NEWS

On the Eve of Protein Destruction: Ubiquitin Research Begins To Pay Off

Ken Garber

For the last three decades, many cancer researchers have focused on the abnormal production of proteins. An overexpressed oncogene or an underexpressed tumor suppressor gene can start cells on the road to cancer. The mechanisms of this malignant transformation have gradually emerged: alteration of signaling cascades, enhancement of angiogenesis, DNA repair defects, cell cycle dysregulation, and inhibition of apoptosis.

Now, protein destruction is vying with protein synthesis for the spotlight. An increasing number of laboratories are studying how cancer is related to ubiquitination, the tagging of proteins by a chain of tiny proteins called ubiquitins for destruction in the proteasome, the cell’s protein shredder. The ubiquitin field "has gone nuts" in the last 5 years, said Allan Weissman, M.D., chief of the Regulation of Protein Function Laboratory at the National Cancer Institute. In many cases, it is now clear, malignancy results when cells stop degrading a protein, or degrade too much, triggering cancer-promoting defects in the cell cycle, DNA repair, apoptosis or signaling—virtually the entire gamut of malignant mechanisms.



View larger version (136K):
[in this window]
[in a new window]
 
Dr. Allan Weissman

 
Laboratories and drug companies are eagerly targeting parts of the ubiquitin pathway for drug development. Even the proteasome itself has emerged as an unlikely but important drug target, with a proteasome inhibitor from Millennium Pharmaceuticals in advanced clinical development against a variety of cancers, particulary multiple myeloma.

The ubiquitin story began in the early 1970s, when Israeli biochemist Avram Hershko, M.D., Ph.D., noticed that the breakdown of a protein he was studying required energy. At the time, protein degradation was thought to be simply a waste disposal process taking place mainly in the lysosome, an organelle that contains enzymes that degrade proteins without the consumption of energy. In 1980 Hershko and postdoc Aaron Ciechanover, M.D., D.Sc., purified ubiquitin, the key component of the mysterious new energy-dependent protein destruction pathway.

Over the next decade Hershko and Ciechanover worked out the main elements of ubiquitin-mediated protein breakdown, or proteolysis, in vitro. Three classes of enzymes, known as ubiquitin ligases and designated E1, E2, and E3, that in turn bind to a ubiquitin chain, transport it, and then attach it to the protein substrate, tagging it for destruction by the proteasome.

Hershko’s work was "very original," commented cell biologist Michele Pagano, M.D., of New York University. "People were very much distracted by the discovery of DNA, of the genetic code, [by] how proteins are made, rather than how proteins are destroyed."

During the 1980s, Russian émigré scientist Alexander Varshavsky, Ph.D., at the Massachusetts Institute of Technology (also with Ciechanover), demonstrated that the same processes function in vivo. Hershko, Ciechanover, and Varshavsky shared the 2000 Lasker Award, the most prestigious prize in the medical sciences.

The discovery of the proteasome by Martin Rechsteiner, Ph.D., at the University of Utah in 1986, added a vital piece to the puzzle. Proteasome inhibitors later helped accelerate the field by making it possible to easily detect ubiquitinated proteins. The number of identified ubiquitin ligases and their substrates has exploded in the last few years, with defects in the ubiquitin system now implicated in many forms of cancer.

But in 1995, when ProScript, a tiny company leasing a basement office in Cambridge, Mass., discovered a drug that blocks the proteasome and decided to see if it could treat cancer, the idea was met with almost universal skepticism, because the treatment seemed likely to kill patients along with their tumors. The proteasome is essential for routine protein turnover, so it seemed as if blockage would shut down much normal biology and cause disastrous side effects. However, testing in the NCI’s 60-cell-line panel showed strong anticancer potential, and toxicity studies in animals were encouraging. ProScript eventually asked the NCI to fund clinical trials of its drug, PS-341. "Our budgets were strapped," recalled Julian Adams, Ph.D., ProScript’s chief scientist. "We were down to fumes."

The NCI agreed. Proteasome inhibitors were "a new group of agents aimed at a novel target," explained John Wright, M.D., Ph.D., of the NCI’s Cancer Therapy Evaluation Program. "And there was preclinical activity that was impressive."

In the clinic, PS-341 has been a surprising success. At the annual meeting of the American Society of Hematology last December, Millennium (which acquired ProScript in 1999) announced phase II clinical trial results for myeloma. The drug halted tumor growth in most of the 54 patients treated, with 33 experiencing at least a 50% reduction in levels of M protein, the standard tumor marker.

