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The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Retinoblastoma is the most common intraocular cancer in childhood. Loss of function in both copies of the RB1 gene is the causal mutation of retinoblastoma. Current treatment for retinoblastoma includes the use of chemotherapeutic agents, such as the DNA damaging agent etoposide, which is a topoisomerase II poison that mainly generates DNA double-strand breaks (DSBs) and genome instability. Unfaithful repairing of DSBs could lead to secondary cancers and serious side effects. Previously, we found that RB knocked-down mammalian cells depend on a highly mutagenic pathway, the micro-homology mediated end joining (MMEJ) pathway, to repair DSBs. Poly ADP ribose polymerase 1 (PARP1) is a major protein in promoting the MMEJ pathway. In this study, we explored the effects of olaparib, a PARP inhibitor, in killing retinoblastoma cells. Retinoblastoma cell line Y79 and primary retinoblastoma cells expressed the cone-rod homeobox protein (CRX), a photoreceptor-specific marker. No detectable RB expression was found in these cells. The co-treatment of olaparib and etoposide led to enhanced cell death in both the Y79 cells and the primary retinoblastoma cells. Our results demonstrated the killing effects in retinoblastoma cells by PARP inhibitor olaparib after inducing DNA double-strand breaks. The use of olaparib in combination with etoposide could improve the cell-killing effects. Thus, lower dosages of etoposide can be used to treat retinoblastoma, which would potentially lead to a lower level of DSBs and a relatively more stable genome.

Retinitis pigmentosa (RP) is a major cause of inherited blindness, and there is presently no cure for RP. Rd1 mouse is the most commonly used RP animal model. Re-expression of cell cycle proteins in post-mitotic neurons is considered an important mechanism of neurodegenerative diseases, including RP. The retinoblastoma tumor suppressor (Rb) is a major regulator of cell cycle progression, yet its role in rd1 mouse retina and related signaling pathways have never been analyzed. By crossing α-Cre, Rb f/f mice with rd1 mice, p21cip1 -/- mice, Cdk1 f/f mice and Cdk2 f/f mice, we established multiple rd1 mouse models with deletions of Rb gene, Cdkn1a (p21cip1) gene, Cdk1 and Cdk2 gene in the retina. Cdk inhibitor CR8 was injected into the vitreous of rd1 mouse to investigate its effects on photoreceptor survival. Rb gene knockout (KO) induces cell death in excitatory retinal neurons (rods, rod bipolar and ganglions) and ectopic proliferation of retinal cells; but it paradoxically delays the rod death of rd1 mice, which is primarily mediated by the Cdk inhibitor Cdkn1a (p21cip1). Interestingly, p21cip1 protects the ectopic dividing rd1 rod cells by inhibiting Cdk1 and Cdk2. However, inhibiting Cdk1 and Cdk2 in rd1 mice with non-dividing rods only has limited and transient protective effects. Our data suggest that there is no ectopic division of rd1 rod cells, and RbKO induces ectopic division but delays the death of rd1 rod cells. This reveals the important protective role of Rb-p21cip1-Cdk axis in rd1 rod cells. P21cip1 is a potential target for future therapy of RP., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer., Competing Interests: The authors have declared that no competing interests exist.

Erythropoiesis is intimately coupled to cell division, and deletion of the cell cycle regulator retinoblastoma protein (pRb) causes anemia in mice. Erythroid-specific deletion of pRb has been found to result in inefficient erythropoiesis because of deregulated coordination of cell cycle exit and mitochondrial biogenesis. However, the pathophysiology remains to be fully described, and further characterization of the link between cell cycle regulation and mitochondrial function is needed. To this end we further assessed conditional erythroid-specific deletion of pRb. This resulted in macrocytic anemia, despite elevated levels of erythropoietin (Epo), and an accumulation of erythroid progenitors in the bone marrow, a phenotype strongly resembling refractory anemia associated with myelodysplastic syndromes (MDS). Using high-fractionation fluorescence-activated cell sorting analysis for improved phenotypic characterization, we illustrate that erythroid differentiation was disrupted at the orthochromatic stage. Transcriptional profiling of sequential purified populations revealed failure to upregulate genes critical for mitochondrial function such as Pgc1β, Alas2, and Abcb7 specifically at the block, together with disturbed heme production and iron transport. Notably, deregulated ABCB7 causes ring sideroblastic anemia in MDS patients, and the mitochondrial co-activator PGC1β is heterozygously lost in del5q MDS. Importantly, the anemia could be rescued through enhanced PPAR signaling in vivo via either overexpression of Pgc1β or bezafibrate administration. In conclusion, lack of pRb results in MDS-like anemia with disrupted differentiation and impaired mitochondrial function at the orthochromatic erythroblast stage. Our findings reveal for the first time a role for pRb in heme and iron regulation, and indicate that pRb-induced anemia can be rescued in vivo through therapeutic enhancement of PPAR signaling., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

