck squamous cell carcinoma (HNSCC).

Methods and Materials: The tolerability and response to bortezomib 0.6 mg/m2 and 0.9 mg/m2 given twice weekly concurrent with daily reirradiation to 50–70 Gy was explored. Blood proteasome inhibition and NF-κB–modulated cytokines and factors were measured. Proteasome inhibition, nuclear localization of NF-κB phospho-p65, apoptosis, and expression of NF-κB–modulated mRNAs were compared in serial biopsies from accessible tumors.

Results: The maximally tolerated dose was exceeded, and study was limited to 7 and 2 patients, respectively, given bortezomib 0.6 mg/m2 and 0.9 mg/m2/dose with reirradiation. Grade 3 hypotension and hyponatremia were dose limiting. Mucositis was Grade 3 or less and was delayed. The mean blood proteasome inhibition at 1, 24, and 48 h after 0.6 mg/m2 was 32%, 16%, and 7% and after 0.9 mg/m2 was 56%, 26%, and 14%, respectively. Differences in proteasome and NF-κB activity, apoptosis, and expression of NF-κB–modulated cell cycle, apoptosis, and angiogenesis factor mRNAs were detected in 2 patients with minor tumor reductions and in serum NF-κB–modulated cytokines in 1 patient with a major tumor reduction.

Conclusions: In combination with reirradiation, the maximally tolerated dose of bortezomib was exceeded at a dose of 0.6 mg/m2 and the threshold of proteasome inhibition. Although this regimen with reirradiation is not feasible, bortezomib induced detectable differences in NF-κB localization, apoptosis, and NF-κB–modulated genes and cytokines in tumor and serum in association with tumor reduction, indicating that other schedules of bortezomib combined with primary radiotherapy or reirradiation may merit future investigation.

Introduction

Head-and-neck squamous cell carcinoma (HNSCC) affecting the oral cavity, pharynx, and larynx are diagnosed in >40,000 new patients in the United States and 500,000 patients worldwide each year (1). For advanced Stages III and IV HNSCC, surgery and radiotherapy (RT) have been the standard therapy for many years, but they are often accompanied by significant deformity and functional deficits. In the past decade, combined RT and chemotherapy have achieved preservation of organ function with comparable survival (2). Regardless of the treatment used, about 50% of patients with HNSCC have recurrence. In selected patients with recurrent HNSCC not amenable to additional surgery, local disease control and prolonged survival have been reported with reirradiation to 60 Gy, albeit with significant toxicity (3, 4, 5, 6). The identification of the molecular mechanisms contributing to the pathogenesis and therapeutic resistance of HNSCC in the primary and recurrent setting is needed to develop more effective therapy and improve survival.

We previously showed that many molecules expressed by human HNSCC and murine SCC that promote angiogenesis, cell cycle progression, and inhibition of cell death (apoptosis) share regulatory sites for transcription factor nuclear factor-κB (NF-κB) (7, 8, 9, 10, 11). The NF-κB heterodimer p50/p65 is constitutively activated in human and murine SCC (7, 8, 9). Blockade of NF-κB activation with signal phosphorylation mutants of inhibitor-κBα (IκB) resulted in inhibition of expression of the several molecules implicated in the oncogenesis of HNSCC, including cyclin D1, antiapoptotic gene inhibitor of apoptosis-1, and cytokine and angiogenesis factors interleukin (IL)-6, IL-8, and Growth Regulated Oncogene-1 (GRO-1) (8, 11). The inhibition of NF-κB and these genes was accompanied by inhibition of tumor cell proliferation, survival, angiogenesis, and tumorigenesis (8, 11), as well as sensitization of HNSCC to tumor necrosis factor-α and RT (12, 13). These observations provided evidence that NF-κB is a possible target for molecular therapy in combination with RT in patients with HNSCC.

A key step in NF-κB activation involves the 26S proteasome (14). NF-κB is normally complexed in an inactive form in the cytoplasm by IκB. Nuclear translocation of NF-κB and gene promoter binding and activation involve signal-induced phosphorylation, ubiquitination, and degradation of IκB by the proteasome, as well as phosphorylation of the NF-κB p65 (Rel A) subunit (14). The essential role of the proteasome in degradation of IκB and activation of NF-κB has suggested that proteasome inhibitors might be useful in therapy for HNSCC and other cancers. In preclinical studies, the proteasome inhibitor bortezomib (VELCADE, formerly PS-341) has been shown to modulate activation of NF-κB, as well as turnover of other important proteins involved in oncogenesis, such as tumor suppressors p53, p21Cip1, and p27 (14, 15, 16). In SCC cell lines, bortezomib was shown to inhibit activation of NF-κB and expression of several genes important in oncogenesis, including cyclin D1, IAP-1, BCL-XL, GRO-1, and vascular endothelial growth factor (VEGF) (17, 18). Consistent with its effects on NF-κB and these targets, bortezomib inhibited cell proliferation, survival, angiogenesis, and tumorigenesis and promoted radiosensitization in preclinical SCC models (17, 19). Bortezomib has also been shown to inhibit RT- and chemotherapy-induced activation of NF-κB and to sensitize cancers to RT in other preclinical models (20, 21, 22). Together, these findings provided a rationale for examining the effects of bortezomib on NF-κB and its target genes, in combination with RT, for therapy for HNSCC patients.

Bortezomib has been studied in several Phase I and II trials and was recently approved for use in patients with therapy-resistant multiple myeloma (23). To date, the clinical and molecular effects of bortezomib in patients with HNSCC and the feasibility of combined therapy with RT have not been determined. Because standard chemotherapy and RT regimens are already established for patients with newly diagnosed advanced HNSCC, we designed a pilot study to examine the feasibility of combining bortezomib and reirradiation in patients with recurrent HNSCC, for whom standard therapy options have been exhausted. On the basis of Phase I pharmacodynamic and toxicity data (23), bortezomib was administered twice weekly as an intravenous bolus beginning at a dose of 0.6 mg/m2 to provide proteasome inhibition without breaks for the duration of daily RT (Fig. 1). Bortezomib alone was given on Day 1 to examine molecular correlates with the drug alone during the first 24 h before initiation of RT. Molecular correlates were examined again with combined bortezomib and RT. Blood proteasome inhibition and NF-κB–modulated cytokines and factors were measured in all patients, and tumor proteasome inhibition, nuclear localization of NF-κB phospho-p65, apoptosis, and expression of NF-κB–modulated mRNAs were compared in 2 patients with tumors accessible for serial biopsy. The clinical toxicities and responses to bortezomib in combination with reirradiation were evaluated.

Section snippets

Consent and eligibility

The Institutional Review Board and Cancer Treatment Evaluation Program of the National Cancer Institute (NCI) approved Protocol 01-C-0104. All patients provided informed consent before the study. Patients with histologically documented, unresectable, persistent, recurrent, or second primary HNSCC were eligible. An interval of at least 6 months since prior RT and 4 weeks since the last major surgery or chemotherapy was required for entry. Patients with distant metastases were eligible if RT for

Patient characteristics and treatment

The characteristics of patients entered in the study are shown in Table 1. Seven patients presented with local recurrence and two had regional and distant recurrence. All 9 had previously undergone surgery and RT; 6 also had had prior chemotherapy. Seven patients had an Eastern Cooperative Oncology Group performance status of 1; 2 had an Eastern Cooperative Oncology Group performance status of 2. Six patients had Grade 2 or 3 baseline dysphagia.

The cumulative dose of reirradiation and

Discussion

This is the first clinical study to examine the feasibility of combining proteasome inhibitor bortezomib with RT and to demonstrate the effects of bortezomib on NF-κB and target genes in HNSCC patients. Bortezomib was given concurrently with reirradiation to patients with recurrent HNSCC, on the basis of evidence that genetic- or proteasome-mediated inhibition of NF-κB activation is associated with inhibition of proliferation, cell survival, angiogenesis, and radiation resistance (8, 11, 13, 19

Acknowledgments

The clinical coordination provided by Donna Headlee, Christine Muir, Linda McCullough, and Theresa Cooley-Zgela, the data management services of Mikhail Kalnitskiy and Jeannette Wanzer, and the technical assistance of Ning Yeh and Xinping Yang are much appreciated.

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    Supported by NIDCD intramural project Grant Z01-DC-00016 (C.V.W.) and NIH Clinical Research Training Program (A.A.C., C.H.D.).

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