The ubiquitin-proteasome pathway plays an important role in cell cycle regulation, neoplastic growth, and metastasis 12. At the heart of this degradative pathway is the 26S proteasome, an adenosine triphosphate-dependent protease. The 26S proteasome consists of a core 20S particle, which contains the catalytic proteinase functions, symmetrically bound to two copies of the regulatory 19S particle. Proteolytic removal of damaged or misfolded ubiquitinated proteins is an important part of the homeostatic function of the 26S proteasome. Additionally, the 26S proteasome is vital in degrading regulatory proteins that govern cell cycle, transcription factor activation, cell trafficking, and apoptosis.

The ubiquitin-proteasome pathway is responsible for the ordered degradation of several regulatory proteins necessary for cells to progress through the cell cycle. The tumor suppressor p53, which acts as a negative regulator of cell growth, is one example of targeted ubiquitin-proteasome mediated degradation. p53 is required for the transcription of a number of genes involved in cell cycle control and DNA synthesis. p53 also plays an important role in apoptosis induced by cellular damage 3. Cyclins and the cyclin-dependent kinase inhibitors p21 and p27 are also regulated by proteasome-dependent proteolysis 4. Both p21 and p27 can induce cell cycle arrest by inhibiting the cyclin D-, E-, and A-dependent kinases 5.

The ubiquitin-proteasome pathway also plays an important role in transcriptional regulation. Nuclear factor-κB (NF-κB) is a key transcription factor, whose activation is regulated by proteasome-mediated degradation of the inhibitor protein I kappa B alpha-associated protein kinase (IκBa) 45. Cell adhesion molecules such as E-selectin, ICAM-1, and VCAM-1, are regulated by NF-κB and are involved in the development of angiogenesis and tumor metastasis in vivo 4. During metastasis, these molecules direct the adhesion and extravasation of tumor cells from the vasculature to distant tissue sites within the body. Additionally, many cell types require NF-κB to maintain cell viability as an anti-apoptotic controlling factor or growth factor 4. Inhibiting NF-κB activation by stabilizing the IκBa protein potentiates apoptosis of cancer cells to environmental stresses and cytotoxic agents.

Another enzyme recently targeted for cancer prevention and treatment, cyclooxygenase (COX)-2, has garnered increasing interest due to substantial epidemiologic, experimental, pathologic, and clinical evidence suggesting that nonsteroidal anti-inflammatory drugs (NSAIDs) possess anticancer properties 678. NSAIDs are thought to exert their anti-inflammatory effects by inhibiting prostaglandin synthesis through nonspecific inhibition of COX enzymes. Prostaglandins, in particular prostaglandin E2, appear to be important in oncogenesis due to their effects on cellular adhesion, immune surveillance, and apoptosis 9. Compared with normal tissues, cancers have been shown to over-express prostaglandins 1011121314151617. In several animal and human models, inhibiting prostaglandin synthesis by blocking COX-2 appears to protect against oncogenesis in many tissue types including: breast, colon, esophageal, lung, skin, and head and neck cancers 6710111213141516.

COX-2 is undetectable in most normal tissues; however, it is induced at sites of inflammation by cytokines, growth factors, tumor promoters, and is over-expressed in neoplasia 17. Several mechanisms have been proposed to explain the role of COX-2 in tumorigenesis, including stimulating cancer cell proliferation, enhancing angiogenesis, and inhibiting apoptosis 1819. In vitro inhibition of COX-2 with targeted molecules is thought to promote apoptosis of cancer cells through inhibition of NF-kB activation 20.

Both COX-2 inhibitors and ubiquitin-proteasome pathway inhibitors down-regulate NF-κB and their combination is an interesting opportunity to explore clinical synergy with these two classes of agents. The ubiquitin-proteasome pathway is involved in NF-κB regulation through its impact on degradation of IκB and release of NF-κB for translocation from the cytoplasm to the nucleus 21. Inhibition of IκB degradation by proteasome inhibition can limit the actions of NF-κB rendering tumor cells more prone to apoptosis. The combination of the NSAID sulindac and bortezomib was found to be synergistic both in vitro, in colon cancer cell lines, and in vivo, in a xenograft model 22. Additionally, the COX-2 inhibitor celecoxib has been reported to induce apoptosis independent of its COX-2 effects using NF-κB as a probably target 23. Consequently, given the relevance of the COX-2 oncogene to numerous solid tumors, clinical evaluation of a COX-2 inhibitor/proteasome inhibitor combination is warranted in patients with solid tumors.

We therefore hypothesized that the combination of the COX-2 inhibitor celecoxib and the ubiquitin-proteasome pathway inhibitor bortezomib is a potentially active clinical combination in the treatment of advanced solid tumors. Therefore, we conducted a phase I study to determine the maximum tolerated dose (MTD) and toxicity profile of bortezomib in combination with celecoxib.

Both COX-2 inhibitors and ubiquitin-proteasome pathway inhibitors down-regulate NF-κB. Combining these two classes of agents is an interesting opportunity to investigate potential clinical synergy with a combination of drugs not known to have overlapping toxicities.

This is the first phase I study to combine bortezomib and celecoxib in patients with advanced solid tumors. The primary endpoint of this study was to determine the maximal tolerated doses of these agents when taken in combination. No dose-limiting toxicities were observed during the first cycle of treatment at any dose-level and cumulative toxicities were manageable.

In our phase I study, two patients with previously progressive disease experienced stable disease on therapy. A patient with metastatic adrenocortical cancer, previously intolerant of mitotane, achieved stable disease for 4.3 months until progression. A second patient with papillary renal cell carcinoma achieved stable disease for 5.1 months. This patient had documented tumor progression on multi-agent chemotherapy prior to enrolling on this trial.

Interesting new data have been reported for celecoxib and bortezomib since our study began. Celecoxib has been shown to potentially have single-agent activity in men with PSA recurrence after local prostate cancer therapy 28. Also, patients with non-small cell lung cancer receiving single-agent bortezomib recently reported an 8% response rate 29. Similarly, a phase II trial of patients with metastatic renal cell carcinoma reported a response rate of 11% and 38% of patients reported stable disease with bortezomib 30. Conversely, no tumor responses were seen in recent studies of bortezomib in metastatic melanoma, breast cancer, or colorectal cancer 313233.

In conclusion, the combination of bortezomib and celecoxib was well tolerated across the dose levels investigated. No dose-limiting toxicities were witnessed and we plan to proceed with dose level six (bortezomib 1.3 mg/m² days 1, 4, 8, and 11 repeated every 21 days in combination with celecoxib 400 mg orally twice daily) in a subsequent phase II study.

The pharmaceutical company Millenium provided bortezomib and provided funding for the conduct of this trial. The authors declare that there are no other competing interests.

JH has made substantial contributions to data analysis, interpretation, and article composition and revision; UC has been involved in acquisition of data; MG has been involved in conception and design; MM has been involved in conception and design; SD has been involved with acquisition of data; CS has been involved with acquisition of data; SS has been involved with acquisition of data; AK has been involved in acquisition of data; AJM has made substantial contributions with acquisition of data, analysis and interpretation of data, and article composition and revision. All authors have read and approved the final manuscript.