David Cobrinik

Pocket proteins and cell cycle control

The retinoblastoma protein (pRB) and the pRB-related p107 and p130 comprise the ‘pocket protein’ family of cell cycle regulators. These proteins are best known for their roles in restraining the G1–S transition through the regulation of E2F-responsive genes. pRB and the p107/p130 pair are required for the repression of distinct sets of genes, potentially due to their selective interactions with E2Fs that are engaged at specific promoter elements. In addition to regulating E2F-responsive genes in a reversible manner, pocket proteins contribute to silencing of such genes in cells that are undergoing senescence or differentiation. Pocket proteins also affect the G1–S transition through E2F-independent mechanisms, such as by inhibiting Cdk2 or by stabilizing p27Kip1, and they are implicated in the control of G0 exit, the spatial organization of replication, and genomic rereplication. New insights into pocket protein regulation have also been obtained. Kinases previously thought to be crucial to pocket protein phosphorylation have been shown to be redundant, and new modes of phosphorylation and dephosphorylation have been identified. Despite these advances, much remains to be learned about the pocket proteins, particularly with regard to their developmental and tumor suppressor functions. Thus continues the story of the pocket proteins and the cell cycle.

Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring V600E BRAF

Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring V600EBRAF mutations. RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.

Retinoblastoma Has Properties of a Cone Precursor Tumor and Depends Upon Cone-Specific MDM2 Signaling

Retinoblastomas result from the inactivation of the RB1 gene and the loss of Rb protein, yet the cell type in which Rb suppresses retinoblastoma and the circuitry that underlies the need for Rb are undefined. Here, we show that retinoblastoma cells express markers of postmitotic cone precursors but not markers of other retinal cell types. We also demonstrate that human cone precursors prominently express MDM2 and N-Myc, that retinoblastoma cells require both of these proteins for proliferation and survival, and that MDM2 is needed to suppress ARF-induced apoptosis in cultured retinoblastoma cells. Interestingly, retinoblastoma cell MDM2 expression was regulated by the cone-specific RXRgamma transcription factor and a human-specific RXRgamma consensus binding site, and proliferation required RXRgamma, as well as the cone-specific thyroid hormone receptor-beta2. These findings provide support for a cone precursor origin of retinoblastoma and suggest that human cone-specific signaling circuitry sensitizes to the oncogenic effects of RB1 mutations.

Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1+/- mice

Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied retinoblastoma protein (pRb) targets partially inhibited tumorigenesis in Rb1+/− mice. Here we report that inactivation of pRb target Skp2 (refs. 7,8) completely prevents spontaneous tumorigenesis in Rb1+/− mice. Targeted Rb1 deletion in melanotrophs ablates the entire pituitary intermediate lobe when Skp2 is inactivated. Skp2 inactivation does not inhibit aberrant proliferation of Rb1-deleted melanotrophs but induces their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knock-in mice reproduces the effects of Skp2 knockout, identifying p27 ubiquitination by SCFSkp2 ubiquitin ligase as the underlying mechanism for Skp2s essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also undergo apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induces apoptosis. These results reveal that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency.

The cyclin-dependent kinase inhibitor p57 Kip2 mediates proliferative actions of PTHrP in chondrocytes

Parathyroid hormone-related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57(Kip2) is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrPs proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression.

Rho regulates p21 CIP1 , cyclin D1, and checkpoint control in mammary epithelial cells

