A Little pRB Can Lead to Big Problems Philip W. Hinds Cancer Discovery 2014;4:764-765.
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VIEWS
IN THE SPOTLIGHT
A Little pRB Can Lead to Big Problems Philip W. Hinds Summary: Germline deletion of RB1, the gene encoding the retinoblastoma tumor-suppressor protein pRB, predisposes to eye tumor formation upon loss of the remaining wild-type allele. Many functions affecting cellcycle control, cell-cycle exit, and numerous other processes involved in the transformed phenotype have been ascribed to pRB, and deregulation of these processes is generally thought to result from complete loss of pRB in both hereditary and sporadic tumors in multiple tissues. Loss of just one allele of RB1 is now shown to lead to replication stress and aneuploidy in both mouse and human cells, and the mechanism through which this haploinsufficient phenotype is achieved may open up new opportunities for interceding both in tumor initiation and in treatment of extant tumors. Cancer Discov; 4(7); 764–5. ©2014 AACR. See related article by Coschi et al., p. 840 (7).
The retinoblastoma tumor-suppressor protein pRB, encoded by the first cloned tumor-suppressor gene, RB1, is also the archetype of hereditary cancer genes, predisposing its heterozygous carriers not only to high rates of the pediatric eye tumor retinoblastoma, but also to several secondary tumors, including sarcomas and melanoma (1). The hereditary nature of disease caused by cytogenetically detectable, germline mutation of one allele of RB1 gave rise to the now-classic “Knudson hypothesis,” which predicted that loss of the second allele in affected individuals is necessary and sufficient to produce retinoblastoma with very high penetrance (2). Many years of work subsequent to the cloning of the RB1 gene and identification and characterization of pRB has led to the “textbook” picture of pRB as a mediator of proliferation, predominantly through transcriptional regulation of cell-cycle transitions and/or cell-cycle exit (3), albeit that “new,” nontranscriptional functions such as regulation of chromosome stability and cellular metabolism have emerged more recently (4, 5). Left unanswered by these numerous, elegant studies, however, is the mechanism through which loss of one allele of RB1 apparently leads so efficiently to loss of the second allele, and possibly to tumor progression as a consequence of additional genetic and epigenetic changes. In the absence of an effect of RB1 heterozygosity on the transformed state of a cell, one is forced to conclude that rates of mutation must be sufficiently high to essentially ensure the second “hit” predicted by Knudson (2) in retinoblastoma, or in tumors other than retinoblastoma that somatic RB1 heterozygosity awaits the chance appearance of mutator alleles that ensure second RB1 allele loss. Although these events are certainly plausible, the high rates of spontaneous tumorigenesis observed in RB1 heterozygous humans and mice (albeit highly tissue specific), coupled with emerging Author’s Affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine and Molecular Oncology Research Institute, Tufts Medical Center, Boston, Massachusetts Corresponding Author: Philip W. Hinds, Tufts University School of Medicine, 150 Harrison Avenue, Jaharis 512D, Boston, MA 02111. Phone 617636-2734; Fax 617-636-9230; E-mail: [email protected] doi: 10.1158/2159-8290.CD-14-0518
©2014 American Association for Cancer Research. 764 | CANCER DISCOVERYJULY 2014
evidence that Rb1 heterozygous mice may indeed be predisposed to tumorigenesis even when the wild-type Rb1 allele is retained (6), support the concept that pRB may be haploinsufficient for some aspect of tumor suppression. This function could be an important clue to prevention of tumorigenesis at a preneoplastic stage. In strong support of a profound effect of RB1 heterozygosity on the propensity of certain cell types to suffer chromosomal instability and genomic aberrations, potentially predisposing them to tumorigenic progression, Coschi and colleagues (7) now show that pRB plays an important, dosage-sensitive role in maintaining the fidelity of DNA replication and accurate chromosome segregation in mitosis. This function of pRB is shown to involve recruitment of E2F1 and Condensin II to satellite repeats at pericentromeres and at least one origin of DNA replication, but not at promoters typically associated with “classical” E2F1 transcriptional regulatory function. Specific loss of this function of pRB, even in the heterozygous state, leads to loss of Condensin II and E2F1 binding to these repeat sequences, with a concomitant increase in focal binding of γH2AX. Consistent with the role of Condensin II in replication and chromosome condensation, cells heterozygous for a pRB protein specifically defective in pRB’s LXCXE domain required for Condensin II binding showed increased DNA replication fork progression but reduced replication protein A (RPA) binding (as assessed by chromatin immunoprecipitation), consistent with defective replication and thereby increased replication stress. Surprisingly, and most importantly, mouse embryo fibroblasts expressing just one copy of the LXCXE-defective pRB (while retaining the wild-type allele) showed CAP-D3 (a Condensin II subunit) binding defects equivalent to those seen in homozygous mutant cells, and, moreover, these fibroblasts displayed chromosome defects and aneuploidy that strongly suggest that Rb1 is haploinsufficient for replication and chromosome segregation control in this cell type. Finally, and compellingly, human fibroblasts derived from patients with retinoblastoma who are germline for one defective RB1 allele also displayed increased γH2AX foci and evidence of defective mitoses. This phenotype correlates with the observation that human sarcomas (and mouse lymphomas) that retain
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Downloaded from cancerdiscovery.aacrjournals.org on August 16, 2014. © 2014 American Association for Cancer Research.
