Philip W. Hinds

A common polymorphism acts as an intragenic modifier of mutant p53 behaviour.

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.

Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.

The Retinoblastoma Protein Acts as a Transcriptional Coactivator Required for Osteogenic Differentiation

The incidence of osteosarcoma is increased 500-fold in patients who inherit mutations in the RB gene. To understand why the retinoblastoma protein (pRb) is specifically targeted in osteosarcoma, we studied its function in osteogenesis. Loss of pRb but not p107 or p130 blocks late osteoblast differentiation. pRb physically interacts with the osteoblast transcription factor, CBFA1, and associates with osteoblast-specific promoters in vivo in a CBFA1-dependent fashion. Association of pRb with CBFA1 and promoter sequences results in synergistic transactivation of an osteoblast-specific reporter. This transactivation function is lost in tumor-derived pRb mutants, underscoring a potential role in tumor suppression. Thus, pRb functions as a direct transcriptional coactivator promoting osteoblast differentiation, which may contribute to the targeting of pRb in osteosarcoma.

Cyclins and cdks in development and cancer: a perspective

A fundamental aspect of cancer is dysregulated cell cycle control. Unlike normal cells that only proliferate when compelled to do so by developmental or other mitogenic signals in response to tissue growth needs, the proliferation of cancer cells proceeds essentially unchecked. This does not mean that cancer cell cycles are necessarily different from those found in normal cycling cells, but rather implies that cancer cells proliferate because they are no longer subject to proliferation-inhibitory influences arising from the stroma or from gene expression pattern changes consequent to ‘terminal’ differentiation, nor do they necessarily require extrinsic growth factors to recruit them into or maintain their proliferative state. Finally, cancer cells have also often avoided normal controls linked to cell cycle progression that halt proliferation in the presence of damaged DNA or other physiological insults. The result of these alterations is the inappropriate proliferation commonly associated with cancerous tumor formation. This review will summarize the current understanding of dysregulation of the G0/G1-to-S-phase transition in cancer cells, with particular emphasis on recent in vivo studies that suggest a need to rethink existing models of cell cycle control in development and tumorigenesis.

Terminal Osteoblast Differentiation, Mediated by runx2 and p27KIP1, Is Disrupted in Osteosarcoma

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A

In this study, we show that NANOG, a master transcription factor, regulates S-phase entry in human embryonic stem cells (hESCs) via transcriptional regulation of cell cycle regulatory components. Chromatin immunoprecipitation combined with reporter-based transfection assays show that the C-terminal region of NANOG binds to the regulatory regions of CDK6 and CDC25A genes under normal physiological conditions. Decreased CDK6 and CDC25A expression in hESCs suggest that both CDK6 and CDC25A are involved in S-phase regulation. The effects of NANOG overexpression on S-phase regulation are mitigated by the down-regulation of CDK6 or CDC25A alone. Overexpression of CDK6 or CDC25A alone can rescue the impact of NANOG down-regulation on S-phase entry, suggesting that CDK6 and CDC25A are downstream cell cycle effectors of NANOG during the G1 to S transition.

Requirement for p27KIP1 in Retinoblastoma Protein-Mediated Senescence

ABSTRACT In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB −/−tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27KIP1 that is accompanied by an increase in p27KIP1 specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27KIP1 and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27KIP1 and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21CIP1 or p27KIP1 induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27KIP1 expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27KIP1 is critical for pRb-induced senescence.

P53 is a tumor suppressor gene

DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.

Melanocytic nevus-like hyperplasia and melanoma in transgenic BRAFV600E mice

BRAF, a cellular oncogene and effector of RAS-mediated signaling, is activated by mutation in ∼60% of melanomas. Most of these mutations consist of a V600E substitution resulting in constitutive kinase activation. Mutant BRAF thus represents an important therapeutic target in melanoma. In an effort to produce a pre-clinical model of mutant BRAF function in melanoma, we have generated a mouse expressing BRAF V600E targeted to melanocytes. We show that in these transgenic mice, widespread benign melanocytic hyperplasia with histological features of nevi occurs, with biochemical evidence of senescence. Melanocytic hyperplasia progresses to overt melanoma with an incidence dependent on BRAF expression levels. Melanomas show CDKN2A loss, and genetic disruption of the CDKN2A locus greatly enhances melanoma formation, consistent with collaboration between BRAF activation and CDKN2A loss suggested from studies of human melanoma. The development of melanoma also involves activation of the Mapk and Akt signaling pathways and loss of senescence, findings that faithfully recapitulate those seen in human melanomas. This murine model of mutant BRAF-induced melanoma formation thus provides an important tool for identifying further genetic alterations that cooperates with BRAF and that may be useful in enhancing susceptibility to BRAF-targeted therapeutics in melanoma.

Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.

Transplantation studies have demonstrated the existence of mammary progenitor cells with the ability to self-renew and regenerate a functional mammary gland. Although these progenitors are the likely targets for oncogenic transformation, correlating progenitor populations with certain oncogenic stimuli has been difficult. Cyclin D1 is required for lobuloalveolar development during pregnancy and lactation as well as MMTV-ErbB2- but not MMTV-Wnt1-mediated tumorigenesis. Using a kinase-deficient cyclin D1 mouse, we identified two functional mammary progenitor cell populations, one of which is the target of MMTV-ErbB2. Moreover, cyclin D1 activity is required for the self-renewal and differentiation of mammary progenitors because its abrogation leads to a failure to maintain the mammary epithelial regenerative potential and also results in defects in luminal lineage differentiation.