RB1 gene is often homozygously deleted or mutated in prostate adenocarcinomas following acquirement of castration resistance and/or metastatic ability. We found that SUCLA2 gene is frequently involved in the deletion of the RB1 gene region in advanced prostate cancer. SUCLA2 constitutes the β-subunit of succinate CoA ligase heterodimer that reversibly converts succinyl CoA into succinate. We sought the possibility that deletion of SUCLA2 gives rise to a metabolic vulnerability that could be targeted therapeutically. We found a significant metabolic shift in SUCLA2-deleted prostate cancer cells, including lower mitochondrial respiratory activity. By screening a number of libraries for compounds that induce cell death selectively in SUCLA2-deficient prostate cancer cells, we identified thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) and PMA (phorbol-12-myristate-13-acetate) from a natural compound library. These findings indicate that the metabolic vulnerability in SUCLA2-deficient prostate cancer cells is pharmacologically targetable.

The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53 , is required and sufficient for tumorigenesis. While RB1 -mutant cells fail to arrest at G 1 -S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1 -deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53 -knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1 -deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1 , inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment., (©2020 American Association for Cancer Research.)

Cardiovascular disease (CVD) is a major cause of global mortality. The proper functioning of the endothelial layer of arteries is crucial to cardiovascular health. Retinoblastoma protein (Rb), encoded by the Rb1 gene, has been shown to offer vasoprotective effects. Herein, we investigated endothelial Rb's effects on arterial function using an endothelial-specific conditional Rb1 knockout (Rb cKO) mouse model. We found that Rb deficiency reduced dihydrofolate reductase (DHFR) activity and downstream NO production in mouse aortic endothelial cells and blocked arterial vasodilation in an endothelial DHFR-dependent manner. Rb deficiency also increased phenylephrine-triggered arterial vasoconstriction, BP levels, and pathological aortic remodeling without significantly affecting prostanoid synthesis. Employing an angiotensin II (AngII)-stimulated apolipoprotein E knockout (apoE -/-) mice fed a standard, non-atherogenic diet, Rb deficiency increased aortic diameter, stimulated abdominal aortic aneurysm (AAA) development, and reduced survival. These pathological responses to Rb deficiency in AngII-stimulated apoE-/- mice were rescued by DHFR overexpression. Cumulatively, our findings reveal that endothelial Rb positively impacts arterial function by supporting vasoprotective endothelial DHFR/NO pathway activity, leading to reduced AAA development.

Cancer cell-intrinsic properties caused by oncogenic mutations have been well characterized; however, how specific oncogenes and tumor suppressors impact the tumor microenvironment (TME) is not well understood. Here, we present a novel non-cell-autonomous function of the retinoblastoma (RB) tumor suppressor in controlling the TME. RB inactivation stimulated tumor growth and neoangiogenesis in a syngeneic and orthotropic murine soft-tissue sarcoma model, which was associated with recruitment of tumor-associated macrophages (TAM) and immunosuppressive cells such as Gr1 + CD11b + myeloid-derived suppressor cells (MDSC) or Foxp3 + regulatory T cells (Treg). Gene expression profiling and analysis of genetically engineered mouse models revealed that RB inactivation increased secretion of the chemoattractant CCL2. Furthermore, activation of the CCL2-CCR2 axis in the TME promoted tumor angiogenesis and recruitment of TAMs and MDSCs into the TME in several tumor types including sarcoma and breast cancer. Loss of RB increased fatty acid oxidation (FAO) by activating AMP-activated protein kinase that led to inactivation of acetyl-CoA carboxylase, which suppresses FAO. This promoted mitochondrial superoxide production and JNK activation, which enhanced CCL2 expression. These findings indicate that the CCL2-CCR2 axis could be an effective therapeutic target in RB-deficient tumors. SIGNIFICANCE: These findings demonstrate the cell-nonautonomous role of the tumor suppressor retinoblastoma in the tumor microenvironment, linking retinoblastoma loss to immunosuppression., (©2019 American Association for Cancer Research.)

Transforming growth factor β1 (TGFβ1) is a potent inhibitor of cell growth that targets gene-regulatory events, but also inhibits the function of CDC45-MCM-GINS helicases (CMG; MCM, Mini-Chromosome Maintenance; GINS, Go-Ichi-Ni-San) through multiple mechanisms to achieve cell-cycle arrest. Early in G 1 , TGFβ1 blocks MCM subunit expression and suppresses Myc and Cyclin E/Cdk2 activity required for CMG assembly, should MCMs be expressed. Once CMGs are assembled in late-G 1 , TGFβ1 blocks CMG activation using a direct mechanism involving the retinoblastoma (Rb) tumor suppressor. Here, in cells lacking Rb, TGFβ1 does not suppress Myc, Cyclin E/Cdk2 activity, or MCM expression, yet growth arrest remains intact and Smad2/3/4-dependent. Such arrest occurs due to inhibition of MCM hexamer assembly by TGFβ1, which is not seen when Rb is present and MCM subunit expression is normally blocked by TGFβ1. Loss of Smad expression prevents TGFβ1 suppression of MCM assembly. Mechanistically, TGFβ1 blocks a Cyclin E-Mcm7 molecular interaction required for MCM hexamer assembly upstream of CDC10-dependent transcript-1 (CDT1) function. Accordingly, overexpression of CDT1 with an intact MCM-binding domain abrogates TGFβ1 arrest and rescues MCM assembly. The ability of CDT1 to restore MCM assembly and allow S-phase entry indicates that, in the absence of Rb and other canonical mediators, TGFβ1 relies on inhibition of Cyclin E-MCM7 and MCM assembly to achieve cell cycle arrest. IMPLICATIONS: These results demonstrate that the MCM assembly process is a pivotal target of TGFβ1 in eliciting cell cycle arrest, and provide evidence for a novel oncogenic role for CDT1 in abrogating TGFβ1 inhibition of MCM assembly., (©2018 American Association for Cancer Research.)

