pRb Function Research Articles

The combination of metformin and high glucose increased longevity of Caenorhabditis elegans a DAF-16/FOXO-independent manner: cancer/diabetic model via C. elegans.

Sedentary lifestyles and diets with high glycemic indexes are considered to be contributing factors to the development of obesity, type 2 diabetes in humans. Metformin, a biguanide medication commonly used to treat type 2 diabetes, has been observed to be associated with longevity; however, the molecular mechanisms underlying this observation are still unknown. The effects of metformin and high glucose, which have important roles in aging-related disease such as diabetes and cancer, were studied in lin-35 worms because they are associated with cancer-associated pRb function in mammals and have a tumour suppressor property. According to our results, the negative effect of high glucose on egg production of lin-35 worms was greater than that of N2 worms. High glucose shortened lifespan and increased body length and width in individuals of both strains. Metformin treatment alone extended the lifespan of N2 and lin-35 worms by reducing fertilization efficiency. However, when metformin was administered in the presence of high glucose, the lifespan of lin-35 worms was clearly longer compared to N2 worms. Additionally, we conclude that glucose and metformin in lin35 worms can extend life expectancy through a DAF-16/FOXO-independent mechanism. Furthermore, the results of this study will provide a new perspective on extending mammalian lifespan through the model organism C. elegans.

The N-Terminal Region of the Transcription Factor E2F1 Contains a Novel Transactivation Domain and Recruits General Transcription Factor GTF2H2.

The transcription factor E2F1 is the principal target of the tumor suppressor pRB. E2F1 promotes cell proliferation by activating growth-promoting genes upon growth stimulation. In contrast, E2F1 contributes to tumor suppression by activating tumor suppressor genes, such as ARF, upon loss of pRB function, a major oncogenic change. The transactivation domain of E2F1 has previously been mapped to the C-terminal region. We show here that the N-terminal region of E2F1 is critical for the activation of tumor suppressor genes. Deletion of the N-terminal region dramatically compromised E2F1 activation of tumor suppressor genes. The N-terminal region showed transactivation ability when fused to the DNA-binding domain of GAL4. A search for novel interacting factors with the N-terminal region, using a yeast two-hybrid system, identified the general transcription factor GTF2H2. Overexpression of GTF2H2 enhanced E2F1 activation of tumor suppressor genes and induction of cell death. Conversely, the knockdown of GTF2H2 compromised both. E2F1 binding enhanced the binding of GTF2H2 to target promoters depending on the integrity of the N-terminal region. Taken together, these results suggest that the N-terminal region of E2F1 contains a novel transactivation domain that mediates the activation of tumor suppressor genes, at least in part, by recruiting GTF2H2.

Insulin and IGF-1 extend the lifespan of Caenorhabditis elegans by inhibiting insulin/insulin-like signaling and mTOR signaling pathways: C. elegans - Focused cancer research

The mutations in Caenorhabditis elegans (C. elegans) that extend lifespan slow down aging by interfering with several signaling pathways, including the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR). The tumor suppressor pRb (retinoblastoma protein) is believed to be involved in almost all human cancers. Lin-35, the C. elegans orthologue of the tumor suppressor pRb, was included in the study to explore the effects of insulin and IGF-1 because it has been linked to cancer-related pRb function in mammals and exhibits a tumor suppressor effect by inhibiting mTOR or IIS signaling. According to our results, IGF-1 or insulin increased the lifespan of lin-35 worms compared to N2 worms by increasing fertilization efficiency, also causing a significant increase in body size. It was concluded that the expression of daf-2 and rsks-1 decreased after insulin or IGF-1 administration, thus extending the lifespan of C. elegans lin-35 worms through both IIS and mTOR-dependent mechanisms. This suggests that it was mediated by the combined effect of the TOR and IIS pathways. These results, especially obtained in cancer-associated mutant lin-35 worms, will be useful in elucidating the C. elegans cancer model in the future.

Expanding Roles of the E2F-RB-p53 Pathway in Tumor Suppression.

The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology.

