MUTAGENS AND MITOSIS:
Courtesy of Thea Goepfert
Section cut through the mammary gland of a rat that had been treated with the carcinogen MNU. Centrosomes (green) are arranged at the base of each nucleus (red), and 45% of cells show amplified chromosomes.
Comments from journal article reviewers often surprise or frusrate. But the reviewer response that cell biologist William Brinkley received six years ago left him stunned.
Brinkley and his collaborator, Subrata Sen at the University of Texas M.D. Anderson Cancer Center, were looking to demonstrate that a protein found on centrosomes, the organizing centers for microtubules during mitosis, disrupted cell division and possibly triggered carcinogenesis. Reviewers praised the work, but declared it incomplete. Brinkley and his colleagues, they instructed, needed to experimentally demonstrate that the protein of interest, when inserted into a diploid cell and induced to produce in excess, actually generated more centrosomes and converted the cell to...
Protein localization studies have revealed a bevy of centrosome-associated proteins that may help control centrosome number, or they may be getting modified in a way that helps regulate cell-cycle progression. Both possibilities could signify a role in carcinogenesis.
After satisfying the reviewer's request, Brinkley and Sen showed in the late 1990s that aurora kinase A localized specifically to centrosomes in mouse cells.1 Aurora kinase A, already found in fruit flies and yeast, is overexpressed in many cancers. Loss of control at the locus leads to overproduction and centrosome anomalies, suggests Brinkley.
At about the same time, Steve Doxsey of the University of Massachusetts in Worcester used antibodies to a centrosome-associated protein called pericentrin to demonstrate that most human tumors have centrosome defects.2 Pericentrin was initially discovered using serum from a patient with a rare autoimmune disease called scleroderma; the serum contained autoantibodies targeting centrosomes.
"I always wrote in my grants that centrosomes organized spindles, and if you have centrosome problems you'll have spindle problems and chromosome missegregation that may lead to cancer," says Doxsey. "But I never really believed that it'd really be true." Nevertheless, his work suggested that several different tumor types had centrosome defects. "To actually find it and to find it to be so pervasive was a surprise to me."
Recent findings in yeast from John Kilmartin at the Medical Research Council Laboratory of molecular biology in Cambridge, UK, suggest that a centrin-binding protein called Sfi1, which has homologs in humans, has a major role in centrosome duplication. Doxsey's conclusions were echoed in another 1998 paper in which Salisbury and colleagues found that a set of 35 high-grade breast tumors had major centrosome defects.3
CAUSE OR CONSEQUENCE
Despite progress in characterizing proteins on the centrosome, no one knows precisely the causative role of centrosomes and centrosome proteins in the onset of cancer. But much of the evidence points to an early role for centrosomes gone awry.
Circumstantial evidence for a causative link came in 2001.4 Doxsey showed that pericentrin, when elevated in vitro, can induce tumor-like features, including increased cell proliferation, the formation of extra chromosomes, as well as multipolar spindles and subsequent chromosome missegregation. "That sort of closes the circle," says Doxsey. "It does not prove that it's involved in tumorigenesis, but it's a better connection."
Last year came what Doxsey calls the biggest surprise thus far. His group found that 20% to 50% of all early lesions in breast, prostate, and cervix that they examined had centrosome defects.5 Salisbury found similar results for breast cancer.6 The findings, says Doxsey, were important for two major reasons: They showed that centrosome defects are one of the earliest changes in carcinogenesis, and that these defects occur together with genetic instability.
Between 75% and 95% of the cells that had one defect had the other. This result, according to Doxsey, suggests that centrosome defects could cause both chromosomal missegregation and genetic instability that can lead to or exacerbate cancer. It's still unclear, however, as to whether the genetic instability comes before the centrosome problems or vice versa.
Further elucidation of the centrosome connection may come from examining its interaction with the tumor suppressor p53. Past work has suggested that p53 localizes to centrosomes. And earlier this year, Sen identified a serine residue on p53 that's phosphorylated by aurora A.7 But, Sen points out, investigators don't yet know how much of this actually happens on centrosomes, or how this relates to centrosome structure and/or function.
Some remain skeptical of the tumor suppressor-centrosome link. Though the elimination of p53 as well as BRCA1 and BRCA2 result in centrosome aberrations, Erich Nigg, professor of cell biology at the Max-Planck Institute for Biochemistry in Martinsried, Germany, offers what he calls an equally plausible explanation: Centrosome aberrations in cells that lack tumor suppressors arise through other mechanisms, such as cell-division failures. That is, without intact tumor suppressors, aberrant cells with multiple centrosomes are able to survive. "It'll look as if the absence of p53 has caused the multiple centrosomes," he says. "But I think it simply has allowed survival of the cell which would otherwise had been eliminated."
BURDEN OF PROOF
Determining whether centrosome aberration is the major causal factor in carcinogenesis may turn out to be a moot point. Even if their effects are somewhat downstream, centrosomes may play a major role in how and when cancers proliferate. "I feel absolutely no urge, no burden of proof to show that the centrosome aberration is the first spark that sets the fire ablaze," says Nigg, adding that he'd be surprised if that were the case. "But once you have a little flame there for whatever reason, a centrosome abnormality in the tumor will make the tumor more unstable [and] the genome more unstable."
Indeed, one model favored by Brinkley, Doxsey, and others suggests that centrosome aberrations spread as part of a sort of Darwinian selection process. Cells with centrosome defects create progeny that missegregate chromosomes. Those cells with advantageous features – for example, anti-apoptotic activity – survive and thrive, potentially as part of carcinogenesis. It's a model that could help explain the pace and pattern of cancer proliferation.
Research progress in centrosome biology, while impressive, has not yet yielded therapeutic candidates. But scientists and companies are starting to look to centrosomes for possible novel prognostics. Doxsey is working with researchers at Cytyc in Boxborough, Mass., to develop a means for earlier cervical cancer diagnoses; they'll be looking for centrosome defects in Pap smear samples. Doxsey and his collaborators also hope to improve on prostate-specific antigen tests for prostate cancer. A centrosome-based diagnostic agent might detect the cancer earlier to enable aggressive treatments for some, and to help others avoid unnecessary prostate removal.
"This whole thing for me, as a basic centrosome biologist, has been a great ride," says Doxsey, referring to his work's unexpected clinical relevance. "It really has put us in a position where we can ask about prognostics and diagnostics and maybe treatments."