The small GTPase Rho is important for cell cycle progression and Ras transformation in fibroblasts. However, it is unclear whether Rho is needed for proliferation in other cell types, and its targets in promoting normal cell cycle progression are unknown. Here, we demonstrate that Rho is required for G1 to S progression in MCF10A mammary epithelial cells, both in response to EGF and in response to oncogenic Ras. We describe two effects of Rho, the repression of p21CIP1 and the induction of cyclin D1, that may underlie its role in promoting S phase entry. The Rho inhibitor, C3 exotransferase, induced p21CIP1 both in EGF-stimulated and V12Ras-expressing cells. In addition, C3 blocked EGF-stimulated cyclin D1 promoter activity whereas V14RhoA induced the cyclin D1 promoter and cooperated with V12Ras in cyclin D1 induction. Finally, a high proportion of cells co-expressing V14RhoA and V12Ras displayed lobulated, polyploid nuclei that were actively synthesizing DNA. Our results demonstrate that Rho plays a fundamental role in promoting Ras-dependent S phase entry in mammary epithelial cells, whether in response to normal or oncogenic signaling, and indicate that in cells expressing oncogenic Ras, the activation of Rho diminishes p21CIP1 expression, increases cyclin D1 promoter activity, and uncouples DNA synthesis from mitosis.

Growth factor-dependent induction of p21CIP1 by the green tea polyphenol, epigallocatechin gallate

Tea polyphenols inhibit tumorigenesis and cell proliferation in rodent models, but their effects on cell signaling and cell cycle control pathways are undefined. Here, we show that the major polyphenol in green tea, epigallocatechin gallate (EGCG), inhibits S phase entry in epidermal growth factor (EGF) - stimulated MCF10A breast epithelial cells when provided in G0 or mid G1, but not when provided after the late G1 restriction point. EGCG induced p21(CIP1/WAF1/SDI1), inhibited cyclin D1-associated pRB kinase activity, and impaired pRB phosphorylation. The ability of EGCG to induce p21 depended upon the addition of EGF, indicating that EGCG synergizes with growth factor-dependent signals to induce p21 and impair cell cycle progression.

Rb suppresses human cone-precursor-derived retinoblastoma tumours.

Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest

Unregulated FGF signaling affects endochondral ossification and long bone growth, causing several genetic forms of human dwarfism. One major mechanism by which FGFs regulate endochondral bone growth is through their inhibitory effect on chondrocyte proliferation. Because mice with targeted mutations of the retinoblastoma (Rb)-related proteins p107 and p130 present severe endochondral bone defects with excessive chondrocyte proliferation, we have investigated the role of the Rb family of cell cycle regulators in the FGF response. Using a chondrocyte cell line, we found that FGF induced a rapid dephosphorylation of all three proteins of the Rb family (pRb, p107, and p130) and a blockade of the cells in the G1 phase of the cell cycle. This cell cycle block was reversed by inactivation of Rb proteins with viral oncoproteins such as polyoma large T (PyLT) antigen and Adenovirus E1A. Expression of a PyLT mutant that efficiently binds pRb, but not p107 and p130, allowed the cells to be growth inhibited by FGF, suggesting that pRb itself is not involved in the FGF response. To investigate more precisely the role of the individual Rb family proteins in FGF-mediated growth inhibition, we used chondrocyte micromass culture of limb bud cells isolated from mice lacking Rb proteins individually or in combination. Although wild-type as well as Rb −/− chondrocytes were similarly growth inhibited by FGF, chondrocytes null for p107 and p130 did not respond to FGF. Furthermore, FGF treatment of metatarsal bone rudiments obtained from p107 −/ −;p130 − / − embryos failed to inhibit proliferation of growth plate chondrocytes, whereas rudiments from p107-null or p130-null embryos showed only a slight inhibition of growth. Our findings indicate that p107 and p130, but not pRb, are critical effectors of FGF-mediated growth inhibition in chondrocytes.

Cdk2-dependent Phosphorylation and Functional Inactivation of the pRB-related p130 Protein in pRB(−), p16INK4A(+) Tumor Cells

The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G1 phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16INK4A in most pRB(−) cells suggests that p107 and p130 may be underphosphorylated and remain associated with E2Fs during G1-S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(−), p16(+) Saos-2 osteosarcoma cells. p130, but not p107, was phosphorylated and released from E2F-4 in late G1 and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(−), p16(+) cells as compared with pRB(+), p16(−) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin E- and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins.