Chronic exposure to carcinogens represents the major risk factor for head and neck squamous cell carcinoma (HNSCC). Beverages derived from broccoli sprout extracts (BSE) that are rich in glucoraphanin and its bioactive metabolite sulforaphane promote detoxication of airborne pollutants in humans. Herein, we investigated the potential chemopreventive activity of sulforaphane using in vitro models of normal and malignant mucosal epithelial cells and an in vivo model of murine oral cancer resulting from the carcinogen 4-nitroquinoline-1-oxide (4NQO). Sulforaphane treatment of Het-1A, a normal mucosal epithelial cell line, and 4 HNSCC cell lines led to dose- and time-dependent induction of NRF2 and the NRF2 target genes NQO1 and GCLC, known mediators of carcinogen detoxication. Sulforaphane also promoted NRF2-independent dephosphorylation/inactivation of pSTAT3, a key oncogenic factor in HNSCC. Compared with vehicle, sulforaphane significantly reduced the incidence and size of 4NQO-induced tongue tumors in mice. A pilot clinical trial in 10 healthy volunteers evaluated the bioavailability and pharmacodynamic activity of three different BSE regimens, based upon urinary sulforaphane metabolites and NQO1 transcripts in buccal scrapings, respectively. Ingestion of sulforaphane-rich BSE demonstrated the greatest, most consistent bioavailability. Mucosal bioactivity, defined as 2-fold or greater upregulation of NQO1 mRNA, was observed in 6 of 9 evaluable participants ingesting glucoraphanin-rich BSE; 3 of 6 ingesting sulforaphane-rich BSE; and 3 of 9 after topical-only exposure to sulforaphane-rich BSE. Together, our findings demonstrate preclinical chemopreventive activity of sulforaphane against carcinogen-induced oral cancer, and support further mechanistic and clinical investigation of sulforaphane as a chemopreventive agent against tobacco-related HNSCC. Cancer Prev Res; 9(7); 547–57. ©2016 AACR.

The destruction of proteins via the ubiquitin–proteasome system is a multi-step, complex process involving polyubiquitination of substrate proteins, followed by proteolytic degradation by the macromolecular 26S proteasome complex. Inhibitors of the proteasome promote the accumulation of proteins that are deleterious to cell survival, and represent promising anti-cancer agents. In multiple myeloma and mantle cell lymphoma, treatment with the first-generation proteasome inhibitor, bortezomib, or the second-generation inhibitor, carfilzomib, has demonstrated significant therapeutic benefit in humans. This has prompted United States Food and Drug Administration (US FDA) approval of these agents and development of additional second-generation compounds with improved properties. There is considerable interest in extending the benefits of proteasome inhibitors to the treatment of solid tumor malignancies. Herein, we review progress that has been made in the preclinical development and clinical evaluation of different proteasome inhibitors in solid tumors. In addition, we describe several novel approaches that are currently being pursued for the treatment of solid tumors, including drug combinatorial strategies incorporating proteasome inhibitors and the targeting of components of the ubiquitin–proteasome system that are distinct from the 26S proteasome complex.

Little is known regarding molecular markers in head and neck squamous cell carcinoma (HNSCC) that predict responsiveness to different therapeutic regimens or predict HNSCC progression. Mutations in procaspase‐8 occur in 9% of HNSCC primary tumors, but the functional consequences of these mutations are poorly understood. In this study, we examined the impact of four, representative, HNSCC‐associated procaspase‐8 mutations on activation of the extrinsic apoptosis pathway, as well as cellular migration and invasion, and in vivo tumor growth. All four mutant proteins acted to potently inhibit activation of apoptosis following treatment with TRAIL or agonistic anti‐Fas. In contrast to wild‐type procaspase‐8, the mutant proteins were not recruited to FADD following treatment with TRAIL or anti‐Fas, but may be constitutively bound by FADD. Three of the four procaspase‐8 mutants promoted enhanced cellular migration and invasion through matrigel, relative to that seen with the wild‐type procaspase‐8 protein. Procaspase‐8 mutation also stimulated the growth of HNSCC xenograft tumors. These findings indicate that HNSCC‐associated procaspase‐8 mutations inhibit activation of the extrinsic apoptosis pathway and are likely to represent markers for resistance to therapeutic regimens incorporating death receptor activators. Moreover, procaspase‐8 mutations may serve as markers of HNSCC tumor progression, as exemplified by enhanced migration, invasion, and tumor growth.

