The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.Key words: breast cancer, senescence, retinoblastoma, p130, p107     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.     

The Rb2/p130 gene product is a nuclear protein whose phosphorylation is cycle regulated

The Rb2/p130 protein has been shown to have a high sequence homology with the retinoblastoma gene product (pRb), one of the most well-characterized tumor suppressor genes, and with pRb-related p107, especially in their conserved pocket domains, which display a primary role in the function of these proteins. In this study, we report on the biochemical and immunocytochemical characterization of the Rb2/p130 protein, using a polyclonal antibody developed against its “spacer” region included in the pocket domain of the whole protein. We show that pRb/p130 is a phosphoprotein located at the nuclear level and that its phosphorylation pathway can be dramatically reduced by phosphatase treatment. Moreover pRb/p130, with p107, with p107, is one of the major targets of the E1A viral oncoprotein-associated kinase activity, showing a phosphorylation pattern which is modulated during the cell cycle, reaching a peak of activation at the onset of S-phase. © 1995 Wiley-Liss, Inc.     

pRb or its cousins: Who controls the family business?

Comment on: Bazarov A, et al. Cell Cycle 2012; 11:1008–1013More than 90% of human cancers are of epithelial origin. Cellular senescence of human mammary epithelial cells (HMECs) is an important barrier that protects cells from immortalization; the first step in breast cancer development.¹ Although induction of tumor suppressor p16 is not evident in some types of normal human fibroblasts undergoing senescence,² in cultured HMECs, senescence occurs by a robust p16 induction, and cells that acquire silencing of p16Ink4a locus eventually proliferate and undergo senescence again by telomere shortening in a p53-dependent manner.¹ Therefore, p16 induction is a critical barrier to immortalize HMECs in culture. p16 inhibits kinase activity of Cdk4/6-cyclinD complexes, which inactivate three pRb family proteins: pRb, p107 and p130. However, the relative contribution of these three pRb family proteins to HMEC senescence is not well understood.In a recent issue of Cell Cycle, Bazarov et al. examined the role of each pRb family protein in p16-mediated senescence in breast cancer cell lines and in HMECs (Fig. 1).³ They showed that knockdown of each of the three pRb family proteins individually did not abrogate senescence mediated by ectopically expressed p16 in the breast cancer cell lines MDA-MB-231 and MCF7. However, the senescence induced by ectopic p16 was abrogated if they introduced E7, which inactivates all three pRb family proteins. Their data suggest that two of pRb family proteins can compensate for the loss of each pRb family protein to induce p16-mediated senescence in these cancer cells. The remaining question is whether all three pRb family members play an additive role, and whether the inactivation of at least two members of the pRb family is required to overcome p16-induced senescence in breast cancer cells. On the other hand, they showed that abrogation of pRb, but not of p107 and/ or p130, attenuates senescence in HMECs, suggesting a non-redundant critical role of pRb in HMEC senescence. These data are consistent with a recent report demonstrating that pRb has a non-redundant role in repressing DNA replication during H-ras-induced senescence of human fibroblasts,⁴ and explain why pRb, but not p107 or p130, is frequently mutated in cancer. Interestingly, although abrogation of pRb is critical for HMECs escaping senescence, simultaneous depletion of pRb together with either p107, p130 or both accelerates bypass of senescence. This suggests that p107 and p130 help pRb to trigger/maintain HMEC senescence in culture and possibly in vivo. Although each pRb family protein preferentially binds to different members of the E2F family,⁵ the contribution of each E2F family protein in escaping p16-mediated senescence remains unclear. Therefore, it will be interesting to see whether the critical role of pRb, and a supportive role of p130 and p107, in p16-mediated HMEC senescence depend on how each pRb family protein interacts with an E2F family protein.Open in a separate windowFigure 1. Contribution of pRb family proteins to p16-mediated senescence in breast cancer cells and HMECs. Knockdown of each of the three pRb family proteins in breast cancer cells does not abrogate ectopic p16-induced senescence, suggesting that either two of pRb family proteins can compensate for the loss of each pRb family proteins or all three of pRb family proteins play an additive role in p16-mediated senescence in breast cancer cells. On the other hand, knockdown of pRb, but not of p107 or p130, abrogates HMEC senescence, suggesting a non-redundant critical role for pRb in senescence of HMECs. However, the knockdown of either p107 or p130, in conjunction with pRb depletion, abrogates HMEC senescence more efficiently than pRb knockdown alone. This suggests a supporting role for p107 and p130 in maintaining HMEC senescence.Bazarov et al. also showed that even aggressive p53-negative breast cancer cells undergo cellular senescence upon ectopic p16 expression. These results are quite encouraging from an epigenetic therapy point of view. Silencing of p16 often occurs in breast cancer cells via promoter methylation. During DNA replication, cells require new p16 promoter methylation to keep p16 silenced. The observations of Bazarov et al. suggest that we may be able to stop the growth of even aggressive p53-negative breast cancers in patients by inducing p16 expression in cancer cells using DNA methylation inhibitors. Back to the question of running family business: “it appears that pRb is still the boss, but in some cases, it may get a helping hand from his cousins- p107 and p130.”     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

