Role of the Tripartite Motif Protein 27 in Cancer Development

Background The tripartite motif family protein 27 (TRIM27) is a transcriptional repressor that interacts with, and attenuates senescence induction by, the retinoblastoma-associated protein (RB1). High expression of TRIM27 was noted in several human cancer types including breast and endometrial cancer, where elevated TRIM27 expression predicts poor prognosis. Here, we investigated the role of TRIM27 expression in cancer development. Methods We assessed TRIM27 expression in human cancer using cancer profiling arrays containing paired tumor and normal cRNA (n=261) as well as in murine skin cancer induced by 7, 12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA). We generated mice with disrupted expression of murine TRIM27 (Trim27(-/-)) and assessed their susceptibility to DMBA/TPA-induced skin tumor development compared with isogenic littermates (n=26 mice per group). We assessed the effect of Trim27 loss on senescence propensity in mouse embryonic fibroblasts (MEFs) by quantifying cell proliferation alongside senescence markers (senescence-associated beta-galactosidase [SA-beta-gal] activity and hypertrophic cell morphology). The contribution of RB1 on senescence and cancer susceptibility (n>20 mice per group) in Trim27(-/-) backgrounds was also assessed. Data were analyzed using the Student’s t, chi(2), or log-rank test as indicated. All statistical tests were two-sided. Results TRIM27 transcript levels are statistically significantly increased in common human cancers, including colon and lung, vs normal tissues (TRIM27 expression relative to ubiquitin: cancers vs normal tissues, mean=0.59, 95% confidence interval [CI]=0.55 to 0.63 vs mean=0.46, 95% CI=0.43 to 0.49, P<.001) as well as in chemically induced mouse skin cancer compared with matched normal tissue (Trim27 expression relative to Gapdh control: tumor vs normal skin, mean=4.2, 95% CI=3.97 to 4.43 vs mean=0.96, 95% CI=0.69 to 1.2, P<.001). Trim27(-/-) mice (n=14) were resistant to chemically induced skin cancer development (eight [57.2%] of 14 mice were tumor free) compared with Trim27(+/+) wild-type littermates (n=13) (one [7.7%] of 13 mice was tumor free). Trim27(-/-) MEFs show enhanced senescence propensity in response to replicative (percentage of SA-beta-gal-positive cells: Trim27(+/+) MEFs vs Trim27(-/-) MEFs, mean=14.2%, 95% CI=11.1% to 17.4% vs mean=53.3%, 95% CI=48.7% to 57.9%, P<.001) or oncogenic stress (percentage of SA-beta-gal-positive cells: Trim27(+/+) MEFs + Ras vs Trim27(-/-) MEFs + Ras, mean=24.0%, 95% CI=19.9% to 28.1% vs mean=37.3%, 95% CI=32.2% to 42.4%, P<.05) compared with Trim27(+/+) MEFs. These responses were alleviated following inactivation of murine RB1 (Rb1). Furthermore, Trim27(-/-) mice are not protected from cancers arising as a consequence of Rb1 deletion (median survival: Trim27(-/-)Rb(+/-) vs Trim27(-/-) Rb+/-, 14 vs 13 months; difference=1.0 month, 95% CI=0.5 to 1.6 months, P=.14). Conclusion TRIM27 expression is a modifier of disease incidence and progression relevant to the development of common human cancers and is a potential target for intervention in cancer. J Natl Cancer Inst 2012;104:9412-95

A Single Nucleotide Polymorphism within the Novel Sex-Linked Testis-Specific Retrotransposed PGAM4 Gene Influences Human Male Fertility

The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes.We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo.These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility

Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

Mechanism-Based Screen Establishes Signalling Framework for DNA Damage-Associated G1 Checkpoint Response

DNA damage activates checkpoint controls which block progression of cells through the division cycle. Several different checkpoints exist that control transit at different positions in the cell cycle. A role for checkpoint activation in providing resistance of cells to genotoxic anticancer therapy, including chemotherapy and ionizing radiation, is widely recognized. Although the core molecular functions that execute different damage activated checkpoints are known, the signals that control checkpoint activation are far from understood. We used a kinome-spanning RNA interference screen to delineate signalling required for radiation-mediated retinoblastoma protein activation, the recognized executor of G1 checkpoint control. Our results corroborate the involvement of the p53 tumour suppressor (TP53) and its downstream targets p21CIP1/WAF1 but infer lack of involvement of canonical double strand break (DSB) recognition known for its role in activating TP53 in damaged cells. Instead our results predict signalling involving the known TP53 phosphorylating kinase PRPK/TP53RK and the JNK/p38MAPK activating kinase STK4/MST1, both hitherto unrecognised for their contribution to DNA damage G1 checkpoint signalling. Our results further predict a network topology whereby induction of p21CIP1/WAF1 is required but not sufficient to elicit checkpoint activation. Our experiments document a role of the kinases identified in radiation protection proposing their pharmacological inhibition as a potential strategy to increase radiation sensitivity in proliferating cancer cells

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

RB activation defect in tumor cell lines

Activation of the retinoblastoma (RB) protein through dephosphorylation arises in cells upon exit from M phase and in response to environmental stresses, including DNA damage. We provide here for the first time evidence that these responses are co-ordinately affected in a subset of tumor derived cell lines. We find that RB dephosphorylation is not apparent in these cells during progression into G(1). Importantly these cells also do not respond with RB activation after DNA damage during S phase. Moreover and as a consequence they display phenotypes classically associated with RB(−) cells, showing accelerated apoptosis after DNA damage and DNA re-replication after spindle-checkpoint activation. A large body of literature provides evidence that controls governing inactivation of RB are lost in tumors. The results presented here indicate that the reverse reaction, namely the activation of RB from an inactive precursor, may also be compromised. Our findings indicate that this type of defect may be coupled with hypersensitivity to DNA damage and an increase in genomic instability in response to spindle-checkpoint activation thus bearing potentially important medical implications

An Overlapping Kinase and Phosphatase Docking Site Regulates Activity of the Retinoblastoma Protein

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking