Bortezomib-induced Apoptosis with Limited Clinical Response is Accompanied by Inhibition of Canonical but Not Alternative Nuclear Factor-{kappa}B Subunits in Head and Neck Cancer

Purpose: Nuclear factor-kappaB (NF-kappaB)/REL transcription factors promote cancer cell survival and progression. The canonical (NF-kappaB1/RELA or cREL) and alternate (NF-kappaB2/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-kappaB/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/m(2)) 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-kappaB 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-kappaB subunits in HNSCC cell lines.

Results: HNSCC showed increased nuclear staining for all five NF-kappaB 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-kappaB 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-alpha-induced and partially inhibiting basal activation of NF-kappaB1/RELA, but not NF-kappaB2/RELB.

Conclusions: Although low-dose bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-kappaB or other prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC.