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Comment on "Low-penetrant RB allele in small-cell cancer shows geldanamycin instability and discordant expression with mutant ras" by Park et al.

It has now been over twenty years since the cloning and characterization of the retinoblastoma gene (RB), the first tumor suppressor gene to be identified. Although it was original identified in the rare eye cancer retinoblastoma, RB is almost ubiquitously affected in human cancer, either through direct mutation of the gene or functional inactivation of the protein. This central role for RB in cancer biology has led to intense scrutiny of its function. Among its biochemical properties, the RB protein can bind to E2F transcription factors, chromatin remodeling proteins such as histone deacetylases, SWI/SNF ATPases, DNA methyltransferases and histone methyltransferases, and viral oncoproteins such as SV40 large T antigen, human papillomavirus E7 and adenovirus E1a. 1 The protein can be inhibited by phosphorylation by cyclin-dependent kinases on up to 16 consensus phosphoacceptor sites. These properties have been useful for mapping the functional domains of the protein and for determining how mutations affect protein function. Physiologically, the RB protein was classically characterized as a transcriptional repressor that inhibited the expression of genes involved in cell cycle progression into S phase. More recently, it has been recognized that the RB protein also has important roles in repressing apoptotic genes and co-activation of genes involved in differentiation. A major question in the field has been whether these physiologic functions all result from the same biochemical activities of the RB protein or whether they constitute separable activities. Low penetrance RB mutants have been especially valuable in this regard. These mutations result in decreased penetrance and expressivity in families with hereditary retinoblastoma. Low penetrance RB mutations can be classified into two broad categories: those that result in reduced expression of the wildtype protein and those that result in an altered amino acid sequence.2 The second category of low penetrance mutants has been of particular value for dissecting the biochemical and physiologic functions of RB. For instance, the ability of the RB protein to bind E2Fs, repress transcription and inhibit S phase entry have been shown to be at least partly distinct from the transcriptional co-activation and differentiation functions.3, 4 An important question that has remained is why low penetrance RB mutants lead to cancer formation in some patients but not others. While the variable penetrance and expressivity have suggested for many years that there must be other cellular factors that modify the effect of low penetrance alleles, such factors have heretofore been unidentified. In an article in this issue of Cell Cycle, Park and co-authors provide the first evidence for such a modifying factor by showing that low penetrance RB mutants may be dependent on chaperones to maintain protein stability.5 For this study, the authors used an extrapulmonary small cell carcinoma line (ECC4) that expresses abundant endogenous levels of a low penetrance RB mutant (R661W). This cell line provided a rare opportunity to examine the effects of a low penetrance RB mutant in cancer cells that have spontaneously acquired the mutation, thereby allowing the mutation and its cellular consequences to be studied without the need for exogenous overexpression of the protein. This particular mutant was already known to be defective for E2F1 binding and for inducing G1 cell cycle arrest, whereas it retained the ability to partially suppress colony growth of RB(-) cells, to induce cell differentiation, and to weakly bind the SV40 large T antigen. In this study, the authors additionally found that low penetrance RB mutants showed greater binding activity when measured in mammalian cells as compared to in vitro GST-based binding assays. This suggested to the authors that factors present in vivo, such as chaperone-like proteins, may be responsible for the enhanced protein stability. In support of this possibility, serial freeze-thaw cycles of ECC4 cell extracts resulted in a depletion of cellular R661W protein, and the Hsp90 inhibitor geldanamycin caused a marked decrease in steady-state R661W protein. Taken together, these findings raise the possibility that the Hsp90 chaperone complex not only stabilizes a wide range of mutant gain-of-function oncogenes but it may also stabilize mutant tumor suppressor proteins. This study provides important insights but also raises new questions. Will Hsp90 inhibitors, which are currently being evaluated in clinical trials as cancer therapeutic agents, have unexpected and undesirable effects by destabilizing tumor suppressor proteins? Is the chaperone-dependent protein stability of R661W a general phenomenon of low penetrance RB mutants? These and other questions will need to be addressed in future studies. References Harbour JW, Dean DC. The Rb/E2F pathway: emerging paradigms and expanding roles. Genes Dev 2000; 14:2545-62. Otterson GA, Chen Wd, Coxon AB, Khleif SN, Kaye FJ. Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function. Proc Natl Acad Sci U S A 1997; 94:12036-40. Otterson GA, Modi S, Nguyen K, Coxon AB, Kaye FJ. Temperature-Sensitive RB Mutations Linked to Incomplete Penetrance of Familial Retinoblastoma in 12 Families. Am J Hum Genet 1999; 65:1040-6. Sellers WR, Novitch BG, Miyake S, Heith A, Otterson GA, Kaye FJ, et al. Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth. Genes Dev 1998; 12:95-106. Park Y, KuboA , Komiya T, Coxon A, Beebe K, Neckers L, Meltzer PS, Kaye FJ. Low-penetrant RB allele in small-cell cancer shows geldanamycin instability and discordant expression with mutant ras. Cell Cycle 2008; 7: In this issue.