Prognostic and Clinicopathological Significance of the Loss of Expression of Retinoblastoma Protein (pRb) in Oral Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis

This systematic review and meta-analysis aims to evaluate the scientific evidence on the implications of retinoblastoma protein (pRb) alterations in oral cancer, in order to determine its prognostic and clinicopathological significance. PubMed, Embase, Web of Science, and Scopus were searched for studies published before February 2022, with no restrictions by publication date or language. The quality of the studies using the Quality in Prognosis Studies tool (QUIPS tool). Meta-analysis was conducted to achieve the proposed objectives, as well as heterogeneity, subgroup, meta-regression, and small study-effects analyses. Twenty studies that met the inclusion criteria (2451 patients) were systematically reviewed and meta-analyzed. Our results were significant for the association between the loss of pRb expression and a better overall survival (HR = 0.79, 95%CI = 0.64–0.98, p = 0.03), whereas no significant results were found for disease-free survival or clinico-pathological parameters (T/N status, clinical stage, histological grade). In conclusion, our evidence-based results demonstrate that loss of pRb function is a factor associated with improved survival in patients with OSCC. Research lines that should be developed in the future are highlighted.

Deregulated E2F Activity as a Cancer-Cell Specific Therapeutic Tool.

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells.

Expanding the prostate cancer cell line repertoire with ACRJ-PC28, an AR-negative neuroendocrine cell line derived from an African-Caribbean patient.

Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.

Relevance of pRB Loss in Human Malignancies.

The retinoblastoma tumor suppressor protein (pRB) is a known regulator of cell-cycle control; however, recent studies identified critical functions for pRB in regulating cancer-associated gene networks that influence the DNA damage response, apoptosis, and cell metabolism. Understanding the impact of these pRB functions on cancer development and progression in the clinical setting will be essential, given the prevalence of pRB loss of function across disease types. Moreover, the current state of evidence supports the concept that pRB loss results in pleiotropic effects distinct from tumor proliferation. Here, the implications of pRB loss (and resultant pathway deregulation) on disease progression and therapeutic response will be reviewed, based on clinical observation. Developing a better understanding of the pRB-regulated pathways that underpin the aggressive features of pRB-deficient tumors will be essential for further developing pRB as a biomarker of disease progression and for stratifying pRB-deficient tumors into more effective treatment regimens.

Abstract PO-138: The first Caribbean cell line, a prostate cancer cell line: ACRJ-PC28

Abstract Background Prostate cancer cell lines from diverse backgrounds are believed to hold the key to addressing current disparities in prostate cancer incidence and mortality rates among Black men. Despite decades of established cell line protocols, developing new cell lines, especially from the prostate, continues to attract less than ideal success rates (<10%). Methods ACRJ-PC28 was developed from a transrectal needle biopsy (TRNB) that was mechanically dissociated and grown on 3T3 STO fibroblast feeder cells followed by establishment via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays were carried out using growth curve analysis, immunoblots, immunohistochemistry, 3D cultures, immunofluorescence imaging, confocal microscopy, WGS and, RNA-Seq. Results ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirms the novelty and human origin of the cell line while western blots indicate the cell line is of basal-luminal type; expresses p53, pRB and is AR negative. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by PCA analysis where ACRJ-PC28 cells cluster alongside other PCa tumour tissues. WGS confirms the absence of exonic mutations and the presence of intronic variants that seem to not affect function of AR, p53 and pRB. Meanwhile, RNA-Seq confirmed the expression of several neuroendocrine markers, most relative to the PC-3 cell line and numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Cytotoxic assay indicated that a Cannabis extract was 3 times more potent in reducing the viability of ACRJ-PC28 compared to PC-3 cells. Conclusions This is the first cell line from the Caribbean and the novel methodology used in its establishment should help develop additional prostate cancer cell lines from the region in hopes of addressing the disparity in PCa among Black men. Citation Format: Henkel Valentine, William Aiken, Belinda Morrison, Ziran Zhao, Holly Fowle, Jason S. Wasserman, Elon Thompson, Warren Chin, Mark Young, Shannique Clarke, Denise Gibbs, Sharon Harrison, Wayne McLaughlin, Tim Kwok, Fang Jin, Kerry Campbell, Anelia Horvath, Rory Thompson, Norman Lee, Yan Zhou, Xavier Graña, Camille Ragin, Simone Badal. The first Caribbean cell line, a prostate cancer cell line: ACRJ-PC28 [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-138.

