Last May, the US Food and Drug Administration approved the first weapon in what could become a brand new arsenal against cancer. That weapon is Velcade (bortezomib), a drug that inhibits an intracellular protein-disposal system known as ubiquitin-mediated proteolysis (UMP). Made by Cambridge, Mass.-based Millennium Pharmaceuticals, Velcade improves and prolongs the lives of some patients with relapsed and refractory multiple myeloma.

As a succession of companies tested Velcade (also called PS-341) over the past eight years, this boronic acid compound aroused skepticism and puzzlement. Besides oncogenic proteins, "there are numerous other proteins whose degradation is controlled by the [UMP] pathway," says I. Bernard Weinstein, professor of medicine at Columbia University. "So it's not intuitively obvious that this would be a mechanism that you'd want to target."

Julian Adams, head of the team that discovered Velcade, confesses: "I wasn't surprised it had an effect on cancer. My surprise--and what took me a long time to understand--was the therapeutic index"--the fact that malignant cells are 1,000-10,000 times more sensitive to the drug than normal cells.

Velcade's unexpected success should spur efforts to develop other drugs targeting UMP. It also further validates a field of research that has ballooned over the past five years. This field explores the relationship between cancer and components of the UMP pathway, particularly enzymes involved in degrading signal transduction and cell cycle proteins. Experiments elucidate how activating or inhibiting these UMP components can affect tumor growth.

MYSTERY MECHANISM Ubiquitin is a highly conserved 76-residue polypeptide found in animals, plants, bacteria, and yeast. In a series of reactions whose final step is catalyzed by enzymes known as E3 ubiquitin ligases, this polypeptide attaches covalently to a substrate protein. When that protein is destined for degradation, another ubiquitin attaches to the substrate-bound ubiquitin. Further ubiquitin-to-ubiquitin linkages form a chain.

The assemblage is then recognized by one of a cell's 30,000 proteasomes, massive complexes whose molecular weight can reach two megadaltons. A proteasome contains sites with chymotryptic, tryptic, or caspase-like activity, which mince a protein substrate into short peptides. The attached ubiquitin molecules are recycled.

Velcade reversibly inhibits the proteasome's chymotryptic site, whose cleaving element is a threonine hydroxyl group. The drug's "boron warhead interacts directly with the threonine hydroxyl group to form a transition-state complex," explains Adams, now senior vice president of drug discovery and development at Millennium. Patients receive Velcade intravenously twice a week. Treatment inhibits 70-80% of a cell's proteasomes.

In a Phase II, 202-patient trial, the drug had some success against difficult cases of multiple myeloma, in which the bone marrow overproduces harmful antibody-forming cells. Tumors responded to varying degrees in 35% of patients, and the disease stabilized in another 24%. "People survived 16 months who were predicted to survive only 6 months," reports Kenneth C. Anderson, director of Harvard Medical School's Jerome Lipper Multiple Myeloma Center.

The big mystery about Velcade is how it triggers apoptosis in patients' tumor cells. Adams acknowledges that the answer will be "difficult to pinpoint," pending analyses of microarray data from the Phase II trial. Preclinical studies, however, suggest several possibilities. The reigning theory is that certain cancers, such as multiple myeloma, depend inordinately on NF-aB. This transcription factor is inhibited when it binds to the protein IaB. Velcade prevents the proteasome from degrading IaB, and NF-aB consequently remains inhibited.

Adams says another hypothesis is being tested. The proteasome's "housekeeping function," he notes, is to remove defective and misfolded proteins, which abound in a cancer cell because of its many genetic lesions. As a result, properly functioning proteasomes are more crucial to the viability of a cancer cell than a normal cell.

Further treatment strategies are under consideration. Immunoblots and cDNA microarrays reveal that Velcade-treated myeloma cells transiently express heat shock protein-90 (hsp-90). This effect, which appears to be part of a stress response to protect the cells against the drug, "provides the rationale for combining Velcade with hsp-90 inhibitors in clinical trials," Anderson points out. "That's going to happen later this year." Studies also show that Velcade somehow induces the cleavage of DNA repair enzymes in myeloma cells. The result--DNA is no longer efficiently repaired--suggests to Anderson that Velcade "may enhance the sensitivity or even overcome the resistance of tumor cells to conventional DNA-damaging alkylating agents."

CULPABLE LIGASES If Velcade were a more selective agent, tumors might display resistance to it after they acquired a few mutations. Instead, the drug's indiscriminate inhibition of an essential cellular complex likely stalls the onset of drug resistance. A proteasome inhibitor, however, has a downside: It stabilizes both pro-oncogenic and anti-oncogenic proteins, which presumably weakens its net effect. So cancer researchers are seeking compounds that selectively inhibit particular E3 ubiquitin ligases. The human genome encodes an estimated 500-600 such ligases, each ubiquitinating a limited set of substrates.

So far, relatively few E3 ligases have been linked to tumors. VHL, for example, is a ligase subunit associated with Von Hippel-Lindau disease, a cause of familial kidney cancer. Germline mutations of the VHL gene lead to accumulation of VHL's substrate, hypoxia-induced factor 1a. This factor promotes angiogenesis, which tumors need to grow.

Another well-examined ligase is murine double minute 2 (Mdm2). Its carboxyterminal domain ubiquitinates the tumor suppressor protein p53. Perhaps 5-10% of malignancies, particularly sarcomas, overexpress Mdm2, often because of gene amplification. The p53 gene typically is not mutated in these tumors, a fact that eliminates a competing explanation for their growth.

Skp2 is an adaptor subunit--a so-called "F box protein"--that recruits substrates to a ubiquitin ligase complex. In lymphomas and in breast, prostate, and oral cancers, skp2 is overproduced for reasons that remain unclear. Skp2 binds p27, leading to the destruction of this negative regulator of the cell cycle. Studies have detected reduction of p27 in epithelial cancers, brain tumors, and lymphomas.

Michele Pagano, associate pathology professor at New York University School of Medicine, investigates the skp2/p27 relationship. He characterizes skp2 as a proto-oncogene because it can transform mouse fibroblasts and promote tumors in transgenic mice. "If you put the data in humans, which are only correlative, and the data in mice and in cells together, the case becomes compelling," he contends.

Recently skp2 was implicated in the ubiquitination--and also, surprisingly, the transcriptional coactivation--of the oncoprotein c-Myc. "I think the reason why skp2 is seen so much in cancer is because of this Myc connection," says J. Wade Harper, a Harvard Medical School pathology professor. Nevertheless, that connection remains controversial among researchers.

UNPREDICTABLE EFFECTS In 2001, two teams of scientists independently reported discovering an F box protein that helps ubiquitinate cyclin E. One group dubbed the protein hCDC4; the other named it Fbw7. Subsequently, Steven I. Reed, leader of one of the groups and a molecular biology professor at Scripps Research Institute, detected disabling hCDC4 mutations in eight of 51 frozen endometrial adenocarcinomas. "The tumors that were mutated for CDC4 were more aggressive," he recalls.

Cyclin E plays a critical role in cell division by helping catalyze the transition from G1 phase to S phase. Reed has found that this cyclin, which is normally expressed briefly, is never downregulated in tumors with hCDC4 mutations. His theory: Cyclin E deregulation causes genomic instability, which "leads to carcinogenesis because it accelerates a loss of heterozygosity." Elimination of a chromatid, in turn, exposes the baleful influence of mutated tumor suppressor genes (normally recessive) on the surviving sister chromatid. Reed is searching now for hCDC4 mutations in 200 breast cancer specimens.

Just because an E3 ubiquitin ligase has a limited set of substrates does not mean that its effect on cancer is predictable. Consider the F box protein b-Trcp (for "transducin repeat containing protein"). This adaptor targets IaB, which is then ubiquitinated and destroyed. In myeloma cells treated with Velcade, IaB appears to act anti-oncogenically, and therefore b-Trcp is presumably pro-oncogenic.

Yet b-Trcp also ubiquitinates the oncoprotein b-catenin and thus might behave anti-oncogenically. Pagano's recent study of knockout mice lacking b-Trcp leads to a similar conclusion. The animals displayed various mitotic defects characteristic of tumor cells. Experiments indicated that this phenotype reflects the accumulation of Emi1, another b-Trcp substrate. To reconcile such pleiotropic effects, Pagano suggests, "It is possible that the same ligase functions as an oncogene in some tissues and as a tumor suppressor in others."

MANIPULATING THE SYSTEM Millennium Pharmaceuticals' Velcade proves that the manipulation of UMP can shrink or stabilize tumors. Currently, at least two other companies are developing drugs that target this system: Rigel Pharmaceuticals of South San Francisco, Calif.; and Celgene, of Warren, NJ.

	PROTEIN PUREE Á LA UBIQUITIN: Through a sequence of reactions, three ubiquitin ligases (E1, E2, and E3) attach a ubiquitin polypeptide to a protein… Click to view an enlarged diagram (24K)

Rigel has focused much of its initial efforts on the anaphase-promoting complex (APC), whose E3 ubiquitin ligase activity prompts the degradation of several mitotic regulators. "We have done a lot of in-house validation [showing] that there is a very strong case to be made for APC being a critical regulator of cell division," says chief scientific officer Donald G. Payan. "And it turns out that the critical subunits of APC are significantly elevated in a number of different cancers," notably those of the lung and breast.

The company has learned that cell-cycle arrest and apoptosis result when RNA interference downregulates these APC subunits in several tumor cell lines. APC inhibitors shrink tumor xenografts in nude mice. Payan discloses that Rigel hopes to start clinically testing an inhibitor by late 2004.

Celgene has discovered inhibitors of the F box protein, skp2, according to Frank Mercurio, senior director of target discovery. These agents cause cell-cycle arrest and apoptosis in cell lines derived from breast, cervical, prostate, and lung cancers, and from gliomas and multiple myeloma. The company is also developing compounds that both inhibit and activate smurf-1 and -2, very similar ubiquitin ligases that target elements of the TGF-b signal-transduction pathway. Smurfs are known to promote turnover of the TGF-b receptor.

Mercurio proposes that smurf activators work by repressing an auto-inhibitory function intrinsic to the ligase. "We found them by the fact that we would put these compounds in a cell, and they would promote degradation of the TGF-b receptor within about two hours," he recalls. Smurf inhibitors and activators might both prove clinically valuable. Inhibitors would prolong signaling along the TGF-b pathway, historically considered a tumor suppressor; activators would downregulate the pathway in some late-stage cancers, where Mercurio calls it a "bad" influence.

Raymond J. Deshaies, an associate biology professor at California Institute of Technology, is taking an unusual approach toward manipulating UMP--altering the substrates instead of interfering with the enzymes. In collaboration with UCLA's Kathleen M. Sakamoto and Yale's Craig M. Crews, Deshaies has fashioned a chimeric molecule containing the IaB site, which is recognized by the F box protein b-Trcp. The researchers attach this chimeric molecule covalently or noncovalently to a potential oncoprotein, such as the estrogen or androgen receptor, and the combination is ubiquitinated and degraded. Says Deshaies: "We've clearly shown that we could achieve that objective in a test tube, and now we've shown we could achieve it inside cells." One challenge is ferrying the chimeric molecule across the cell membrane.

Deshaies and another set of colleagues have also identified Rpn11, a new target for inhibiting proteasome function. This enzyme, which has a novel metalloprotease-active site, must remove ubiquitin molecules attached to a protein; otherwise, the proteasome cannot degrade that protein. Deshaies hopes to develop Rpn11 inhibitors with more favorable pharmacokinetics and a more convenient dosing schedule than he attributes to Velcade.

Will UMP-targeted anticancer agents besides Velcade be available soon? Payan estimates that ubiquitin ligase inhibitors are about five years behind kinase inhibitors, such as Gleevec, in the product pipeline (see The Current Status of Cancer Treatment). The kinds of selectivity and potency questions raised about kinase inhibitors in the late 1980s and early 1990s, he adds, "are going to dog these [ligase] programs until somebody shows up with something that works." Velcade, currently being tested on a host of tumors, might quell some of these doubts.

In the basic science arena, "two things are going on," observes Harper. The first is that screens of various systems seemingly unrelated to UMP are stumbling across more and more genes implicated in ubiquitination. The second development is the use of high-throughput approaches that, Harper says, aim "to look, in a unified way, at the whole family of genes and to map out pathways on a genomic scale."

Douglas Steinberg (dougste@attglobal.net) is a freelance writer in New York City