Interaction of c-Myc with the pRb-related protein p107 results in inhibition of c-Myc-mediated transactivation.

The product of the c-myc proto-oncogene, c-Myc, is a sequence-specific DNA binding protein with an N-terminal transactivation domain and a C-terminal DNA binding domain. Several lines of evidence indicate that c-Myc activity is essential for normal cell cycle progression. Since the abundance of c-Myc during the cell cycle is constant, c-Myc's activity may be regulated at a post-translational level. We have shown previously that the N-terminus of c-Myc can form a specific complex with the product of the retinoblastoma gene, pRb, in vitro. These data suggested a model in which pRb, or pRb-related proteins, regulate c-Myc activity through direct binding. We show here that the pRb-related protein p107, but not pRb itself, forms a specific complex with the N-terminal transactivation domain of c-Myc in vivo. Binding of p107 to c-Myc causes a significant inhibition of c-Myc transactivation. Expression of c-Myc releases cells from a p107-induced growth arrest, but not from pRb-induced growth arrest. Our data suggest that p107 can control c-Myc activity through direct binding to the transactivation domain and that c-Myc is a target for p107-mediated growth suppression.     

Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.     

Cooperation between p53 and p130(Rb2) in induction of cellular senescence

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.     

Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.     

Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107- deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb- deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.     

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.     

The switch from pRb/p105 to Rb2/p130 in DNA damage and cellular senescence

Cellular senescence is a response to genotoxic stress that results in an irreversible cell cycle arrest. Activation of this pathway relies on the activity of the retinoblastoma proteins and proteins of the DNA damage response cascade. Here, we discuss the functional relevance of the switch from pRb/p105 to Rb2/p130 that becomes apparent when cells enter senescent arrest.     

An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis

Simian virus 40 large T antigen transforms cells by sequestration and inactivation of the tumor suppressor proteins p53, retinoblastoma gene product (pRb), and the pRb-related proteins p107 and p130. Thus, the absence of functional p53 is expected to promote T antigen-mediated tumorigenesis. However, in a transgenic mouse model of T antigen-mediated beta cell carcinogenesis (Rip1Tag2), tumor volumes are significantly diminished when these mice are intercrossed with p53-deficient mice. Whereas the incidence of beta tumor cell apoptosis is unaffected, their proliferation rate is reduced in p53-deficient beta cell tumors in vivo and in cell lines established from these tumors in vitro. Biochemical analyses reveal higher levels of T antigen in wild-type tumor cells as compared to p53-deficient tumor cells. The data indicate that p53 stabilizes SV40 large T antigen, thereby augmenting its oncogenic potential as manifested by increased proliferation rates in wild-type beta tumor cells as compared to p53-deficient beta tumor cells.