VIEWS one wild-type RB1 allele contain as many whole chromosome changes as those lacking both RB1 alleles, and both had far more of these changes that did tumors that are wild-type for RB1. Thus, although the tumor data alone cannot assess whether there is a role for aneuploidy before tumorigenesis or after tumor initiation, the fact that RB1 heterozygosity leads to replication stress and mitotic defects that are recapitulated in tumors strongly suggests a tumorigenic role for this process in both human and mouse cells heterozygous for RB1. As with many studies of chromosome instability, defective replication, and aneuploidy in mammalian cells, the results here remain correlative with tumor formation, rather than incontrovertibly causative. However, overall the study is compelling in its implication that RB1 heterozygosity is a driver of both aneuploidy and tumorigenesis, with the former very likely promoting the latter. That the mechanistic basis of this rests squarely on Condensin II recruitment is well supported by the data, and this in turn provides a superb example of the power of genetic manipulation of the mouse genome in unraveling complex mechanisms affected by multifunctional proteins like pRb. In this case, mice bearing specific, point-mutant knockin alleles of Rb1 allowed a clear demonstration that E2F1 association with pRb is required for Codensin II recruitment, but “classic” interaction of pRb with E2F1 that allows transcriptional regulation is dispensable for CAP-D3 binding and protection from replication stress. These fine-tuned mechanistic insights also immediately raise the possibility of therapeutic intervention, given the recent report that manipulation of Cohesin function can suppress RB1-dependent chromosome abnormalities (8). Coupled with methods to detect cells defective in this function of pRB, one can begin to design strategies to lessen the rate of progression of such defective cells to the neoplastic state. In addition to providing clarity to the probable mechanism through which pRB protects from replication stress when present in sufficient levels, this work raises a number of intriguing questions related to the potential impact of this function on tumorigenesis. Although it seems likely that elevated propensity to undergo chromosome aberrations could drive the neoplastic transition, both mouse and human genetics strongly suggest that this is a tissue-specific phenomenon, because the types of tumors that readily arise in RB1 heterozygous humans and mice are limited. Here, the authors show that replication stress occurs in fibroblasts and is persistent in sarcomas (but absent when both alleles of RB1 are intact), but in the absence of data interrogating this effect in a wide variety of cell types, it remains unclear whether replication stress in the heterozygous condition is cell-type specific, or whether this is universal but confers differential susceptibility to tumor formation in different tissues. A second intriguing implication from the analysis of sarcomas in humans and lymphomas in mice presented here arises from the observation of a low level of aneuploidy in pRB-sufficient tumors. Given that tumors with intact pRB very often deregulate pRB function through activation of pRB kinases (often consequent to loss of p16INK4a), these data imply that loss or
reduction of pRB protein has very different consequences on tumor formation compared with kinase activation, and further exploration of the regulation of pRB–E2F1–Condensin II complex formation is warranted. Finally, this study raises the possibility that RB1 heterozygosity carries with it an innate ability to enhance bypass of tumor-suppressive mechanisms that otherwise would have to be overcome by two or more low-frequency mutations in tumors driven by other genetic events. That is, one might imagine that replication stress arising from RB1 heterozygosity would be met with induction of senescence or apoptosis in the likely event that aberrancies are detrimental to the cell. However, given the high frequency of discrete mutational events that arise in an individual that lacks one copy of RB1 in all of its cells, the probability of selection for mutations that bypass these tumor-protective processes would seem to be very high. Thus, tumor suppressors that have the capacity to raise mutation rates in a haploinsufficient manner might be expected to lead to an increased rate of senescence or apoptosis, a finding seemingly at odds with increased susceptibility to cancer, but this can be reconciled by the very mutational process itself. It will be very interesting to see if RB1 heterozygous tissues behave differently in regard to the ability to bypass such protective mechanisms, and if other hereditary tumor suppressors involved with genome integrity, such as BRCA1, might also lead to such a paradoxical duality by inducing high rates of senescence or apoptosis concomitant with increased tumor formation in individuals with single allele loss in the germline.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Published online July 7, 2014.
REFERENCES 1. Woo KI, Harbour JW. Review of 676 second primary tumors in patients with retinoblastoma: association between age at onset and tumor type. Arch Opthalmol 2010;128:865–70. 2. Knudson AGJ. Mutation and cancer: statistical study of retinoblastomas. Proc Natl Acad Sci U S A 1971;68:820–3. 3. Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 2013;14:518–28. 4. Manning AL, Dyson NJ. RB: mitotic implications of a tumour suppressor. Nat Rev Cancer 2012;12:220–6. 5. Hilgendorf KI, Leschiner ES, Nedelcu S, Maynard MA, Calo E, Ianari A, et al. The retinoblastoma protein induces apoptosis directly at the mitochondria. Genes Dev 2013;27:1003–15. 6. Williams BO, Remington L, Albert DM, Mukai S, Bronson RT, Jacks T. Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat Genet 1994;7:480–4. 7. Coschi CH, Ishak CA, Gallo D, Marshall A, Talluri S, Wang J, et al. Haploinsufficiency of an RB–E2F1–Condensin II complex leads to aberrant replication and aneuploidy. Cancer Discov 2014;4:840–53. 8. Manning AL, Yazinski SA, Nicolay B, Bryll A, Zou L, Dyson NJ. Suppression of genome instability in pRB-deficient cells by enhancement of chromosome cohesion. Mol Cell 2014;53:993–1004.
JULY 2014CANCER DISCOVERY | 765
Downloaded from cancerdiscovery.aacrjournals.org on August 16, 2014. © 2014 American Association for Cancer Research.
Updated version
Cited Articles
E-mail alerts Reprints and Subscriptions Permissions
Access the most recent version of this article at: http://cancerdiscovery.aacrjournals.org/content/4/7/764
This article cites by 8 articles, 3 of which you can access for free at: http://cancerdiscovery.aacrjournals.org/content/4/7/764.full.html#ref-list-1
Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected].
Downloaded from cancerdiscovery.aacrjournals.org on August 16, 2014. © 2014 American Association for Cancer Research.
VIEWS
IN THE SPOTLIGHT
A Little pRB Can Lead to Big Problems Philip W. Hinds Summary: Germline deletion of RB1, the gene encoding the retinoblastoma tumor-suppressor protein pRB, predisposes to eye tumor formation upon loss of the remaining wild-type allele. Many functions affecting cellcycle control, cell-cycle exit, and numerous other processes involved in the transformed phenotype have been ascribed to pRB, and deregulation of these processes is generally thought to result from complete loss of pRB in both hereditary and sporadic tumors in multiple tissues. Loss of just one allele of RB1 is now shown to lead to replication stress and aneuploidy in both mouse and human cells, and the mechanism through which this haploinsufficient phenotype is achieved may open up new opportunities for interceding both in tumor initiation and in treatment of extant tumors. Cancer Discov; 4(7); 764–5. ©2014 AACR. See related article by Coschi et al., p. 840 (7).
The retinoblastoma tumor-suppressor protein pRB, encoded by the first cloned tumor-suppressor gene, RB1, is also the archetype of hereditary cancer genes, predisposing its heterozygous carriers not only to high rates of the pediatric eye tumor retinoblastoma, but also to several secondary tumors, including sarcomas and melanoma (1). The hereditary nature of disease caused by cytogenetically detectable, germline mutation of one allele of RB1 gave rise to the now-classic “Knudson hypothesis,” which predicted that loss of the second allele in affected individuals is necessary and sufficient to produce retinoblastoma with very high penetrance (2). Many years of work subsequent to the cloning of the RB1 gene and identification and characterization of pRB has led to the “textbook” picture of pRB as a mediator of proliferation, predominantly through transcriptional regulation of cell-cycle transitions and/or cell-cycle exit (3), albeit that “new,” nontranscriptional functions such as regulation of chromosome stability and cellular metabolism have emerged more recently (4, 5). Left unanswered by these numerous, elegant studies, however, is the mechanism through which loss of one allele of RB1 apparently leads so efficiently to loss of the second allele, and possibly to tumor progression as a consequence of additional genetic and epigenetic changes. In the absence of an effect of RB1 heterozygosity on the transformed state of a cell, one is forced to conclude that rates of mutation must be sufficiently high to essentially ensure the second “hit” predicted by Knudson (2) in retinoblastoma, or in tumors other than retinoblastoma that somatic RB1 heterozygosity awaits the chance appearance of mutator alleles that ensure second RB1 allele loss. Although these events are certainly plausible, the high rates of spontaneous tumorigenesis observed in RB1 heterozygous humans and mice (albeit highly tissue specific), coupled with emerging Author’s Affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine and Molecular Oncology Research Institute, Tufts Medical Center, Boston, Massachusetts Corresponding Author: Philip W. Hinds, Tufts University School of Medicine, 150 Harrison Avenue, Jaharis 512D, Boston, MA 02111. Phone 617636-2734; Fax 617-636-9230; E-mail: [email protected] doi: 10.1158/2159-8290.CD-14-0518
©2014 American Association for Cancer Research. 764 | CANCER DISCOVERYJULY 2014
evidence that Rb1 heterozygous mice may indeed be predisposed to tumorigenesis even when the wild-type Rb1 allele is retained (6), support the concept that pRB may be haploinsufficient for some aspect of tumor suppression. This function could be an important clue to prevention of tumorigenesis at a preneoplastic stage. In strong support of a profound effect of RB1 heterozygosity on the propensity of certain cell types to suffer chromosomal instability and genomic aberrations, potentially predisposing them to tumorigenic progression, Coschi and colleagues (7) now show that pRB plays an important, dosage-sensitive role in maintaining the fidelity of DNA replication and accurate chromosome segregation in mitosis. This function of pRB is shown to involve recruitment of E2F1 and Condensin II to satellite repeats at pericentromeres and at least one origin of DNA replication, but not at promoters typically associated with “classical” E2F1 transcriptional regulatory function. Specific loss of this function of pRB, even in the heterozygous state, leads to loss of Condensin II and E2F1 binding to these repeat sequences, with a concomitant increase in focal binding of γH2AX. Consistent with the role of Condensin II in replication and chromosome condensation, cells heterozygous for a pRB protein specifically defective in pRB’s LXCXE domain required for Condensin II binding showed increased DNA replication fork progression but reduced replication protein A (RPA) binding (as assessed by chromatin immunoprecipitation), consistent with defective replication and thereby increased replication stress. Surprisingly, and most importantly, mouse embryo fibroblasts expressing just one copy of the LXCXE-defective pRB (while retaining the wild-type allele) showed CAP-D3 (a Condensin II subunit) binding defects equivalent to those seen in homozygous mutant cells, and, moreover, these fibroblasts displayed chromosome defects and aneuploidy that strongly suggest that Rb1 is haploinsufficient for replication and chromosome segregation control in this cell type. Finally, and compellingly, human fibroblasts derived from patients with retinoblastoma who are germline for one defective RB1 allele also displayed increased γH2AX foci and evidence of defective mitoses. This phenotype correlates with the observation that human sarcomas (and mouse lymphomas) that retain
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on August 16, 2014. © 2014 American Association for Cancer Research.
VIEWS one wild-type RB1 allele contain as many whole chromosome changes as those lacking both RB1 alleles, and both had far more of these changes that did tumors that are wild-type for RB1. Thus, although the tumor data alone cannot assess whether there is a role for aneuploidy before tumorigenesis or after tumor initiation, the fact that RB1 heterozygosity leads to replication stress and mitotic defects that are recapitulated in tumors strongly suggests a tumorigenic role for this process in both human and mouse cells heterozygous for RB1. As with many studies of chromosome instability, defective replication, and aneuploidy in mammalian cells, the results here remain correlative with tumor formation, rather than incontrovertibly causative. However, overall the study is compelling in its implication that RB1 heterozygosity is a driver of both aneuploidy and tumorigenesis, with the former very likely promoting the latter. That the mechanistic basis of this rests squarely on Condensin II recruitment is well supported by the data, and this in turn provides a superb example of the power of genetic manipulation of the mouse genome in unraveling complex mechanisms affected by multifunctional proteins like pRb. In this case, mice bearing specific, point-mutant knockin alleles of Rb1 allowed a clear demonstration that E2F1 association with pRb is required for Codensin II recruitment, but “classic” interaction of pRb with E2F1 that allows transcriptional regulation is dispensable for CAP-D3 binding and protection from replication stress. These fine-tuned mechanistic insights also immediately raise the possibility of therapeutic intervention, given the recent report that manipulation of Cohesin function can suppress RB1-dependent chromosome abnormalities (8). Coupled with methods to detect cells defective in this function of pRB, one can begin to design strategies to lessen the rate of progression of such defective cells to the neoplastic state. In addition to providing clarity to the probable mechanism through which pRB protects from replication stress when present in sufficient levels, this work raises a number of intriguing questions related to the potential impact of this function on tumorigenesis. Although it seems likely that elevated propensity to undergo chromosome aberrations could drive the neoplastic transition, both mouse and human genetics strongly suggest that this is a tissue-specific phenomenon, because the types of tumors that readily arise in RB1 heterozygous humans and mice are limited. Here, the authors show that replication stress occurs in fibroblasts and is persistent in sarcomas (but absent when both alleles of RB1 are intact), but in the absence of data interrogating this effect in a wide variety of cell types, it remains unclear whether replication stress in the heterozygous condition is cell-type specific, or whether this is universal but confers differential susceptibility to tumor formation in different tissues. A second intriguing implication from the analysis of sarcomas in humans and lymphomas in mice presented here arises from the observation of a low level of aneuploidy in pRB-sufficient tumors. Given that tumors with intact pRB very often deregulate pRB function through activation of pRB kinases (often consequent to loss of p16INK4a), these data imply that loss or
reduction of pRB protein has very different consequences on tumor formation compared with kinase activation, and further exploration of the regulation of pRB–E2F1–Condensin II complex formation is warranted. Finally, this study raises the possibility that RB1 heterozygosity carries with it an innate ability to enhance bypass of tumor-suppressive mechanisms that otherwise would have to be overcome by two or more low-frequency mutations in tumors driven by other genetic events. That is, one might imagine that replication stress arising from RB1 heterozygosity would be met with induction of senescence or apoptosis in the likely event that aberrancies are detrimental to the cell. However, given the high frequency of discrete mutational events that arise in an individual that lacks one copy of RB1 in all of its cells, the probability of selection for mutations that bypass these tumor-protective processes would seem to be very high. Thus, tumor suppressors that have the capacity to raise mutation rates in a haploinsufficient manner might be expected to lead to an increased rate of senescence or apoptosis, a finding seemingly at odds with increased susceptibility to cancer, but this can be reconciled by the very mutational process itself. It will be very interesting to see if RB1 heterozygous tissues behave differently in regard to the ability to bypass such protective mechanisms, and if other hereditary tumor suppressors involved with genome integrity, such as BRCA1, might also lead to such a paradoxical duality by inducing high rates of senescence or apoptosis concomitant with increased tumor formation in individuals with single allele loss in the germline.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Published online July 7, 2014.
REFERENCES 1. Woo KI, Harbour JW. Review of 676 second primary tumors in patients with retinoblastoma: association between age at onset and tumor type. Arch Opthalmol 2010;128:865–70. 2. Knudson AGJ. Mutation and cancer: statistical study of retinoblastomas. Proc Natl Acad Sci U S A 1971;68:820–3. 3. Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 2013;14:518–28. 4. Manning AL, Dyson NJ. RB: mitotic implications of a tumour suppressor. Nat Rev Cancer 2012;12:220–6. 5. Hilgendorf KI, Leschiner ES, Nedelcu S, Maynard MA, Calo E, Ianari A, et al. The retinoblastoma protein induces apoptosis directly at the mitochondria. Genes Dev 2013;27:1003–15. 6. Williams BO, Remington L, Albert DM, Mukai S, Bronson RT, Jacks T. Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat Genet 1994;7:480–4. 7. Coschi CH, Ishak CA, Gallo D, Marshall A, Talluri S, Wang J, et al. Haploinsufficiency of an RB–E2F1–Condensin II complex leads to aberrant replication and aneuploidy. Cancer Discov 2014;4:840–53. 8. Manning AL, Yazinski SA, Nicolay B, Bryll A, Zou L, Dyson NJ. Suppression of genome instability in pRB-deficient cells by enhancement of chromosome cohesion. Mol Cell 2014;53:993–1004.
JULY 2014CANCER DISCOVERY | 765
Downloaded from cancerdiscovery.aacrjournals.org on August 16, 2014. © 2014 American Association for Cancer Research.