Abstract 2928: Evaluating Cks1-Skp2 interface as an effective therapeutic target for RB1-deficient metastatic castration-resistant prostate cancer (mCRPC)

Abstract Most prostate cancers are locally confined and can be cured by surgery, radiation therapy, and androgen deprivation therapy (ADT). Many prostate cancers initially respond to ADT but become resistant over time and ultimately relapse with a more aggressive, chemotherapy-resistant, and often metastatic disease, termed metastatic castration-resistant prostate cancer (mCRPC), with five-year survival rate <30%. Compared with regional and castration-sensitive prostate cancer, mCRPC harbor higher frequency of inactivating RB1 mutations. Unfortunately, no targeted therapy is available for RB1-deficient tumors, as it is not possible to restore this tumor suppressor in tumor cells. Therefore, we aim to identify downstream signaling targets of pRb which could serve as drug targets and thereby restore the critical functions of pRb. Our group previously identified the E3 ubiquitin ligase SCF-Skp2 as a direct repression target of pRb. Skp2 knockout completely blocks prostate tumorigenesis and cancer-related deaths in an Rb1 and Trp53 double deficient mouse model. As a substrate of Skp2, the CDK inhibitor p27 is upregulated upon Skp2 deletion, which slows cell cycle progression and thus cell proliferation. Our research findings indicate that Skp2 is a promising target for RB1 deficient mCRPC treatment. Skp2 post-translationally regulates a large number of proteins, while Cks1 serves as an essential adaptor protein only for Skp2-mediated p21/p27/p57 ubiquitination, and its Asn45 plays a critical role in binding with Skp2. A Cks1 N45R mutant has been reported to abolish the binding of Cks1 with Skp2. In order to specifically and effectively target Skp2's function in p27 degradation, we generated Cks1 N45R knockin mice and evaluated its anti-tumorigenesis effect. Here we show Cks1 N45R knockin completely inhibits tumorigenesis in the Rb1 and Trp53 double deficient prostate cancer mouse model, with p27 being elevated in the prostate to the comparable amount of that in Skp2 knockout. Surprisingly, Cks1 N45R leads to reduced Skp2 protein stability and consequently the total protein level of Skp2 decreases by more than 50%. We found that the CDK1/2 inhibitor Dinaciclib downregulates Skp2 protein levels by promoting Skp2's interaction and ubiquitination with the E3 ligase Cdh1. Our preliminary data suggests that increased p27 facilitates Cdh1-mediated Skp2 degradation by suppressing Cdk2/CyclinA-mediated Skp2 and Cdh1 phosphorylation. This study highlights the potential of developing small molecule inhibitors targeting the Cks1-Skp2 interface for RB1-mutant mCPRC, it also shows the regulation of Skp2 protein levels may be a viable strategy to phenocopy the antitumor effect of Skp2 knockout. Furthermore, we expect that the combination of Cdk1/2 inhibitors and Cks1-Skp2 interface inhibitors would increase the efficacy and selectivity of targeted therapy in mCRPC. Citation Format: Yingjiao Xue, Liang Zhu, Hongling Zhao, Edward L. Schwartz. Evaluating Cks1-Skp2 interface as an effective therapeutic target for RB1-deficient metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2928.

Retinoblastoma Tumor Suppressor Protein Roles in Epigenetic Regulation.

Simple SummaryLoss of function of the retinoblastoma gene (RB1) is the rate-limiting step in the initiation of both the hereditary and sporadic forms of retinoblastoma tumor. Furthermore, loss of function of the retinoblastoma tumor suppressor protein (pRB) is frequently found in most human cancers. In retinoblastoma, tumor progression is driven by epigenetic changes following pRB loss. This review focuses on the diverse functions of pRB in epigenetic regulation.Mutations that result in the loss of function of pRB were first identified in retinoblastoma and since then have been associated with the propagation of various forms of cancer. pRB is best known for its key role as a transcriptional regulator during cell cycle exit. Beyond the ability of pRB to regulate transcription of cell cycle progression genes, pRB can remodel chromatin to exert several of its other biological roles. In this review, we discuss the diverse functions of pRB in epigenetic regulation including nucleosome mobilization, histone modifications, DNA methylation and non-coding RNAs.

Involvement of HPV Infection in the Release of Macrophage Migration Inhibitory Factor in Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV) infection has been well-established as a risk factor in head and neck squamous cell carcinoma (HNSCC). The carcinogenic effect of HPV is mainly due to the E6 and E7 oncoproteins, which inhibit the functions of p53 and pRB, respectively. These oncoproteins could also play a role in the Warburg effect, thus favoring tumor immune escape. Here, we demonstrated that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is expressed at higher levels in HPV-negative patients than in HPV-positive patients. However, the secretion of MIF is higher in HPV-positive human HNSCC cell lines, than in HPV-negative cell lines. In-HPV positive cells, the half inhibitory concentration (IC50) of MIF inhibitor (4-iodo-6-phenylpyrimidine (4-IPP)) is higher than that in HPV-negative cells. This result was confirmed in vitro and in vivo by the use of murine SCCVII cell lines expressing either E6 or E7, or both E6 and E7. Finally, to examine the mechanism of MIF secretion, we conducted proton nuclear magnetic resonance (1H-NMR) experiments, and observed that lactate production is increased in both the intracellular and conditioned media of HPV-positive cells. In conclusion, our data suggest that the stimulation of enzymes participating in the Warburg effect by E6 and E7 oncoproteins increases lactate production and hypoxia inducible factor 1α (HIF-1α) expression, and finally induces MIF secretion.

Abstract B04: Novel Methods to Target RB Pathway Disruption in Osteosarcoma

Abstract Osteosarcoma, prevalently a pediatric disease, is the most common primary non-hematologic bone tumor. The five-year survival rates range from 24-62% depending on age. A deeper understanding of the disease and new therapeutic approaches are crucial to reducing osteosarcoma's morbidity and mortality. Data from our and other laboratories point to interconnections between bone development and cancer. Specifically, RB1 gene mutation (encoding retinoblastoma protein or pRB) is observed in 60% of sporadic osteosarcomas, and the pRB pathway is altered in osteosarcomas that retain pRB function. Mouse data suggests that loss of pRB can change osteoprogenitor function and increase osteosarcoma formation. Our work shows that: pRB loss leads to the expansion of bipotent (adipogenic/osteogenic) progenitor cells, properties also found in osteosarcomas; pRB binds to and activates the transcriptional regulator of osteogenesis Runx2, suggesting that pRB could alter promoter binding by chromatin modifying enzymes and regulate osteoblast differentiation; pRB can act as a transcriptional coactivator with Runx2 inducing Indian hedgehog (Ihh) expression late in osteoblast differentiation (Ihh can drive osteoprogenitor cells to the osteoblast lineage, potentially mediating the pRB phenotype loss in osteoblasts). Our studies also show a role for CDK6 in Runx2 activation and bone development, and a requirement for CDK6 activity in p53-dependent osteosarcoma. Thus, here we (a) analyze the role of pRB in fate specification of osteoblasts; (b) test the function of Ihh as a mediator of altered differentiation and tumorigenesis in pRB-null cells; and (c) test the role of CDK6 as a therapeutic target in osteosarcoma. Citation Format: Philip W. Hinds, Miriam Enos, Elizabeth Kong, Elisabeth Dignan, Volkan Gunduz, Jodie Pietruska. Novel Methods to Target RB Pathway Disruption in Osteosarcoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B04.

MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation.

Retinoblastomas can arise from cone photoreceptor precursors in response to the loss of pRB function. Cone precursor-specific circuitry cooperates with pRB loss to initiate this process and subsequently contributes to the malignancy. Intrinsic high level MDM2 expression is a key component of the cone precursor circuitry and is thought to inactivate p53-mediated tumor surveillance that could otherwise be induced in response to pRB loss. However, the MDM2-related MDM4 has also been proposed to abrogate p53-mediated tumor surveillance in the absence of detectable MDM2 in retinoblastoma cells, bringing into question the importance of high-level MDM2 versus MDM4 expression. Here we report that high-level MDM2 but not MDM4 has a consistent critical role in retinoblastoma cell proliferation in vitro as well as in orthotopic xenografts. Reduction of either MDM2 or MDM4 weakly induced p53, yet reduction of MDM2 but not MDM4 severely impaired proliferation and survival through a p53-independent mechanism. Specifically, MDM2 up-regulated the mRNA expression and translation of another component of the cone circuitry, MYCN, in retinoblastoma cells. Moreover, MYCN was essential to retinoblastoma cell growth and tumor formation, and ectopic MYCN partially reversed the effects of MDM2 depletion, indicating that MYCN is an important MDM2 target. These findings indicate that high-level MDM2 expression is needed in order to perform a critical p53-independent function and may obviate the need for genomic alterations to the p53 pathway during retinoblastoma tumorigenesis.

Abstract 3691: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

Abstract Retinoblastomas can arise from cone photoreceptor precursors in response to the loss of pRB function. Some aspects of cone precursor-specific circuitry cooperate with pRB loss to initiate this process and subsequently continue to contribute to the malignancy. Intrinsic high level MDM2 expression is a key component of this circuitry and is thought to inactivate p53-mediated tumor surveillance that could otherwise be induced by aberrant cell cycle entry. However, the MDM2-related MDM4 has also been proposed to abrogate p53-mediated tumor surveillance in the absence of detectable MDM2. Here we report that high-level MDM2 but not MDM4 has a consistent, critical role in retinoblastoma cell proliferation. Reduction of MDM2 and MDM4 only weakly induced p53, yet reduction of MDM2 but not MDM4 severely impaired proliferation and survival through a p53-independent mechanism. Specifically, MDM2 up-regulated the translation of another component of the cone circuitry, MYCN, in retinoblastoma cells. These findings indicate that high-level MDM2 expression is needed in order to perform a critical p53-independent function and may obviate the need for genomic alterations to the p53 pathway in retinoblastoma cells. Citation Format: Donglai Qi, David E. Cobrinik. MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3691.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to causeAML.

Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells.

The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype.

Identification of novel target genes specifically activated by deregulated E2F in human normal fibroblasts.

The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth-related genes but also in tumor suppression by activating pro-apoptotic and growth-suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27(Kip1) and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth-related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F-responsive promoter elements and to contribute to E2F1-mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function.

Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer Treatment.

Cancer cells bypass normal controls over mitotic cell-cycle progression to achieve a deregulated state of proliferation. The retinoblastoma tumor suppressor protein (pRb) governs a key cell-cycle checkpoint that normally prevents G1-phase cells from entering S-phase in the absence of appropriate mitogenic signals. Cancer cells frequently overcome pRb-dependent growth suppression via constitutive phosphorylation and inactivation of pRb function by cyclin-dependent kinase (CDK) 4 or CDK6 partnered with D-type cyclins. Three selective CDK4/6 inhibitors, palbociclib (Ibrance; Pfizer), ribociclib (Novartis), and abemaciclib (Lilly), are in various stages of development in a variety of pRb-positive tumor types, including breast cancer, melanoma, liposarcoma, and non-small cell lung cancer. The emerging, positive clinical data obtained to date finally validate the two decades-old hypothesis that the cyclin D-CDK4/6 pathway is a rational target for cancer therapy.

Pleiotropic Functions of RB Protein in Tumor Suppression

RB tumor suppressor gene product (pRB) has been proposed to interact with more than 200 cellular proteins. In addition to the phosphorylation, carried out by cyclindependent kinases (CDKs), pRB undergoes various types of post-translational modifications. The post-translational modifications of pRB affect the binding status with numerous effector molecules including the E2F family transcription factors, proteins with LxCxE motifs, and proteasome-related proteins. This variability of effector molecules enables pRB to exert pleiotropic functions. These functions also differ remarkably depending on the cell lineage and cellular context. Therefore, many recent reviews have referred to pRB as an adaptor protein based on its functions, in addition to as a pocket protein based on its structure. E2F family members have been critically implicated in pRB functions to drive cell cyclerelated events, yet consists minority of effector molecules in terms of number. In addition, E2Fs also target numerous genes those are not directly involved in the cell cycle control. Therefore, we speculate that pRB performs numerous cell cycle-independent functions in both E2F-dependent and -independent manners. This review, although without strictly discriminating between E2F-dependent and -independent functions, aims to describe the canonical and non-canonical functions of pRB.