By David N. Leff
The Drosophila melanogaster fruit fly ¿ pioneer in vivo model of genetic research ¿ is famous for its quirkily named genes. One of them is kr|ppel ¿ the German word for ¿cripple.¿ But despite this disability-associated moniker, kr|ppel has jumped that reservation, and now denotes a ubiquitous mammalian gene.
¿It encodes a growing family of transcription factors,¿ observed translational researcher Scott Friedman, ¿the first of which, identified in Drosophila, was called the kr|ppel. But its sequence ultimately was closely related to one assigned to a growing family of these factors. Now these have been found in man, with 14 or 15 identified.¿
Friedman, who heads the Division of Liver Diseases at the Mount Sinai School of Medicine in New York, is senior author of a paper in today¿s Science, dated Dec. 21, 2001. It¿s titled: ¿KLF6, a candidate tumor suppressor gene mutated in prostate cancer.¿
¿The key finding in this article,¿ Friedman told BioWorld Today, ¿is that prostate cancer is a widely prevalent disease, which seems to be growing. Despite that, and unlike many other tumors, there has been no dominant tumor-suppressor gene that seemed to be effective. In many other tumors, like colon cancer,¿ he continued, ¿genes like p53 are frequently mutated. Yet in prostate cancer, there was no such tumor suppressor, until now, identified as frequently mutated.
¿Now, as we report, we¿ve identified a new gene, KLF6 ¿ kr|ppel-like factor 6 ¿ which works like p53, but independently of p53. In our hands, it has displayed a mutational frequency of well over 50 percent in sporadic prostate tumors. So this really offers, I think, a potentially important new insight into the signals that go awry when prostate cancer develops.¿
A modicum of serendipity diverted Friedman from his clinical expertise in liver disease to the experimental field of prostate oncology.
¿My interest for many years,¿ he recounted, ¿has not been in cancer, but in scarring of the liver ¿ fibrosis. My colleagues and I were instrumental in identifying a hepatic cell type that makes most of the scar. And in trying to elucidate this effect, we did a molecular approach called substraction cloning to ask what kinds of genes are turned on when this cell becomes active. One of those that we cloned was this Kr|ppel-like factor.
New Gene On Chromosome 10 Block
¿Data that we haven¿t yet published,¿ Friedman went on, ¿led us to identify a growth-suppressive role for this protein. Once we understood that, we were able to discover that it¿s positioned on a particular gene locus in the short arm of human chromosome 10, which is very frequently deleted in prostate cancer.
¿Every gene has two copies,¿ he explained, ¿one on each of the two chromosomal pairs. In order for a tumor suppressor gene to have its function completely lost, it needs to lose both copies. Typically, the way that happens, is for example when p53 sustains the physical loss of a piece of the chromosome that encodes that genomic neighborhood. The second event is a mutation or change in the gene¿s sequence so it¿s no longer encoding a functional protein. Then you¿ve got functional loss of both copies of the gene.
¿So we said, Gee, a growth-suppressive gene that¿s located on a chromosome frequently deleted in prostate cancer may be that its loss contributes to the pathogenesis.¿ So our team immediately started analyzing prostate tumor samples, to identify the two essential features of any tumor suppressor gene.
¿We were able to establish both those criteria for the KLF6 prostate cancer protein ¿ loss of heterozygosity (LOH) ¿ physical loss of one allele and mutation of the other. And the frequency was rather startling. Of all prostate cancers we sampled ¿ we didn¿t analyze a huge number, but enough so it¿s statistically valid ¿ overall, 55 percent of tumors had mutations. With those cancers that had LOH ¿ loss of the KLF6 locus, and how many of these had mutations, the numbers for both were around 70 percent.¿
The co-authors obtained primary tumor tissue specimens from 22 prostate cancer patients. LOH analysis revealed that one KLF6 allele was deleted in 17 samples ¿ 77 percent. Functional studies confirmed that whereas wild-type KLF6 protein upregulated the p21 growth suppressor, and significantly reduced cell proliferation, tumor-derived mutated KLF6 did not.
Hence, their paper suggested, ¿KLF6 is a tumor-suppressor gene involved in human prostate cancer.¿
¿Prostate cancer more than many malignancies,¿ Friedman observed, ¿has a good deal of heterogeneity. We found islands of malignant cells surrounded by normal cells, and the molecular changes in each of those foci could be different. These multiple mutant foci are typical of prostate cancer. So using laser-capture microdissection, we were able to physically carve out different islands of tumor, and analyze the DNA only from those foci independent one of the other, and see whether there was loss, and whether there were mutations. Prostate tumors are known to be highly heterogeneous. No one had ever identified the heterogeneity extended to this now very important tumor suppressor ¿ KLF6. So the novelty is not so much heterogeneity of prostate cancer but the fact that that heterogeneity is true for this protein as well.¿
Patent Pending, Biotech Talks In Progress
Friedman raised the clinical question: ¿How do we use this information? Does the presence of a KLF6 mutation tell us how likely that prostate tumor will be aggressive or not aggressive? To respond to hormonal or chemotherapy, or other treatments? In other words, how do we stratify the risk, knowing now whether a prostate cancer harbors a KLF6 mutation or not? Those are the studies we¿re working on, but have no doubt others will pick up on that as well. The most explicit new therapy,¿ he pointed out, ¿although more of a long-term prospect, would be trying to reintroduce normal KLF6 protein, so it returns to its normal function, in hopes of arresting tumorigenesis.¿
Some three months ago, Mount Sinai applied to patent Friedman¿s finding, under the heading, ¿KLF6, Kr|ppel-like factor 6 suppressor protein involved in p-53 independent transcriptional upregulation with p21 in vivo, and diagnostics and screening based on this protein.¿
¿We haven¿t signed any agreements at all with potential commercial partners,¿ Friedman allowed, ¿but we have had some preliminary discussions with one biotech company in particular that has a great visibility in genomics and genomic testing ¿ mostly for cancers.¿