Since myeloma is incurable, myeloma specialists have greeted PS-341 with jubilation—along with some misgivings. "We have something to whack myeloma with [that] we didn’t have a year ago," said James Berenson, M.D., director of myeloma programs at Cedars-Sinai Medical Center in Los Angeles. "That’s pretty cool." But, he added, "This is not an easy drug." Some patients experience such severe pain after taking PS-341 that they refuse to continue treatment. But enough have benefited that about 30 separate clinical trials are now under way in patients with a wide range of cancers. (See box above.)


View this table:
[in this window]
[in a new window]
 
Selected Clinical Trials of PS-341

 
Although side effects so far seem manageable, no one knows the long-term effects of partial, periodic proteasome inhibition in humans. "We definitely need to understand the effects of partial inhibition before we can make sweeping statements about why it’s not toxic to normal cells," said cell biologist Dave McConkey, Ph.D., of the University of Texas M. D. Anderson Cancer Center, Houston. Primate studies have only have data from 3 months of follow-up.



View larger version (147K):
[in this window]
[in a new window]
 
Dr. Dave McConkey

 
PS-341’s mechanism of action is poorly understood, but it seems to kill tumors in a variety of ways. For example, the cell cycle regulatory proteins known as cyclins must be ubiquitinated and destroyed for the cell cycle to proceed through cell division. Adams originally expected that PS-341, by allowing cyclin buildup, would arrest cell division and halt tumor growth.

PS-341 does seem to have this effect, but other factors are also at work. In particular, PS-341 appears to inhibit NF-{kappa}B, said McConkey. NF-{kappa}B is a transcription factor that promotes expression of genes whose products promote tumor and tumor blood vessel growth and block apoptosis. NF-{kappa}B overactivation is a hallmark of many cancers. PS-341, by forcing the buildup of a protein that prevents NF-{kappa}B activation, may starve tumors of their blood supply and growth stimuli and promote their self-destruction.

On a cellular level, blocking the proteasome stresses cancer cells by jamming them with proteins. Adams believes that cancer cells are selectively vulnerable to PS-341 because, with their dysfunctional cell cycle checkpoints, they cannot handle the stress of protein buildup as easily as normal cells can. This stress causes "catastrophic signaling events in the cell, which drive the tumor cell to die," explained Adams. "A normal, untransformed cell can withstand the stress response, at least for short periods of time." Intermittent dosing apparently allows the proteasome in normal cells time to recover.

But PS-341, because it indiscriminately raises levels for hundreds of proteins without regard to their anticancer effect, is not an ideal cancer drug. Targeting E3 ubiquitin ligases, on the other hand, promises more specificity, because most E3s tag only a few proteins for destruction. Such drugs can, in theory, block degradation of only those proteins thought to have a direct anticancer effect.

One important drug target is the E3 ligase known as Skp2, one of about 70 known "F-box proteins." New York University’s Pagano, in 1999, identified Skp2 as the F-box protein that controls degradation of p27, an important tumor suppressor gene. Skp2 levels are abnormally high in many human tumors.

"A drug blocking Skp2 activity [is] going to be cytostatic, it will block proliferation, and probably together with another drug it could be very efficient," predicted Pagano. Pagano’s laboratory, in collaboration with the New Jersey biotechnology company Celgene, is looking for Skp2 inhibitors, and so is Millennium. Other companies are in the hunt as well.

Another potential target is the anaphase promoting complex (APC), an E3 with several protein substrates, including cyclin. APC controls the final stage of mitosis. Abnormal APC has been implicated in some cancers, and blocking APC should halt cell division, although (as for all ligase inhibitors) the anticancer effect remains to be seen.

The most exciting target may be Mdm2, an E3 that targets the p53 tumor suppressor gene for degradation in the proteasome. "In many cancers that express wild-type p53 there’s actually a gene amplification of Mdm2," said Weissman. "That’s the way those cells lower the p53 levels." Mdm2 inhibitors, in theory, should be effective cancer cell killers, because restoring p53 levels tends to promote apoptosis. The NCI’s Karen Vousden, Ph.D., together with Weissman and Maryland biotech company IGEN, are now screening for such inhibitors.

Other ubiquitin ligases have been implicated in cancer, and the list of drug targets is growing, because the ubiquitin field is still very young. But it has already spawned new daughter fields, including ubiquitin-like proteins (UBLs), and deubiquitinating enzymes, which remove ubiquitins from proteins and keep them from being destroyed. "I think that, after the ligases, the area of deubiquitination is really going to become the next hot area," said Weissman.



             
Copyright © 2002 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement