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Genetic alterations in human cancers and murine models indicate that retinoblastoma (Rb) and p53 have critical tumor suppressive functions in retinoblastoma, a tumor of neural origin, and neuroendocrine tumors including small cell lung cancer and medullary thyroid cancer (MTC). Rb inactivation is the initiating lesion in retinoblastoma and current models propose that induction of apoptosis is a key p53 tumor suppressive function. Genetic studies in mice, however, indicate that other undefined p53 tumor suppressive functions are operative in vivo. How p53 loss cooperates with Rb inactivation to promote carcinogenesis is also not fully understood. In the current study, genetically engineered mice were generated to determine the role of Rb and p53 in MTC pathogenesis and test the hypothesis that p53 suppresses carcinogenesis by inhibiting mammalian target of rapamycin (mTOR) signaling. Conditional Rb ablation resulted in thyroid tumors mimicking human MTC, and additional p53 loss led to rapid tumor progression. p53 suppressed tumorigenesis by inhibiting cell cycle progression, but did not induce apoptosis. On the contrary, p53 loss led to increased apoptosis that had to be overcome for tumor progression. The mTOR activity was markedly increased in p53-deficient tumors and rapamycin treatment suppressed tumor cell growth, identifying mTOR inhibition as a critical p53 tumor suppressive function. Rapamycin treatment did not result in AKT/mitogen-activated protein kinase activation, providing evidence that this feedback mechanism operative in other cancers is not a general response to mTORC1 inhibition. Together, these studies provide mechanistic links between genetic alterations and aberrant signaling pathways critical in carcinogenesis, and identify essential Rb and p53 tumor suppressive functions in vivo. [ABSTRACT FROM AUTHOR]

The processes that control the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain poorly understood. Epidermal growth factor receptor 2 (ErbB2) overexpression is common in DCIS, as is disruption of the retinoblastoma tumor suppressor (RB) pathway. Here, we examined the cooperative impact of ErbB2 and RB deregulation on facets of disease progression. Our studies demonstrate that RB deficiency altered the expression of key molecules needed for proper cellular organization and epithelial cell-cell adhesion as part of a program related to the epithelial-to-mesenchymal transition (EMT). An increase in the invasive potential of ErbB2-overexpressing cells was observed upon RB depletion. Further, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared with DCIS-like lesions developing from RB-proficient cells. Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner. Finally, in a cohort of DCIS cases, we show that, although elevated levels of ErbB2 are associated with increased risk of a subsequent DCIS recurrence, it is not associated with progression to invasive disease. In contrast, RB loss in ErbB2-positive DCIS cases was associated with increased risk for invasive breast cancer. Taken together, these data demonstrate a key role for the RB pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor protein is a major sensor of cellular stresses, and upon stabilization, activates or represses many genes that control cell fate decisions. While the mechanism of p53-mediated transactivation is well established, several mechanisms have been proposed for p53-mediated repression. Here, we demonstrate that the cyclin-dependent kinase inhibitor p21 is both necessary and sufficient for the downregulation of known p53-repression targets, including survivin, CDC25C, and CDC25B in response to p53 induction. These same targets are similarly repressed in response to p16 overexpression, implicating the involvement of the shared downstream retinoblastoma (RB)-E2F pathway. We further show that in response to either p53 or p21 induction, E2F4 complexes are specifically recruited onto the promoters of these p53-repression targets. Moreover, abrogation of E2F4 recruitment via the inactivation of RB pocket proteins, but not by RB loss of function alone, prevents the repression of these genes. Finally, our results indicate that E2F4 promoter occupancy is globally associated with p53-repression targets, but not with p53 activation targets, implicating E2F4 complexes as effectors of p21-dependent p53-mediated repression. [ABSTRACT FROM AUTHOR]

Whole chromosome instability (CIN) is a common feature of cancer cells and has been linked to increased tumor evolution and metastasis. Several studies have shown that the loss of the pRB tumor suppressor causes mitotic defects and chromosome mis-segregation. pRB is inactivated in many types of cancer and this raises the possibility that the loss of pRB may be a general cause of CIN in tumors. Paradoxically, retinoblastoma tumor cells have a relatively stable karyotype and currently the circumstances in which pRB inactivation causes CIN in human cancers are unclear. Here we utilize a fluorescence in situ hybridization-based approach to score numerical heterogeneity in chromosome copy number as a readout of CIN. Using this technique, we show that high levels of CIN correlate with the combined inactivation of pRB and p53 and that this association is evident in two independent panels of cancer cell lines. Retinoblastoma cell lines characteristically retain a wild-type TP53 gene, providing an opportunity to test the relevance of this functional relationship. We show that retinoblastoma cell lines display mitotic defects similar to those seen when pRB is depleted from non-transformed cells, but that the presence of wild-type p53 suppresses the accumulation of aneuploid cells. A similar synergy between pRB and p53 inactivation was observed in HCT116 cells. These results suggest that the loss of pRB promotes segregation errors, whereas loss of p53 allows tolerance and continued proliferation of the resulting, genomically unstable cancer cells. Hence, it is the cooperative effect of inactivation of both pRB and p53 tumor suppressor pathways that promotes CIN. [ABSTRACT FROM AUTHOR]

Src is the founding member of a diverse family of intracellular tyrosine kinases, and Src has a key role in promoting cancer growth, in part, through its association with receptor tyrosine kinases. However, some Src-related proteins have widely divergent physiological roles, and these proteins include the Rak/Frk tyrosine kinase (Frk stands for Fyn-related kinase), which inhibits cancer cell growth and suppresses tumorigenesis. Rak/Frk phosphorylates and stabilizes the Pten tumor suppressor, protecting it from degradation, and Rak/Frk associates with the retinoblastoma (Rb) tumor suppressor. However, the role of Rak/Frk in receptor-mediated signaling is largely unknown. Here, we demonstrate that Rak/Frk associates with epidermal growth factor receptor (EGFR), increasing in activity and EGFR binding after EGF stimulation, when it decreases the pool of EGFR present at the plasma membrane. EGFR-Rak binding is direct, requires the SH2 and SH3 domains of Rak/Frk for efficient complex formation and is not dependent on the Grb2 adaptor protein. EGFR mutations are associated with increased EGFR activity and tumorigenicity, and we found that Rak/Frk associates preferentially with an EGFR exon 19 mutant, EGFRΔ747-749/A750P, compared with wild-type EGFR. Furthermore, Rak/Frk inhibited mutant EGFR phosphorylation at an activating site and dramatically decreased the levels of EGFRΔ747-749/A750P from the plasma membrane. Taken together, the results suggest that Rak/Frk inhibits EGFR signaling in cancer cells and has elevated activity against EGFR exon 19 mutants. [ABSTRACT FROM AUTHOR]

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response. [ABSTRACT FROM AUTHOR]

RB+/− individuals develop retinoblastoma and, subsequently, many other tumors. The Rb relatives p107 and p130 protect the tumor-resistant Rb−/− mouse retina. Determining the mechanism underlying this tumor suppressor function may expose novel strategies to block Rb pathway cancers. p107/p130 are best known as E2f inhibitors, but here we implicate E2f-independent Cdk2 inhibition as the critical p107 tumor suppressor function in vivo. Like p107 loss, deleting p27 or inactivating its Cdk inhibitor (CKI) function (p27CK−) cooperated with Rb loss to induce retinoblastoma. Genetically, p107 behaved like a CKI because inactivating Rb and one allele each of p27 and p107 was tumorigenic. Although Rb loss induced canonical E2f targets, unexpectedly p107 loss did not further induce these genes, but instead caused post-transcriptional Skp2 induction and Cdk2 activation. Strikingly, Cdk2 activity correlated with tumor penetrance across all the retinoblastoma models. Therefore, Rb restrains E2f, but p107 inhibits cross talk to Cdk. While removing either E2f2 or E2f3 genes had little effect, removing only one E2f1 allele blocked tumorigenesis. More importantly, exposing retinoblastoma-prone fetuses to small molecule inhibitors of E2f (HLM006474) or Cdk (R547) for merely 1 week dramatically inhibited subsequent tumorigenesis in adult mice. Protection was achieved without disrupting normal proliferation. Thus, exquisite sensitivity of the cell-of-origin to E2f and Cdk activity can be exploited to prevent Rb pathway-induced cancer in vivo without perturbing normal cell division. These data suggest that E2f inhibitors, never before tested in vivo, or CKIs, largely disappointing as therapeutics, may be effective preventive agents. [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (RB)-E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G1/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB-E2F1 pathway. [ABSTRACT FROM AUTHOR]

Comparing normal colorectal mucosa and adenomas focusing on deregulated pathways obtains insight into the biological processes of early colorectal carcinogenesis. Publicly available microarray expression data from 26 normal mucosa and 47 adenoma samples were analyzed. Biological pathways enriched in adenomas were identified with Gene Set Enrichment Analysis (GSEA). The analysis revealed 10, 11 and 16 gene sets distinguishing adenomas from normal mucosa according to Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Map Annotator and Pathway Profiler (GenMAPP) and Biocarta databases, respectively. Biological pathways known to be involved in colon carcinogenesis such as cell cycle (P=0.002) and Wnt signaling (P=0.007) were enriched in adenomas. In addition, we found enrichment of novel pathways such as retinoblastoma (Rb) pathway (P=0.002), Src pathway (P=0.004), folate biosynthesis (P=0.019) and Hedgehog signaling (P=0.037) in adenomas. Microarray results for Rb and Src pathway genes were validated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on mRNA isolated from an independent set of adenoma and normal colon samples. A high correlation between microarray data and qRT-PCR expression data was found. The relevance of targeting of identified pathways was shown using the Rb pathway inhibitors roscovitine and PD-0332991 and the Src pathway inhibitor dasatinib. All inhibitors used induced cell growth reduction in adenoma cells. This study shows a bioinformatical and functional approach leading to potentially new options for chemoprevention of colorectal cancer. [ABSTRACT FROM AUTHOR]

Serous borderline ovarian tumors (SBOTs) are slow-growing, non-invasive ovarian epithelial neoplasms. SBOTs are considered to be distinct entities that give rise to invasive low-grade serous carcinomas (LGCs), which have a relatively poor prognosis and are unrelated to high-grade serous carcinomas (HGCs). The mechanisms underlying the progression of non-invasive SBOTs to invasive epithelial ovarian carcinomas are not understood. We recently established short-term cultures of SBOT cells from tumor biopsies and showed that inactivation of p53, retinoblastoma (Rb) and/or PP2A by the simian virus 40 (SV40) large (LT) and small T antigens extends the life span of the cells and endows them with the ability to invade Matrigel-coated transwells. In this study, we show that concurrent inhibition of p53 and Rb by the SV40 LT produces cells (referred to as SBOT4-LT) with increased life span and cell invasion. To distinguish the roles of p53 and Rb in the progression from SBOTs to invasive ovarian carcinomas, we performed small interfering RNA-mediated knockdown of endogenous p53 in a spontaneously immortalized SBOT cell line, SBOT3.1, which increased cell invasion. This increased invasive activity was associated with the transcriptional downregulation of E-cadherin, correlated with an increase in PIK3CA levels and the increased activation of Akt. Conversely, in invasive LGC-derived MPSC1 cells, enhancing the levels of p53 decreased cell invasion and diminished the phosphatidylinositol 3-kinase (PI3K)/Akt-mediated downregulation of E-cadherin. Inhibition of Rb also enhanced invasiveness, but did not affect the levels of PIK3CA and E-cadherin in SBOT3.1 cells, suggesting that it functions by a different pathway. To our knowledge, this study is the first to show that p53 has an important role in the progression from SBOTs to invasive carcinomas. In addition, our findings suggest that downregulation of E-cadherin by the PI3K/Akt pathway contributes to this progression. [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (Rb) inhibits both cell division and apoptosis, but the mechanism by which Rb alternatively regulates these divergent outcomes remains poorly understood. Cyclin-dependent kinases (Cdks) promote cell division by phosphorylating and reversibly inactivating Rb by a hierarchical series of phosphorylation events and sequential conformational changes. The stress-regulated mitogen-activated protein kinase p38 also phosphorylates Rb, but it does so in a cell cycle-independent manner that is associated with apoptosis rather than with cell division. Here, we show that p38 phosphorylates Rb by a novel mechanism that is distinct from that of Cdks. p38 bypasses the cell cycle-associated hierarchical phosphorylation and directly phosphorylates Rb on Ser567, which is not phosphorylated during the normal cell cycle. Phosphorylation by p38, but not Cdks, triggers an interaction between Rb and the human homolog of murine double minute 2 (Hdm2), leading to degradation of Rb, release of E2F1 and cell death. These findings provide a mechanistic explanation as to how Rb regulates cell division and apoptosis through different kinases, and reveal how Hdm2 may functionally link the tumor suppressors Rb and p53. [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (pRb) and the pRb-related proteins, p130 and p107, form the 'pocket protein' family of cell cycle regulatory factors. A well characterized function of these proteins is the cell cycle-dependent regulation of E2F-responsive genes. The biological activity of pocket proteins is regulated by phosphorylation and for the founding member pRb it has been shown that acetylation also has an important role in modulating its function during the cell cycle. Here, we show that hyperphosphorylated retinoblastoma 2 (Rb2)/p130 also exists in an acetylated form in NIH3T3 cells. Acetylated p130 is present in the nucleus but not in the cytoplasm. Acetylation is cell cycle dependent, starting in S-phase and persisting until late G2-period. Using recombinant p130 and truncated forms for in vitro acetylation by the acetyltransferase p300, we could identify K1079 in the C-terminal part as the major acetylation site by mass spectrometry. Minor acetylation sites were pinpointed to K1068 and K1111 in the C-terminus, and K128 and K130 in the N-terminus. The human papilloma virus 16 protein-E7 preferentially binds to acetylated p130 and significantly increases in vitro p130 acetylation by p300. [ABSTRACT FROM AUTHOR]

One of the characteristics of gliomas is a decrease in the expression of connexin43, a protein that forms gap junctions. Restoring connexin43 expression in glioma cells reduces their exacerbated rate of cell growth, although it is not yet known how connexin43 modifies the expression of genes involved in cell proliferation. Here, we show that restoring connexin43 to C6 glioma cells impedes their progression from G0/G1 to the S phase of the cell cycle by reducing retinoblastoma phosphorylation and cyclin E expression through the upregulation of p21 and p27. Interestingly, connexin43 diminishes the oncogenic activity of c-Src exhibited by glioma cells. By studying a Tyr247 and Tyr265 mutant connexin43, we show that these residues are required for connexin43 to inhibit c-Src activity and cell proliferation. In conclusion, by acting as a substrate of c-Src, connexin43 reduces its oncogenic activity and decreases the rate of glioma cell proliferation, potentially an early step in the antiproliferative effects of connexin43. Although c-Src is known to phosphorylate connexin43, this study provides the first evidence that connexin43 can also inhibit c-Src activity. [ABSTRACT FROM AUTHOR]

Normal tissue cells survive and proliferate only while anchored to solid substrate. Conversely, transformed cells both survive and proliferate following detachment, having lost attachment context through unclear mechanisms. p66Shc is a focal adhesion-associated protein that reports cell attachment through a RhoA-dependent mechanosensory test. We find that human small cell lung cancer (SCLC) cells and mouse Lewis lung carcinoma (LLC), which display aggressive metastatic behavior, lack both p66Shc and retinoblastoma (pRB) and bypass anoikis. Re-expression of p66Shc in these cells restores anoikis and provides striking protection from metastasis by LLC cells in vivo. Notably, knockdown of p66Shc in normal epithelial cells leads to unrestrained Ras activation, preventing anoikis through downstream suppression of RhoA but blocking proliferation in a pRB-dependent manner, thus mimicking oncogenic Ras. Conversely, LLC and SCLC cells display constitutive Ras activation necessary to bypass anoikis, which is reversed by re-expression of p66Shc. p66Shc therefore coordinates Ras-dependent control of proliferation and anchorage sensation, which can be defeated in the evolution of highly metastatic tumors by combined loss of both p66Shc and pRB. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are produced in growth factor-signaling pathways leading to cell proliferation, but the mechanisms leading to ROS generation and the targets of ROS signals are not well understood. Using a focused siRNA screen to identify redox-related proteins required for growth factor-induced cell cycle entry, we show that two ROS-generating proteins, the NADPH oxidases NOX4 and DUOX2, are required for platelet-derived growth factor (PDGF) induced retinoblastoma protein (Rb) phosphorylation in normal human fibroblasts. Unexpectedly, NOX4 and DUOX2 knockdown did not inhibit the early signaling pathways leading to cyclin D1 upregulation. However, hours after growth factor stimulation, NOX4 and DUOX2 knockdown reduced ERK1 phosphorylation and increased levels of the tumor suppressor protein p53 and a cell cycle inhibitor protein p21 (Waf1/Cip1) that is transcriptionally regulated by p53. Co-knockdown of NOX4 or DUOX2 with either p53 or with p21 overcame the inhibition of Rb phosphorylation that occurred with NOX4 or DUOX2 knockdown alone. Our results argue that rather than primarily affecting growth factor receptor signaling, NOX4 and DUOX2 regulate cell cycle entry as part of a p53-dependent checkpoint for proliferation. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid is a potent promoter of cellular differentiation processes, which is used in cancer therapy. Glioblastoma spheroid cultures are enriched in tumor-initiating cells, and provide a model to test new treatment options in vitro. We investigated the molecular mechanisms of response to exposure to differentiation-promoting conditions in such cultures. Microarray analyses of five independent cultures showed that after induction of differentiation, inhibitors of transforming growth factor-β/bone morphogenetic protein, Wnt/β-catenin and IGF signaling were upregulated, whereas expression of several microRNAs decreased, particularly that of the miR-17-92 cluster. In primary astrocytic gliomas (n=82), expression of several members of miR-17-92 was significantly higher relative to those of normal brain (n=8) and significantly increased with tumor grade progression (P<0.05). A high-level amplification of the miR-17-92 locus was detected in one glioblastoma specimen. Transfection of inhibitors of miR-17-92 induced increased apoptosis and decreased cell proliferation in glioblastoma spheroids. Mir-17-92 inhibition was also associated with increased messenger RNA (mRNA) and/or protein expression of CDKN1A, E2F1, PTEN and CTGF. The CTGF gene was shown to be a target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays. Our results suggest that miR-17-92 and its target CTGF mediate effects of differentiation-promoting treatment on glioblastoma cells through multiple regulatory pathways. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a significant worldwide health concern that is associated with discrete etiological events, encompassing viral infection, metabolic stress and genotoxic compounds. In particular, exposure to the genotoxic hepatocarcinogen aflatoxin B1 (AFB1) is a significant factor in the genesis of human liver cancer. Presumably, genetic events associated with HCC could influence the effect of environmental insults, yielding a predilection for tumor development. The retinoblastoma (RB) tumor suppressor pathway is functionally inactivated in HCC through discrete mechanisms; however, the role of RB in suppressing tumorigenesis in this disease is poorly understood. Therefore, we analysed how RB status affects the response to AFB1 in reference to acute exposures and tumor development reflective of chronic exposure. Liver-specific Rb deletion resulted in an aberrant proliferative response to AFB1. This cell-cycle induction was associated with increased levels of secondary genetic damage and failure in appropriate cell-cycle coupling. This effect of RB loss was unique to AFB1 and involved the induction of a non-canonical proliferative pathway, and was not merely reflective of the overall cell-cycle deregulation or aberrant regenerative responses. The acute responses to AFB1 exposure presaged aberrations in hepatocyte nuclear morphology and ploidy with RB loss. Correspondingly, RB-deficient livers showed significantly enhanced susceptibility to liver tumorigenesis initiated by AFB1. Combined, these studies show that RB has a critical role in mediating checkpoint responses in liver tissue to maintain genome integrity and in suppressing tumorigenesis. [ABSTRACT FROM AUTHOR]

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/β-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/β-catenin pathway as revealed by nuclear β-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another β-catenin target gene, revealing a complex, Wnt/β-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/β-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/β-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.Oncogene (2009) 28, 2245–2256; doi:10.1038/onc.2009.80; published online 4 May 2009 [ABSTRACT FROM AUTHOR]

Mutations of the retinoblastoma tumor suppressor gene RB are frequently observed in human cancers, but rarely in non-small cell lung carcinomas (NSCLCs). Emerging evidence also suggests that the RB-related gene p130 is inactivated in a subset of human NSCLCs. To directly test the specific tumor suppressor roles of RB and p130 in NSCLC, we crossed Rb and p130 conditional mutant mice to mice carrying a conditional oncogenic K-Ras allele. In this model, controlled oncogenic K-Ras activation leads to the development of adenocarcinoma, a major subtype of NSCLC. We found that loss of p130 accelerated the death of mice, providing direct evidence in vivo that p130 is a tumor suppressor gene, albeit a weak one in this context. Loss of Rb increased the efficiency of lung cancer initiation and resulted in the development of high-grade adenocarcinomas and rapid death. Thus, despite the low frequency of RB mutations in human NSCLCs and reports that K-Ras activation and loss of RB function are rarely found in the same human tumors, loss of Rb clearly cooperates with activation of oncogenic K-Ras in lung adenocarcinoma development in mice.Oncogene (2009) 28, 1393–1399; doi:10.1038/onc.2008.491; published online 19 January 2009 [ABSTRACT FROM AUTHOR]

The retinoblastoma protein pRB suppresses tumorigenesis largely through regulation of the E2F transcription factors. E2F4, the most abundant E2F protein, is thought to act in cooperation with pRB to restrain cell proliferation. In this study, we analyse how loss of E2f4 affects the tumorigenicity of pRB-deficient tissues. As Rb−/−;E2f4−/− germline mice die in utero, we generated Rb−/−;E2f4−/− chimeric animals to allow examination of adult tumor phenotypes. We found that loss of E2f4 had a differential effect on known Rb-associated neuroendocrine tumors. It did not affect thyroid and adrenal glands tumors but partially suppressed lung neuroendocrine hyperplasia. The most striking effect was in the pituitary where E2F4 loss delayed the development, and reduced the incidence, of Rb mutant tumors. This tumor suppression increased the longevity of the Rb−/−;E2f4−/− chimeric animals allowing us to identify novel tumor types. We observed ganglionic neuroendocrine neoplasms, lesions not associated earlier with mutation of either Rb or E2f4. Moreover, a subset of the Rb−/−;E2f4−/− chimeras developed either low- or high-grade carcinomas in the urothelium transitional epithelium supporting a key role for Rb in bladder cancer.Oncogene (2009) 28, 500–508; doi:10.1038/onc.2008.406; published online 10 November 2008 [ABSTRACT FROM AUTHOR]

Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide ‘degron’ from hypoxia-inducible factor-1α, which binds to the Von –Hippel–Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-α (ERα; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERα in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERα-dependent breast cancer cells by inducing G1 arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERα expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G1 arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERα and AR, respectively.Oncogene (2008) 27, 7201–7211; doi:10.1038/onc.2008.320; published online 15 September 2008 [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDKs) are important in regulating cell cycle transitions, particularly in coordinating DNA replication. Although the role of CDK2 activity on the replication apparatus has been extensively studied, the role of CDK4/6 in DNA replication control is less understood. Through targeted inhibition of CDK4/6 activity, we demonstrate that CDK4/6 kinase activity promotes cdc6 and cdt1 expression, and pre-replication complex (pre-RC) assembly in cycling cells. Conversely, CDK2 inhibition had no effect on the pre-RC assembly. The inhibition of pre-RC assembly is dependent on a functional retinoblastoma (RB) protein, which mediates downstream effects. As such, CDK4/6 inhibition has minimal effect on the replication apparatus in the absence of RB. The requirement of CDK4/6 was further interrogated using cells lacking D-type cyclins, in which replication complexes form normally, and correspondingly CDK4/6 inhibition had no effect on cell cycle or replication control. However, in the absence of D-type cyclins, CDK2 inhibition resulted in the attenuation of cdc6 and cdt1 levels, suggesting overlapping roles for CDK4/6 and CDK2 in regulating replication protein activity. Finally, CDK4/6 inhibition prevented the accumulation of cdc6 and cdt1 as cells progressed from mitosis through the subsequent G1. Combined, these studies indicate that CDK4/6 activity is important in regulating the expression of these critical mediators of DNA replication.Oncogene (2008) 27, 7083–7093; doi:10.1038/onc.2008.319; published online 8 September 2008 [ABSTRACT FROM AUTHOR]

The E2f transcription factors are key downstream targets of the retinoblastoma protein tumor suppressor that control cell proliferation. E2F3 has garnered particular attention because it is amplified in various human tumors. E2f3 mutant mice typically die around birth and E2f3-deficient cells have a proliferation defect that correlates with impaired E2f target gene activation and also induction of p19Arf and p53. The E2f3 locus encodes two isoforms, E2f3a and E2f3b, which differ in their N-termini. However, it is unclear how E2f3a versus E2f3b contributes to E2f3's requirement in either proliferation or development. To address this, we use E2f3a- and E2f3b-specific knockouts. We show that inactivation of E2f3a results in a low penetrance proliferation defect in vitro whereas loss of E2f3b has no effect. This proliferation defect appears insufficient to disrupt normal development as E2f3a and E2f3b mutant mice are both fully viable and have no detectable defects. However, when combined with E2f1 mutation, inactivation of E2f3a, but not E2f3b, causes significant proliferation defects in vitro, neonatal lethality and also a striking cartilage defect. Thus, we conclude that E2f3a and E2f3b have largely overlapping functions in vivo and that E2f3a can fully substitute for E2f1 and E2f3 in most murine tissues.Oncogene (2008) 27, 6561–6570; doi:10.1038/onc.2008.253; published online 28 July 2008 [ABSTRACT FROM AUTHOR]

E2Fs are key regulators of cell-cycle progression, and their transcriptional activities are regulated by histone acetyltransferases (HATs). Retinoblastoma (Rb) family proteins (pRb, p107 and p130) bind to E2Fs and inhibit their transcriptional activities by disrupting HAT binding and recruitment of histone deacetylases. In this study, we show that IκB kinases (IKKα or IKKβ) activation inhibits cell growth and E2F-dependent transcription in normal human fibroblasts. The inhibition of E2F by IKKs was not observed in cells lacking nuclear factor (NF)-κB/p65; however, it was observed in cells lacking three Rb family genes. p65 disrupted the physical interaction between activator E2Fs (F2F1, E2F2 and E2F3) and the HAT cofactor transactivation/transformation-domain associated protein, resulting in a reduction in E2F-responsive gene expression. Furthermore, IKKα and IKKβ directly phosphorylated E2F4, resulting in nuclear accumulation and enhanced DNA binding of the E2F4/p130 repressor complex. Our study describes a novel growth inhibitory system that functions by Rb-independent suppression of E2Fs by the IKK/NF-κB signaling pathway.Oncogene (2008) 27, 5696–5705; doi:10.1038/onc.2008.184; published online 9 June 2008 [ABSTRACT FROM AUTHOR]

Translocations of the retinoic acid receptor-α (RARα) locus with the promyelocytic leukemia zinc-finger (PLZF) or PML genes lead to expression of oncogenic PLZF–RARα or PML–RARα fusion proteins, respectively. These fusion oncoproteins constitutively repress RARα target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML–RARα have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here, we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (poxvirus and zinc-finger, POZ) and DNA-binding (zinc-finger) domains. In addition, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction of Rb with PML or E2F1, the PLZF–Rb interaction is not dependent on hypophosphorylation of Rb. These data are supported by chromatin immunoprecipitation analysis, which indicates that PLZF associates with the promoter region of CDC6, a known E2F/Rb target gene. Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F/Rb target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to function in stem cells and taken together these data suggest that interactions between PLZF and Rb could be important in stem cell biology.Oncogene (2008) 27, 5260–5266; doi:10.1038/onc.2008.159; published online 26 May 2008 [ABSTRACT FROM AUTHOR]

The retinoblastoma pathway is often inactivated in human tumors resulting in deregulated E2F activity that can induce both proliferation and cell death. Although the role of E2F in apoptosis is well characterized, little is known regarding its putative participation in other cell death pathways. We show here that activation of E2F1 upregulates the expression of four autophagy genes—microtubule-associated protein-1 light chain-3 (LC3), autophagy-related gene-1 (ATG1), ATG5 and damage-regulated autophagy modulator (DRAM). E2F1-mediated induction of LC3, ATG1 and DRAM is direct and indeed, endogenous E2F1 can be found bound to regions encompassing the promoters of these genes. Regulation of ATG5 by E2F1 is indirect. Importantly, we demonstrate that E2F1 activation enhances autophagy and conversely, reducing endogenous E2F1 expression inhibits DNA damage-induced autophagy. These studies identify E2F1 as a transcriptional regulator of autophagy, and for the first time establish a role for E2F1 in DNA damage-induced autophagy.Oncogene (2008) 27, 4860–4864; doi:10.1038/onc.2008.117; published online 14 April 2008 [ABSTRACT FROM AUTHOR]

MDM2 is a key ubiquitin E3 ligase for p53 and its activity is critically regulated by a set of modulators, including ARF, p300, YY1 and recently by gankyrin, an oncoprotein frequently overexpressed in human heptocellular carcinomas. We have previously shown that MDM2 binds to and promotes retinoblastoma protein (Rb) degradation. Here we show that Rb inhibits MDM2 E3 ligase activity resulting in stabilization of p53. In addition, we demonstrated that Rb inhibits MDM2-mediated p53 ubiquitination in a gankyrin-dependent manner and the Rb–gankyrin interaction is critical for Rb-induced p53 stabilization. Furthermore, acute ablation of Rb facilitates gankyrin-mediated p53 destabilization, and desensitizes cancer cells for chemotherapy-induced apoptosis. These results indicate that Rb antagonizes gankyrin to inhibit MDM2-mediate p53 ubiquitination in cancer cells and suggest that the status of both p53 and Rb is important for efficacy of cancer chemotherapy.Oncogene (2008) 27, 4034–4043; doi:10.1038/onc.2008.43; published online 10 March 2008 [ABSTRACT FROM AUTHOR]

Inactivation of the retinoblastoma (RB) tumor suppressor pathway, via elevated cyclin-dependent kinase (CDK) activity, is observed in majority of human cancers. Since CDK deregulation is evident in most cancer cells, pharmacological CDK inhibition has become an attractive therapeutic strategy in oncology. We recently showed that an oncogenic CDK4R24C mutation alters the subcellular localization of the normally nuclear RB phosphoprotein. Here, using 71 human cancer cell lines and over 300 primary human cancer tissues, we investigated whether changes in RB subcellular localization occur during human cancer progression. We uncover that diverse human cancers and their derived cell lines, particularly those with poor tumor differentiation, display significant cytoplasmic mislocalization of ordinarily nuclear RB. The nucleocytoplasmically distributed RB was derived via CDK-dependent and Exportin1-mediated nuclear export. Indeed, cytoplasmically mislocalized RB could be efficiently confined to the nucleus by pharmacologically reducing CDK activity or by inhibiting the Exportin1-mediated nuclear export pathway. Our observations uncover a post-translational CDK-dependent mechanism of RB inactivation and suggest that cytoplasmically localized RB may harbor a tumor promoting function. We propose that RB inactivation, via aberrant nucleocytoplasmic transport, may disrupt normal cell differentiation programs and accelerate the cancer process. These results are evidence that tumor cells modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.Oncogene (2008) 27, 3156–3164; doi:10.1038/sj.onc.1210970; published online 10 December 2007 [ABSTRACT FROM AUTHOR]

p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative ‘brake’ to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21Cip1 in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53V143A) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21Cip1 was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21Cip1 expression.Oncogene (2008) 27, 2763–2771; doi:10.1038/sj.onc.1210937; published online 19 November 2007 [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (pRB) has the dual capability to negatively regulate both E2F-induced cell cycle entry and E2F1-induced apoptosis. In this report, we characterize a unique pRB–E2F1 interaction. Using mutagenesis to disrupt E2F1 binding, we find that the ability of pRB to regulate E2F1-induced apoptosis is diminished when this interaction is lost. Strikingly, this mutant form of pRB retains the ability to control E2F responsive cell cycle genes and blocks cell proliferation. These functional properties are the reciprocal of a previously described E2F binding mutant of pRB that interacts with E2F1, but lacks the ability to interact with other E2Fs. Our work shows that these distinct interactions allow pRB to separately regulate E2F-induced cell proliferation and apoptosis. This suggests a novel form of regulation whereby separate types of binding contacts between the same types of molecules can confer distinct functional outcomes.Oncogene (2008) 27, 1572–1579; doi:10.1038/sj.onc.1210803; published online 24 September 2007 [ABSTRACT FROM AUTHOR]

In the absence of mitogenic stimuli, cells normally arrest in G1/0, because they fail to pass the G1-restriction point. However, abrogation of the G1-restriction point (by loss of the retinoblastoma gene family) reveals a second-restriction point that arrests cells in G2. Serum-starvation-induced G2 arrest is effectuated through inhibitory interactions of p27KIP1 and p21CIP1 with cyclins A and B1 and can be reversed through mitogen re-addition. In this study, we have investigated the pathways that allow cell cycle re-entry from this G2 arrest. We provide evidence that recovery from G2 arrest depends on the rat sarcoma viral oncogene (RAS) and phosphatidylinositol-3 kinase pathways and show that oncogenic hits, such as overexpression of c-MYC or mutational activation of RAS can abrogate the G2-restriction point. Together, our results provide new mechanistic insight into multistep carcinogenesis.Oncogene (2008) 27, 1142–1154; doi:10.1038/sj.onc.1210724; published online 13 August 2007 [ABSTRACT FROM AUTHOR]

Mammalian SWI/SNF-related complexes have been implicated in cancer based on some of the subunits physically interacting with retinoblastoma (RB) and other proteins involved in carcinogenesis. Additionally, several subunits are mutated or not expressed in tumor-derived cell lines. Strong evidence for a role in tumorigenesis in vivo, however, has been limited to SNF5 mutations that result primarily in malignant rhabdoid tumors (MRTs) in humans and MRTs as well as other sarcomas in mice. We previously generated a null mutation of the Brg1 catalytic subunit in the mouse and reported that homozygotes die during embryogenesis. Here, we demonstrate that Brg1 heterozygotes are susceptible to mammary tumors that are fundamentally different than Snf5 tumors. First, mammary tumors are carcinomas not sarcomas. Second, Brg1+/− tumors arise because of haploinsufficiency rather than loss of heterozygosity. Third, Brg1+/− tumors exhibit genomic instability but not polyploidy based on array comparative genomic hybridization results. We monitored Brg1+/−, Brm−/− double-mutant mice but did not observe any tumors resembling those from Snf5 mutants, indicating that the Brg1+/− and Snf5+/− tumor phenotypes do not differ simply because Brg1 has a closely related paralog whereas Snf5 does not. These findings demonstrate that BRG1 and SNF5 are not functionally equivalent but protect against cancer in different ways. We also demonstrate that Brg1+/− mammary tumors have relatively heterogeneous gene expression profiles with similarities and differences compared to other mouse models of breast cancer. The Brg1+/− expression profiles are not particularly similar to mammary tumors from Wap-T121 transgenic line where RB is perturbed. We were also unable to detect a genetic interaction between the Brg1+/− and Rb+/− tumor phenotypes. These latter findings do not support a BRG1–RB interaction in vivo.Oncogene (2008) 27, 460–468; doi:10.1038/sj.onc.1210664; published online 16 July 2007 [ABSTRACT FROM AUTHOR]

C-terminal-binding protein interacting protein (CtIP) was first isolated as a binding partner of C-terminal-binding protein (CtBP). It is considered to contribute to the transcriptional repression and cell cycle regulatory properties of the retinoblastoma (Rb) family of proteins and to have a role in the cellular response to DNA damage. Here, we have shown that CtIP is a novel target for the adenovirus oncoprotein early region 1A (AdE1A). AdE1A associates with CtIP in both Ad5E1-transformed cells and Ad5-infected cells and binds directly in glutathione-S-transferase pull-down assays. Two binding sites have been mapped on Ad5E1A – the N-terminal α-helical region (residues 1–30) and conserved region 3 (CR3) – the transcriptional activation domain. CtIP can bind AdE1A and CtBP independently, raising the possibility that ternary complexes exist in Ad-transformed and -infected cells. Significantly, reduction of CtIP expression with small interfering RNAs results in reduction of the ability of a Gal4 DNA-binding domain-CR3 construct to transactivate a Gal 4-responsive luciferase reporter and this effect is reversed by reduction of CtBP expression. Therefore, in this model, CtIP acts as a transcriptional co-activator of AdE1A when dissociated from CtBP, through the action of AdE1A. These data are consistent with observations that CtIP expression is induced by AdE1A during viral infection and that reduction of CtIP expression with RNA interference can retard virus replication. In addition, AdE1A causes disruption of the CtIP/Rb complex during viral infection by its interaction with CtIP, possibly contributing to transcriptional derepression.Oncogene (2007) 26, 7467–7479; doi:10.1038/sj.onc.1210551; published online 4 June 2007 [ABSTRACT FROM AUTHOR]

