NF-kappaB includes a family of signal-activated transcription factors that normally regulate responses to injury and infection but which are aberrantly activated in many carcinomas (1). Cumulative evidence implicates NF-kappaB in cell survival, inflammation, angiogenesis, spread and therapeutic resistance during tumor development, progression and metastasis of carcinomas. Non-specific natural and synthetic agents that inhibit NF-kappaB have demonstrated activity and safety in prevention or therapy. NF-kappaB-activating kinases and the proteasome are under investigation for targeted prevention and therapy of carcinoma.? ? During this period, we reported on molecular correlative studies from our phase I clinical trial of proteasome inhibitor bortezomib with reirradiation for patients with recurrent HNSCC (01-C-0104; ref. 2). The canonical (NF-kappaB1/RELA or cREL) and alternate (NF-kappaB2/RELB) pathways require the proteasome for cytoplasmic-nuclear translocation. 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. 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. We conclude that 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. Overall, 5/17 patients treated at the initial dose level demonstrated partial responses. This study continues accrual at the next dose level to define the maximally tolerated dose with the new schedule that provides a two week break from drug treatment. ? ? Limited drug penetration in tumors is another problem affecting clinical responsiveness. In a collaborative study with the Department of Radiology, it was found that pulsed high-intensity focused ultrasound (HIFU) could enhance apoptosis and growth inhibition of squamous cell carcinoma xenografts with proteasome inhibitor Bortezomib (3). We found that treatment 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. HIFU in combination with Bortezomib may warrant investigation for control of inoperable local regional disease.? ? A third potential limitation of therapy with drugs such as Bortezomib is the nature of the dynamic response of the targeted pathway to inhibition. In a collaborative study with Dr. Myong-Hee Sung and colleagues of the Laboratory of Receptor Biology, NCI, the dynamic Effect of Bortezomib on NF-kappaB Activity and Gene Expression in Tumor Cells was investigated (4). When administered to pre-activated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway 'blockade', the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways.? ? We wrote an invited commentary for a study based on our previous and continued studies on the role of Inhibitor-kappaB kinases in HNSCC (5). We have obtained evidence that IKK1 (IKKalpha)and IKK 2 (IKKbeta) both contribute to activation of NF-kappaB, and identified a role for protein kinase CK2 in IKK/NF-kappaB activation (Yu et al., Cancer Res., 2006; Van Waes, Clin Cancer Res. 2007). Studies are underway to determine if classical (IKK1/2 NF-kappaB1) and alternative (IKK1 NF-kappaB2) pathways are activated and contribute to gene expression and malignant phenotype of HNSCC, and may be inhibited by known and newly developed agents. In their study, Maeda et al. reported that loss or decreased nuclear expression of IKKalpha occurs in 20 of 64 ( 33%) of oral SCCs and is significantly associated with decreased histologic differentiation and prognosis. Their data indicate that besides its role in NF-kB activation, nuclear IKKalpha may play a role in differentiation, suppressing development of poorly differentiated SCC. Such loss of nuclear IKKalpha is associated with decreased differentiation and epithelial to mesenchymal transition, a phenotype linked with SCC progression, invasion, and worse prognosis. The possible effects of IKK inhibitors in inhibiting versus promoting malignant potential merit investigation.? ? Our collaboration was sought by colleagues at the University of Pittsburgh NCI SPORE in helping characterize survival signaling related to autocrine and paracrine chemokine receptor 7 (CCR7) activation in head and neck cancer, which may relate to NF-kappaB and Akt. CCR7 mediates survival and invasiveness of metastatic HNSCC to regional lymph nodes. Constitutive prosurvival signaling by the phosphoinositide-3 kinase/Akt pathway has been observed in cells independent of epidermal growth factor receptor (EGFR) signaling. In the absence of exogenous ligand treatment, blockade of CCR7 signaling reduced levels of phosphorylated (activated) Akt and decreased HNSCC cell viability, enhancing the effect of EGFR inhibition. CCR7 stimulation protected metastatic HNSCC cells from cisplatin-induced apoptosis in an Akt-dependent manner. Metastatic nodes expressed and secreted higher levels of CCL19 than benign nodes or primary tumors. CCR7-positive murine SCCHN tumors grew more slowly in plt mice than in control BALB/c mice. Thus, secretion of CCL19 and CCL21 by SCCHN cells and by paracrine sources combine to promote activation of CCR7 prosurvival signaling associated with tumor progression and disease relapse. CCR7 and its cognate chemokines may be useful biomarkers of SCCHN progression, and blockade of CCR7-mediated signaling may enhance the efficacy of platinum- and EGFR-based therapies.
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