Treatment of cells with chemotherapy drugs activates the intrinsic mitochondrial pathway of apoptosis and the caspase protease cascade. Recently, the lysosomal protease cathepsin D has been implicated in apoptosis caused by oxidative stress, inhibition of protein kinase C, and stimulation of the TNFR1 and Fas death receptors. However, the role of cathepsin D in chemotherapy-induced cell death has remained largely unexplored. In this report, we show that treatment of U937 leukemia cells with the chemotherapy drug etoposide (VP-16) results in cathepsin D release into the cytosol within 4 hours after initiation of drug treatment. VP-16-induced cathepsin D release was not inhibited by z-VAD-FMK or pepstatin A, suggesting that it occurred independently of the activities of caspase proteases or cathepsin D. Down-regulation of cathepsin D expression in suspension U937 cells or adherent HeLa cells using cathepsin D small interfering RNA partially inhibited cell death resulting from treatment of cells with tumor necrosis factor-α, tumor necrosis factor–related apoptosis inducing ligand, or the chemotherapy drugs VP-16, cisplatin, and 5-fluorouracil. Moreover, cathepsin D down-regulation significantly delayed cytochrome c release and caspase-3 activation in response to chemotherapy treatment. Incubation of isolated mitochondria with cathepsin D–treated cytosolic extracts resulted in potent release of cytochrome c, indicating that a cytoplasmic substrate mediates the effects of cathepsin D on mitochondria. Together, these findings show that cathepsin D plays an important role in chemotherapy-induced cell death, and that cathepsin D lies upstream of cytochrome c release and caspase-3 activation in the chemotherapy-induced execution pathway.

Mapping protein interactions driving cancer: Cancer is a genetic disease, and much cancer research is focused on identifying carcinogenic mutations and determining how they relate to disease progression. Three papers demonstrate how mutations are processed through networks of protein interactions to promote cancer (see the Perspective by Cheng and Jackson). Swaney et al . focus on head and neck cancer and identify cancer-enriched interactions, demonstrating how point mutant–dependent interactions of PIK3CA, a kinase frequently mutated in human cancers, are predictive of drug response. Kim et al . focus on breast cancer and identify two proteins functionally connected to the tumor-suppressor gene BRCA1 and two proteins that regulate PIK3CA. Zheng et al . developed a statistical model that identifies protein networks that are under mutation pressure across different cancer types, including a complex bringing together PIK3CA with actomyosin proteins. These papers provide a resource that will be helpful in interpreting cancer genomic data. —VV

Purpose: Carfilzomib is a selective, irreversible inhibitor of the chymotrypsin-like activity of the proteasome and is undergoing clinical evaluation in myeloma. ONX 0912 (oprozomib) is an orally bioavailable derivative. The activities of carfilzomib and ONX 0912 against solid tumor malignancies are less well understood. We investigated the impact and mechanisms of action of carfilzomib and ONX 0912 in preclinical models of head and neck squamous cell carcinoma (HNSCC). Experimental Design: The effects of carfilzomib and ONX 0912 on HNSCC cell survival and xenograft tumor growth were evaluated. The impact and mechanisms of both agents on apoptosis and autophagy induction were also investigated. The contribution of the unfolded protein response (UPR) to autophagy induction and the role of autophagy in attenuating HNSCC cell death were determined. Results: Carfilzomib and ONX 0912 potently induced apoptosis in HNSCC cell lines via upregulation of pro-apoptotic Bik. Upregulation of Mcl-1 by these agents served to dampen their efficacies. Carfilzomib and ONX 0912 also induced autophagy, mediated, in part, by activation of the UPR pathway involving upregulation of ATF4 transcription factor. Autophagy induction served a prosurvival role. Oral administration of ONX 0912 inhibited the growth of HNSCC xenograft tumors in a dose-dependent manner. Conclusions: These results show that carfilzomib and ONX 0912 are potently active against HNSCC cells, and the activities of these agents can be enhanced via suppression of Mcl-1 or inhibition of autophagy. Oral ONX 0912 exhibits in vivo activity against HNSCC tumors and may represent a useful therapeutic agent for this malignancy. Clin Cancer Res; 18(20); 5639–49. ©2012 AACR.