The 14-3-3σ, upregulated by p53 in response to DNA damage, can have a positive-feedback impact driving p53 activities and is a human cancer epithelial marker downregulated in various tumors. However, the precise roles of 14-3-3σ during tumorigenesis are not well characterized. Here, we show that 14-3-3σ is a critical regulator of murine double minute oncogene (MDM2). 14-3-3σ interacts with MDM2 at the RING domain. The C-terminal region of 14-3-3σ binds to MDM2 very efficiently. Importantly, 14-3-3σ overexpression leads to destabilization of MDM2 through enhancing MDM2 self-ubiquitination and accelerating turnover rate. Conversely, loss of 14-3-3σ results in a significant increase in MDM2 protein. Moreover, live-cell images indicated that 14-3-3σ can affect the location of MDM2 from the nucleus to the cytoplasm, and that MDM2-mediated cytoplasmic localization of p53 can be reversed by the presence of 14-3-3σ. Significantly, we further showed that 14-3-3σ causes MDM2 downregulation, thereby stabilizing p53 and inhibiting tumor growth in animal tumors. Also, 14-3-3σ blocks MDM2-mediated retinoblastoma degradation and p53 NEDDylation. Our results provide evidence that 14-3-3σ is a pivotal MDM2 regulator involved in blocking a variety of activities of MDM2.Oncogene (2007) 26, 7355–7362; doi:10.1038/sj.onc.1210540; published online 4 June 2007 [ABSTRACT FROM AUTHOR]

The INK4A locus encodes two tumor suppressor genes, p16INK4A and p14ARF, transcribed using alternative exons 1α or 1β spliced onto the same exons 2 and 3. Both p16INK4A and p14ARF are capable of inhibiting the cell-cycle progression, albeit in different manner; p16INK4A is phosphorylation of retinoblastoma (pRB) dependent while p14ARF is p53-dependent. In this study, we report the discovery of a novel variant of p16INK4A, termed p16γ, in a primary T-cell acute lymphoblastic leukemia (T-ALL) patient sample and a neuroblastoma cell line, which was expressed at both the transcriptional and translational levels. Cloning and sequencing of the p16γ cDNA revealed that p16γ was identical to p16INK4A, except that it contained an in-frame insertion of 197 bp between exons 2 and 3. p16γ expression was detected in the majority of p16INK4A-expressing primary T-ALL and B-ALL patient samples and other p16INK4A-expressing tumor samples, but was only barely detectable in some normal mononuclear cells and other non-tumor samples. Structural analysis by nuclear magnetic resonance and circular dichroism confirmed that p16γ, like p16INK4A, is also an ankyrin-repeat protein. Functional analysis of p16γ revealed that p16γ protein interacted with cyclin D-dependent kinase4 and inhibited its kinase activity. Using a luciferase reporter assay, the transfection of p16γ repressed the E2F response, the downstream target of pRB, with an efficacy equivalent to that of p16INK4A. Moreover p16γ, like p16INK4A, induced cell-cycle arrest at G0/G1, and inhibited cell growth in colony formation assay.Oncogene (2007) 26, 7017–7027; doi:10.1038/sj.onc.1210507; published online 7 May 2007 [ABSTRACT FROM AUTHOR]

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.Oncogene (2007) 26, 6954–6958. doi:10.1038/sj.onc.1210516; published online 7 May 2007 [ABSTRACT FROM AUTHOR]

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of cis-elements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB. Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.Oncogene (2007) 26, 6307–6318; doi:10.1038/sj.onc.1210450; published online 23 April 2007 [ABSTRACT FROM AUTHOR]

The tumour suppressor genes, TP53 and RB1, and four genes involved in their regulation, INK4a, ARF, MDM2 and MDMX, were analysed in a series of 36 post-radiotherapy radiation-induced sarcomas. One-third of the tumours developed in patients carrying a germline mutation of RB1 that predisposed them to retinoblastoma and radiation-induced sarcomas. The genetic inactivation of RB1 and/or TP53 genes was frequently observed in these sarcomas. These inactivations were owing to an interplay between point mutations and losses of large chromosome segments. Radiation-induced somatic mutations were observed in TP53, but not in RB1 or in the four other genes, indicating an early role of TP53 in the radio-sarcomagenesis. RB1 and TP53 genes were biallelically coinactivated in all sarcomas developing in the context of the predisposition, indicating that both genes played a major role in the formation of these sarcomas. In the absence of predisposition, TP53 was biallelically inactivated in one-third of the sarcomas, whereas at least one allele of RB1 was wild type. In both genetic contexts, the TP53 pathway was inactivated by genetic lesions and not by the activation of the ARF/MDM2/MDMX pathway, as recently shown in retinoblastomas. Together, these findings highlight the intricate tissue- and aetiology-specific relationships between TP53 and RB1 pathways in tumorigenesis.Oncogene (2007) 26, 6106–6112; doi:10.1038/sj.onc.1210404; published online 19 March 2007 [ABSTRACT FROM AUTHOR]

Toxic compounds such as carcinogens are removed from the body by the action of a series of detoxifying enzymes and transporters expressed in the liver and the small intestine. We have found that intestinal epithelial cells expressing the SV40 large T antigen (TAg) contain significantly lower levels of mRNAs, encoding several drug metabolizing/detoxifying enzymes and transporters compared to their non-transgenic littermates. In addition, TAg blocks the induction of these mRNAs by xenobiotics. The repression depends on an intact LXCXE motif in TAg, suggesting that inactivation of the retinoblastoma (Rb) family of tumor suppressors plays a role in the process. These results imply that a functional Rb pathway in the intestine is necessary for the expression of the detoxification system used to clear carcinogens, and suggest that loss of this tumor suppressor might alter susceptibility to chemical injury. In addition, the effect of TAg on the detoxification pathway appears to be tissue-specific, as its ectopic expression in the liver failed to suppress the P450 enzymes. The TAg-mediated suppression of drug metabolizing/detoxifying enzymes may have broad implications in the metabolism and mechanism of action of both carcinogens and prescription drugs.Oncogene (2007) 26, 5124–5131; doi:10.1038/sj.onc.1210310; published online 5 March 2007 [ABSTRACT FROM AUTHOR]

The INK4a/ARF locus, encoding two tumor suppressor proteins, p16INK4a and p14ARF (ARF), plays key roles in many cellular processes including cell proliferation, apoptosis, cellular senescence and differentiation. Inactivation of INK4a/ARF is one of the most frequent events during human cancer development. Although p16INK4a is a critical component in retinoblastoma protein (Rb)-mediated growth regulatory pathway, p14ARF plays a pivotal role in the activation of p53 upon oncogenic stress signals. A body of evidence indicates that ARF also possesses growth suppression functions independent of p53, the mechanism of which is not well understood. We have recently shown that MDM2 interacts with Rb and promotes proteasome-dependent Rb degradation. In this study, we show that ARF disrupts MDM2–Rb interaction resulting in Rb accumulation. Wild-type ARF, but not ARF mutant defective in MDM2 interaction, stabilizes Rb and inhibits colony foci formation independent of p53. In addition, ablation of Rb impairs ARF function in growth suppression. Thus, this study demonstrates that ARF plays a direct role in regulation of Rb and suggests that inactivation of ARF may lead to defects in both p53 and Rb pathways in human cancer development.Oncogene (2007) 26, 4627–4634; doi:10.1038/sj.onc.1210254; published online 5 February 2007 [ABSTRACT FROM AUTHOR]

To identify possible genetic interactions between the mechanisms of tumor suppression of menin and pRb, we intercrossed mice with targeted deletions of Men1 and Rb1, and compared tumor development in cohorts of animals carrying single or dual mutations of these tumor-suppressor genes. In mice lacking one copy of Men1, pancreatic islet and anterior pituitary adenomas are common. In animals lacking one copy of Rb1, intermediate pituitary and thyroid tumors occur at high frequency, with less frequent development of pancreatic islet hyperplasia and parathyroid lesions. In mice heterozygous for both Men1 and Rb1, pancreatic hyperplasia and tumors of the intermediate pituitary and thyroid occurred at high frequency. Serum measurements of calcium and glucose did not vary significantly between genotypic groups. Loss of heterozygosity at the Rb1 locus was common in pituitary and thyroid tumors, whereas loss of menin was observed in pancreatic and parathyroid lesions. The tumor spectrum in the double heterozygotes was a combination of pathologies seen in each of the individual heterozygotes, without decrease in age of onset, indicating independent, non-additive effects of the two mutations. Together with the lack of increased tumor spectrum, this suggests that menin and pRb function in a common pathway of tumor suppression.Oncogene (2007) 26, 4009–4017. doi:10.1038/sj.onc.1210163; published online 18 December 2006 [ABSTRACT FROM AUTHOR]

The retinoblastoma protein RB regulates cell proliferation, differentiation and apoptosis. Homozygous knockout of Rb in mice causes embryonic lethality owing to placental defects that result in excessive apoptosis. RB binds to a number of cellular proteins including the nuclear Abl protein and inhibits its tyrosine kinase activity. Ex vivo experiments have shown that genotoxic or inflammatory stress can activate Abl kinase to stimulate apoptosis. Employing the Rb-null embryos as an in vivo model of apoptosis, we have shown that the genetic ablation of Abl can reduce apoptosis in the developing central nervous system and the embryonic liver. These results are consistent with the inhibitory interaction between RB and Abl, and provide in vivo evidence for the proapoptotic function of Abl.Oncogene (2007) 26, 3868–3877. doi:10.1038/sj.onc.1210157; published online 18 December 2006 [ABSTRACT FROM AUTHOR]

DDB2, a gene mutated in XPE patients, is involved in global genomic repair especially the repair of cyclobutane pyrimidine dimers (CPDs), and is regulated by p53 in human cells. We show that DDB2 is expressed in mouse tissues and demonstrate, using primary mouse epithelial cells, that mouse DDB2 is regulated by E2F transcription factors. Retinoblastoma (Rb), a tumor suppressor critical for the control of cell cycle progression, regulates E2F activity. Using Cre-Lox technology to delete Rb in primary mouse hepatocytes, we show that DDB2 gene expression increases, leading to elevated DDB2 protein levels. Furthermore, we show that endogenous E2F1 and E2F3 bind to DDB2 promoter and that treatment with E2F1-antisense or E2F1-small interfering RNA (siRNA) decreases DDB2 transcription, demonstrating that E2F1 is a transcriptional regulator for DDB2. This has consequences for global genomic repair: in Rb-null cells, where E2F activity is elevated, global DNA repair is increased and removal of CPDs is more efficient than in wild-type cells. Treatment with DDB2-siRNA decreases DDB2 expression and abolishes the repair phenotype of Rb-null cells. In summary, these results identify a new regulatory pathway for DDB2 by E2F, which does not require but is potentiated by p53, and demonstrate that DDB2 is involved in global repair in mouse epithelial cells.Oncogene (2007) 26, 3572–3581. doi:10.1038/sj.onc.1210151; published online 18 December 2006 [ABSTRACT FROM AUTHOR]

The risk of developing ovarian cancer is about 1% over a lifetime, but it is the most deadly gynecologic cancer, in part due to lack of diagnostic markers for early-stage disease and cell model system for studying early neoplastic changes. Most existing immortal human ovarian surface epithelial cells were achieved by using viral protein such as SV40 T/t antigen or E6/E7, which inactivate multiple cellular pathways. In the current study, we used a small interfering RNA (siRNA) against the retinoblastoma gene (pRb) and ectopic expression of human telomerase reverse transcriptase (hTERT) to immortalize the primary ovarian epithelial cell line OSE137 and two additional human ovarian surface epithelial cells. The immortalized OSE137 showed increased telomerase activity, lengthened telomeres, increased G2/M phase, altered cell-cycle regulatory proteins but nontumorigenic. As both Rb and hTERT pathways are commonly altered in human ovarian cancer and these genetic changes are faithfully modeled in these cells without using viral protein, these immortal cells represent an authentic in vitro model system with which to study the initiation and progression of human ovarian cancer.Oncogene (2007) 26, 1492–1498. doi:10.1038/sj.onc.1209905; published online 4 September 2006 [ABSTRACT FROM AUTHOR]