Most retinoblastomas initiate in response to the inactivation of the RB1 gene and loss of functional RB protein. The tumors may form with few additional genomic changes and develop after a premalignant retinoma phase. Despite this seemingly straightforward etiology, mouse models have not recapitulated the genetic, cellular, and stage-specific features of human retinoblastoma genesis. For example, whereas human retinoblastomas appear to derive from cone photoreceptor precursors, current mouse models develop tumors that derive from other retinal cell types. To investigate the basis of the human cone-specific oncogenesis, we compared developmental stage-specific cone precursor responses to RB loss in human and murine retina cultures and in cone-specific Rb1 -knockout mice. We report that RB-depleted maturing (ARR3 + ) but not immature (ARR3 - ) human cone precursors enter the cell cycle, proliferate, and form retinoblastoma-like lesions with Flexner-Wintersteiner rosettes, then form low or nonproliferative premalignant retinoma-like lesions with fleurettes and p16 INK4A and p130 expression, and finally form highly proliferative retinoblastoma-like masses. In contrast, in murine retina, only RB-depleted immature (Arr3 - ) cone precursors entered the cell cycle, and they failed to progress from S to M phase. Moreover, whereas intrinsically highly expressed MDM2 and MYCN contribute to RB-depleted maturing (ARR3 + ) human cone precursor proliferation, ectopic MDM2 and Mycn promoted only immature (Arr3 - ) murine cone precursor cell-cycle entry. These findings demonstrate that developmental stage-specific as well as species- and cell type-specific features sensitize to RB1 inactivation and reveal the human cone precursors' capacity to model retinoblastoma initiation, proliferation, premalignant arrest, and tumor growth., Competing Interests: The authors declare no conflict of interest.

Inactivation of the retinoblastoma gene ( RB1 ) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1 +/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1 +/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1 -null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation., Competing Interests: The authors declare no conflict of interest.

The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.

Purpose: Previously, we demonstrated enhanced adaptation after small bowel resection (SBR) in intestinal-specific retinoblastoma (Rb)-deficient mice along with elevated levels of insulin-like growth factor 2 (IGF2) expression within the villi. The purpose of this study was to verify that the insulin-like growth factor 1 receptor (IGF1R) plays a role in this phenomenon., Methods: Inducible and intestinal specific Rb and IGF1R double knockout mice (iRb/IGF1R-IKO) (n=4) and Rb single knockout mice (iRb-IKO) (n=5) underwent 50% mid SBR. On post-operative day 28, mice were harvested, and structural adaptation was measured as changes in crypt depth and villus height. Rates of enterocyte proliferation were recorded. IGF2 expression within the remnant villi was measured via RT-PCR., Results: Both iRb-IKO and iRb/IGF1R-IKO mice demonstrated enhanced adaptation with at least a 45% increase in both crypt depth and villus height in the proximal and distal remnant bowel. Both groups showed elevation of IGF2 expression in the remnant villi, but there were no differences between the two groups., Conclusion: Epithelial IGF1R is dispensable for IGF2-mediated enhanced intestinal adaptation in retinoblastoma-deficient mice. Our findings suggest that IGF2 signals for enhanced adaptation in cells outside of the epithelium. Further investigation is needed to study the IGF2/IGF1R signaling interaction within the mesenchyme., Level of Evidence: Animal study - not clinical., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Genomic instability (GIN) is a hallmark of most cancer cells. However, compared to most human cancer cell types, the retinoblastoma tumor cells show a relatively stable genome. The fundamental basis of this genomic stability has yet to be elucidated, and the role of certain proteins involved in cell cycle regulation may be the key to the development of these specific genotypes. We examined whether thyroid hormone receptor beta 1 and 2 (TRβ1 and TRβ2), known to regulate tumorigenesis, and PTTG1, a mitotic checkpoint protein, play a role in maintaining genomic stability in retinoblastoma. In order to elucidate the role of these proteins in development of aneuploidy/polyploidy, an indicator of GIN, we first studied comparative GIN in retinoblastomas and multiple RB mutant cancer cell lines using single nucleotide polymorphism (SNP) analysis. We then utilized pLKO lentiviral vectors to selectively modify expression of the targeted cell cycle proteins and interpret their effect on downstream cell cycle proteins and their relative effects on the development of polyploidy in multiple tumor cell lines. The SNP analysis showed that retinoblastomas displayed relatively fewer genomic copy number changes as compared to other RB1-deficient cancer cell lines. Both TRβ1 and TRβ2 knockdown led to accumulation of E2F1 and PTTG1 and increased GIN as demonstrated by an increase in polyploidy. Downregulation of PTTG1 led to a relative decrease in GIN while upregulation of PTTG1 led to a relative increase in GIN. Knockdown of E2F1 led to a downstream decrease in PTTG1 expression. Rb-knockdown also upregulated E2F1 and PTTG1 leading to increased GIN. We showed that Rb is necessary for PTTG1 inhibition and genomic stability. A relatively stable genome in retinoblastoma tumor cells is maintained by TRβ1 and TRβ2-mediated PTTG1 inhibition, counteracting Rb-deficiency-related GIN. TRβ1, TRβ2 and Rb-KD all led to the downstream PTTG1 accumulation, apparently through an activation of E2F1 resulting in extensive genomic instability as seen in other Rb-deficient tumors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c-Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c-Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum-free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS-like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient-derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c-Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS-like cells by Rb knockdown and c-Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512-526., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

SCF Skp2/Cks1 ubiquitinates Thr187-phosphorylated p27 for degradation. Overexpression of Skp2 coupled with underexpression of p27 are frequent characteristics of cancer cells. When the role of SCF Skp2/Cks1 -mediated p27 ubiquitination in cancer was specifically tested by p27 Thr187-to-Ala knockin (p27T187A KI), it was found dispensable for Kras G12D -induced lung tumorigenesis but essential for Rb1-deficient pituitary tumorigenesis. Here we identify pRb and p53 doubly deficient (DKO) prostate tumorigenesis as a context in which p27 ubiquitination by SCF Skp2/Cks1 is required for p27 downregulation. p27 protein accumulated in prostate when p27T187A KI mice underwent DKO prostate tumorigenesis. p27T187A KI or Skp2 knockdown (KD) induced similar degrees of p27 protein accumulation in DKO prostate cells, and Skp2 KD did not further increase p27 protein in DKO prostate cells that contained p27T187A KI (AADKO prostate cells). p27T187A KI activated an E2F1-p73-apoptosis axis in DKO prostate tumorigenesis, slowed disease progression and significantly extended survival. Querying co-occurrence relationships among RB1, TP53, PTEN, NKX3-1 and MYC in TCGA of prostate cancer identified co-inactivation of RB1 and TP53 as the only statistically significant co-occurrences in metastatic castration-resistant prostate cancer (mCRPC). Together, our study identifies Skp2/Cks1 pocket inhibitors as potential therapeutics for mCRPC. Procedures for establishing mCRPC organoid cultures from contemporary patients were recently established. An Skp2/Cks1 pocket inhibitor preferentially collapsed DKO prostate tumor organoids over AADKO organoids, which spontaneously disintegrated over time when DKO prostate tumor organoids grew larger, setting the stage to translate mouse model findings to precision medicine in the clinic on the organoid platform., Competing Interests: The authors declare no conflict of interest.

The long term prognosis of liver cancer patients remains unsatisfactory because of cancer recurrence after surgical interventions, particularly in patients with viral infections. Since hepatitis B and C viral proteins lead to inactivation of the tumor suppressors p53 and Retinoblastoma (Rb), we hypothesize that surgery in the context of p53/Rb inactivation initiate de novo tumorigenesis.We, therefore, generated transgenic mice with hepatocyte and cholangiocyte/liver progenitor cell (LPC)-specific deletion of p53 and Rb, by interbreeding conditional p53/Rb knockout mice with either Albumin-cre or Cytokeratin-19-cre transgenic mice.We show that liver cancer develops at the necrotic injury site after surgical resection or radiofrequency ablation in p53/Rb deficient livers. Cancer initiation occurs as a result of specific migration, expansion and transformation of cytokeratin-19+-liver (CK-19+) cells. At the injury site migrating CK-19+ cells formed small bile ducts and adjacent cells strongly expressed the transforming growth factor β (TGFβ). Isolated cytokeratin-19+ cells deficient for p53/Rb were resistant against hypoxia and TGFβ-mediated growth inhibition. CK-19+ specific deletion of p53/Rb verified that carcinomas at the injury site originates from cholangiocytes or liver progenitor cells.These findings suggest that human liver patients with hepatitis B and C viral infection or with mutations for p53 and Rb are at high risk to develop tumors at the surgical intervention site.

Outcomes for patients diagnosed with the bone cancer osteosarcoma have not improved significantly in the last four decades. Only around 60% of patients and about a quarter of those with metastatic disease survive for more than five years. Although DNA-damaging chemotherapy drugs can be effective, they can provoke serious or fatal adverse effects including cardiotoxicity and therapy-related cancers. Better and safer treatments are therefore needed. We investigated the anti-osteosarcoma activity of IAP antagonists (also known as Smac mimetics) using cells from primary and metastatic osteosarcomas that arose spontaneously in mice engineered to lack p53 and Rb expression in osteoblast-derived cells. The IAP antagonists SM-164, GDC-0152 and LCL161, which efficiently target XIAP and cIAPs, sensitized cells from most osteosarcomas to killing by low levels of TNFα but not TRAIL. RIPK1 expression levels and activity correlated with sensitivity. RIPK3 levels varied considerably between tumors and RIPK3 was not required for IAP antagonism to sensitize osteosarcoma cells to TNFα. IAP antagonists, including SM-164, lacked mutagenic activity. These data suggest that drugs targeting XIAP and cIAP1/2 may be effective for osteosarcoma patients whose tumors express abundant RIPK1 and contain high levels of TNFα, and would be unlikely to provoke therapy-induced cancers in osteosarcoma survivors., Competing Interests: The authors have no conflicts of interest.

Blood cells are receiving an increasing attention as an easily accessible source of transcript-based biomarkers. We studied the feasibility of using mouse whole blood RNA in this context. Several paradigms were studied: (i) metabolism-related transcripts known to be affected in rat tissues and peripheral blood mononuclear cells (PBMC) by fasting and upon the development of high fat diet (HFD)-induced overweight were assessed in whole blood RNA of fasted rats and mice and of HFD-fed mice; (ii) retinoic acid (RA)-responsive genes in tissues were assessed in whole blood RNA of control and RA-treated mice; (iii) lipid metabolism-related transcripts previously identified in PBMC as potential biomarkers of metabolic health in a rat model were assessed in whole blood in an independent model, namely retinoblastoma haploinsufficient (Rb+/-) mice. Blood was collected and stored in RNAlater® at -80°C until analysis of selected transcripts by real-time RT-PCR. Comparable changes with fasting were detected in the expression of lipid metabolism-related genes when RNA from either PBMC or whole blood of rats or mice was used. HFD-induced excess body weight and fat mass associated with expected changes in the expression of metabolism-related genes in whole blood of mice. Changes in gene expression in whole blood of RA-treated mice reproduced known transcriptional actions of RA in hepatocytes and adipocytes. Reduced expression of Fasn, Lrp1, Rxrb and Sorl1 could be validated as early biomarkers of metabolic health in young Rb+/- mice using whole blood RNA. Altogether, these results support the use of whole blood RNA in studies aimed at identifying blood transcript-based biomarkers of nutritional/metabolic status or metabolic health. Results also support reduced expression of Fasn, Lrp1, Rxrb and Sorl1 in blood cells at young age as potential biomarkers of metabolic robustness.

Missense mutations of fibroblast growth factor receptor 3 (FGFR3) occur in up to 80% of low-grade papillary urothelial carcinoma of the bladder (LGP-UCB) suggesting that these mutations are tumor drivers, although direct experimental evidence is lacking. Here we show that forced expression of FGFR3b-S249C, the most prevalent FGFR3 mutation in human LGP-UCB, in cultured urothelial cells resulted in slightly reduced surface translocation than wild-type FGFR3b, but nearly twice as much proliferation. When we expressed a mouse equivalent of this mutant (FGFR3b-S243C) in urothelia of adult transgenic mice in a tissue-specific and inducible manner, we observed significant activation of AKT and MAPK pathways. This was, however, not accompanied by urothelial proliferation or tumorigenesis over 12 months, due to compensatory tumor barriers in p16-pRB and p19-p53-p21 axes. Indeed, expressing FGFR3b-S249C in cultured human urothelial cells expressing SV40T, which functionally inactivates pRB/p53, markedly accelerated proliferation and cell-cycle progression. Furthermore, expressing FGFR3b-S243C in transgenic mouse urothelium expressing SV40T converted carcinoma-in-situ to high-grade papillary urothelial carcinoma. Together, our study provides new experimental evidence indicating that the FGFR3 mutations have very limited urothelial tumorigenicity and that these mutations must collaborate with other genetic events to drive urothelial tumorigenesis.

Chemotherapy-induced myelosuppression continues to represent the major dose-limiting toxicity of cytotoxic chemotherapy, which can be manifested as neutropenia, lymphopenia, anemia, and thrombocytopenia. As such, myelosuppression is the source of many of the adverse side effects of cancer treatment including infection, sepsis, bleeding, and fatigue, thus resulting in the need for hospitalizations, hematopoietic growth factor support, and transfusions (red blood cells and/or platelets). Moreover, clinical concerns raised by myelosuppression commonly lead to chemotherapy dose reductions, therefore limiting therapeutic dose intensity, and reducing the antitumor effectiveness of the treatment. Currently, the only course of treatment for myelosuppression is growth factor support which is suboptimal. These treatments are lineage specific, do not protect the bone marrow from the chemotherapy-inducing cytotoxic effects, and the safety and toxicity of each agent is extremely specific. Here, we describe the preclinical development of G1T28, a novel potent and selective CDK4/6 inhibitor that transiently and reversibly regulates the proliferation of murine and canine bone marrow hematopoietic stem and progenitor cells and provides multilineage protection from the hematologic toxicity of chemotherapy. Furthermore, G1T28 does not decrease the efficacy of cytotoxic chemotherapy on RB1-deficient tumors. G1T28 is currently in clinical development for the reduction of chemotherapy-induced myelosuppression in first- and second-line treatment of small-cell lung cancer. Mol Cancer Ther; 15(5); 783-93. ©2016 AACR., (©2016 American Association for Cancer Research.)

Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non-targeting RNAs, myotubes silenced for Rb (by 80-90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5-fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up-regulated levels of glucose transporter type 4 expression (∼5-fold increase), increased basal glucose uptake, and enhanced insulin-induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise., (© 2015 Wiley Periodicals, Inc.)

The retinoblastoma gene (RB1) has been implicated as a tumor suppressor in multiple myeloma (MM), yet its role remains unclear because in the majority of cases with 13q14 deletions, un-mutated RB1 remains expressed from the retained allele. To explore the role of Rb1 in MM, we examined the functional consequences of single- and double-copy Rb1 loss in germinal center B cells, the cells of origin of MM. We generated mice without Rb1 function in germinal center B cells by crossing Rb1(Flox/Flox) with C-γ-1-Cre (Cγ1) mice expressing the Cre recombinase in class-switched B cells in a p107(-/-) background to prevent p107 from compensating for Rb1 loss (Cγ1-Rb1(F/F)-p107(-/-)). All mice developed normally, but B cells with two copies of Rb1 deleted (Cγ1-Rb1(F/F)-p107(-/-)) exhibited increased proliferation and cell death compared with Cγ1-Rb1(+/+)-p107(-/-) controls ex vivo. In vivo, Cγ1-Rb1(F/F)-p107(-/-) mice had a lower percentage of splenic B220+ cells and reduced numbers of bone marrow antigen-specific secreting cells compared with control mice. Our data indicate that Rb1 loss induces both cell proliferation and death in germinal center B cells. Because no B-cell malignancies developed after 1 year of observation, our data also suggest that Rb1 loss is not sufficient to transform post-germinal center B cells and that additional, specific mutations are likely required to cooperate with Rb1 loss to induce malignant transformation., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

S phase kinase-associated protein 2 (Skp2) has been shown to be required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb) deficient mice. Here we have demonstrated that flavokawain A (FKA), a novel chalcone from the kava plant, selectively inhibited the growth of pRb deficient cell lines and resulted in a proteasome-dependent and ubiquitination-mediated Skp2 degradation. Degradation of Skp2 by FKA was found to be involved in a functional Cullin1, but independent of Cdh1 expression. Further studies have demonstrated that FKA docked into the ATP binding pocket of the precursor cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) complex, inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Finally, dietary feeding of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions (HG-PIN) and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis. Immunohistochemistry studies revealed that dietary FKA feeding resulted in marked anti-proliferative and apoptotic effects via down-regulation of Skp2 and NEDD8 and up-regulation of p27/Kip1 in the prostate of TRAMP mice. Our findings therefore provide evidence that FKA is a promising NEDDylation inhibitor for targeting Skp2 degradation in prostate cancer prevention and treatment.

The Rb and Pten tumor suppressor genes are important regulators of bone development and both are frequently mutated in the bone cancer osteosarcoma (OS). To determine if Rb1 and Pten synergize as tumor suppressor genes for osteosarcoma, we co-deleted them in osteoprogenitor cells. Surprisingly, we observed rapid development of adipogenic but not osteosarcoma tumors in the ΔRb1/Pten mice. ΔPten solo deleted mice also developed lipoma tumors but at a much reduced frequency and later onset than those co-deleted for Rb1. Pten deletion also led to a marked increase in adipocytes in the bone marrow. To better understand the function of Pten in bone development in vivo, we conditionally deleted Pten in OSX(+) osteoprogenitor cells using OSX-Cre mice. μCT analysis revealed a significant thickening of the calvaria and an increase in trabeculae volume and number in the femur, consistent with increased bone formation in these mice. To determine if Pten and Rb1 deletion actively promotes adipogenic differentiation, we isolated calvarial cells from Pten(fl/fl) and Pten(fl/fl); Rb1(fl/fl) mice, infected them with CRE or GFP expressing adenovirus, treated with differentiation media. We observed slightly increased adipogenic, and osteogenic differentiation in the ΔPten cells. Both phenotypes were greatly increased upon Rb1/Pten co-deletion. This was accompanied by an increase in expression of genes required for adipogenesis. These data indicate that Pten deletion in osteoblast precursors is sufficient to promote frequent adipogenic, but only rare osteogenic tumors. Rb1 hetero- or homo-zygous co-deletion greatly increases the incidence and the rapidity of onset of adipogenic tumors, again, with only rare osteosarcoma tumors.

Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture. Here, we investigated how two critical proliferative pathways, Myc and E2f, are integrated to control cell cycles in normal and Rb-deficient cells using a murine intestinal model. We show that Myc and E2f1-3 have little impact on normal G1-S transitions. Instead, they synergistically control an S-G2 transcriptional program required for normal cell divisions and maintaining crypt-villus integrity. Surprisingly, Rb deficiency results in the Myc-dependent accumulation of E2f3 protein and chromatin repositioning of both Myc and E2f3, leading to the 'super activation' of a G1-S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb-deficient cells hijack and redeploy Myc and E2f3 from an S-G2 program essential for normal cell cycles to a G1-S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null cells on Myc.

Activator E2Fs and Myc cooperate as master regulators of proliferation. A new study sheds light on one of the fundamental questions in cancer biology: how do oncogenic changes, such as Retinoblastoma (RB)-mutation, modify E2F and Myc activity?

Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene-induced senescence (OIS). A combination of real-time metabolic monitoring, and metabolome and gene expression analyses showed that OIS-induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB-depleted or downstream glycolytic enzyme-depleted OIS cells, suggesting that RB-mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells., (© 2015 AlphaMed Press.)

Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Cellular reprogramming to iPSCs has uncovered unsuspected links between tumor suppressors and pluripotency factors. Using this system, it was possible to identify tumor suppressor p27 as a repressor of Sox2 during differentiation. This led to the demonstration that defects in the repression of Sox2 can contribute to tumor development. The members of the retinoblastoma family of pocket proteins, pRb, p107 and p130, are negative regulators of the cell cycle with tumor suppressor activity and with roles in differentiation. In this work we studied the relative contribution of the retinoblastoma family members to the regulation of Sox2 expression. We found that deletion of Rb or p130 leads to impaired repression of Sox2, a deffect amplified by inactivation of p53. We also identified binding of pRb and p130 to an enhancer with crucial regulatory activity on Sox2 expression. Using cellular reprogramming we tested the impact of the defective repression of Sox2 and confirmed that Rb deficiency allows the generation of iPSCs in the absence of exogenous Sox2. Finally, partial depletion of Sox2 positive cells reduced the pituitary tumor development initiated by Rb loss in vivo. In summary, our results show that Sox2 repression by pRb is a relevant mechanism of tumor suppression.

Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Retinoblastoma-1 (RB1), and the RB1-related proteins p107 and p130, are key regulators of the cell cycle. Although RB1 is required for normal erythroid development in vitro, it is largely dispensable for erythropoiesis in vivo. The modest phenotype caused by RB1 deficiency in mice raises questions about redundancy within the RB1 family, and the role of RB1 in erythroid differentiation. Here we show that RB1 is the major pocket protein that regulates terminal erythroid differentiation. Erythroid cells lacking all pocket proteins exhibit the same cell cycle defects as those deficient for RB1 alone. RB1 has broad repressive effects on gene transcription in erythroid cells. As a group, RB1-repressed genes are generally well expressed but downregulated at the final stage of erythroid development. Repression correlates with E2F binding, implicating E2Fs in the recruitment of RB1 to repressed genes. Merging differential and time-dependent changes in expression, we define a group of approximately 800 RB1-repressed genes. Bioinformatics analysis shows that this list is enriched for terms related to the cell cycle, but also for terms related to terminal differentiation. Some of these have not been previously linked to RB1. These results expand the range of processes potentially regulated by RB1, and suggest that a principal role of RB1 in development is coordinating the events required for terminal differentiation.

RUNX1 encodes a RUNX family transcription factor (TF) and was recently identified as a novel mutated gene in human luminal breast cancers. We found that Runx1 is expressed in all subpopulations of murine mammary epithelial cells (MECs) except the secretory alveolar luminal cells. Conditional knockout of Runx1 in MECs by MMTV-Cre led to a decrease in luminal MECs, largely due to a profound reduction in the estrogen receptor (ER)-positive mature luminal subpopulation, a phenotype that could be rescued by the loss of either Trp53 or Rb1. Mechanistically RUNX1 represses Elf5, a master regulatory TF gene for alveolar cells, and regulates mature luminal TF/co-factor genes (e.g., Foxa1 and Cited1) involved in the ER program. Collectively, our data identified a key regulator of the ER⁺ luminal lineage whose disruption may contribute to the development of ER⁺ luminal breast cancer when under the background of either TP53 or RB1 loss.

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

The cellular microenvironment plays a relevant role in cancer development. We have reported that mesenchymal stromal/stem cells (MSCs) deficient for p53 alone or together with RB (p53(-/-)RB(-/-)) originate leiomyosarcoma after subcutaneous (s.c.) inoculation. Here, we show that intrabone or periosteal inoculation of p53(-/-) or p53(-/-)RB(-/-) bone marrow- or adipose tissue-derived MSCs originated metastatic osteoblastic osteosarcoma (OS). To assess the contribution of bone environment factors to OS development, we analyzed the effect of the osteoinductive factor bone morphogenetic protein-2 (BMP-2) and calcified substrates on p53(-/-)RB(-/-) MSCs. We show that BMP-2 upregulates the expression of osteogenic markers in a WNT signaling-dependent manner. In addition, the s.c. coinfusion of p53(-/-)RB(-/-) MSCs together with BMP-2 resulted in appearance of tumoral osteoid areas. Likewise, when p53(-/-)RB(-/-) MSCs were inoculated embedded in a calcified ceramic scaffold composed of hydroxyapatite and tricalciumphosphate (HA/TCP), tumoral bone formation was observed in the surroundings of the HA/TCP scaffold. Moreover, the addition of BMP-2 to the ceramic/MSC implants further increased the tumoral osteoid matrix. Together, these data indicate that bone microenvironment signals are essential to drive OS development., (© AlphaMed Press.)

One mechanism of tumour suppression by pRb is repressing E2F1. Hence, E2f1 deletion diminishes tumorigenesis following Rb1 loss. However, E2F1 promotes both proliferation and apoptosis. It therefore remains unclear how de-repressed E2F1 promotes tumorigenesis. Another mechanism of pRb function is repressing Skp2 to elevate p27 to arrest proliferation. However, Skp2 deletion induced apoptosis, not proliferation arrest, in Rb1-deficient pituitary tumorigenesis. Here we show that Rb1 deletion induces higher expression of E2F1 target genes in the absence of Skp2. E2F1 binds less cyclin A but more target promoters when Rb1 is deleted with Skp2 knockout or p27T187A knockin, suggesting that stabilized p27 prevents cyclin A from binding and inhibiting E2F1. In Rb1-deficient pituitary tumorigenesis, Skp2 deletion or p27T187A mutation converts E2F1's role from proliferative to apoptotic. These findings delineate a pRb-Skp2-p27-cyclin A-E2F1 pathway that determines whether E2F1 is proliferative or apoptotic in Rb1-deficient tumorigenesis.

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1(met), fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA(+)/CD24(-/low)/CD44(+) cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.

Increasing evidence suggests that mesenchymal stem/stromal cells (MSCs) carrying specific mutations are at the origin of some sarcomas. We have reported that the deficiency of p53 alone or in combination with Rb (Rb(-/-) p53(-/-)) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53(-/-) and Rb(-/-)p53(-/-) MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53(-/-) and Rb(-/-)p53(-/-) ASCs. In addition, gene expression profiling revealed transcriptome similarities between p53- or Rb-p53-deficient BM-MSCs/ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem to determine the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate toward the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors, which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development.

This study analyzed 140 patients with isolated del13q14 on interphase FISH (I-FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment-naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%-96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5-year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50-69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5-year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I-FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I-FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance., (Copyright © 2012 John Wiley & Sons, Ltd.)

The 'activating' E2fs (E2f1-3) are transcription factors that potently induce quiescent cells to divide. Work on cultured fibroblasts suggested they were essential for division, but in vivo analysis in the developing retina and other tissues disproved this notion. The retina, therefore, is an ideal location to assess other in vivo adenovirus E2 promoter binding factor (E2f) functions. It is thought that E2f1 directly induces apoptosis, whereas other activating E2fs only induce death indirectly by upregulating E2f1 expression. Indeed, mouse retinoblastoma (Rb)-null retinal neuron death requires E2f1, but not E2f2 or E2f3. However, we report an entirely distinct mechanism in dying cone photoreceptors. These neurons survive Rb loss, but undergo apoptosis in the cancer-prone retina lacking both Rb and its relative p107. We show that while E2f1 killed Rb/p107 null rod, bipolar and ganglion neurons, E2f2 was required and sufficient for cone death, independent of E2f1 and E2f3. Moreover, whereas E2f1-dependent apoptosis was p53 and p73-independent, E2f2 caused p53-dependent cone death. Our in vivo analysis of cone photoreceptors provides unequivocal proof that E2f-induces apoptosis independent of E2f1, and reveals distinct E2f1- and E2f2-activated death pathways in response to a single tumorigenic insult.

The specific ablation of Rb1 gene in epidermis (Rb(F/F);K14cre) promotes proliferation and altered differentiation but does not produce spontaneous tumour development. These phenotypic changes are associated with increased expression of E2F members and E2F-dependent transcriptional activity. Here, we have focused on the possible dependence on E2F1 gene function. We have generated mice that lack Rb1 in epidermis in an inducible manner (Rb(F/F);K14creER(TM)). These mice are indistinguishable from those lacking pRb in this tissue in a constitutive manner (Rb(F/F);K14cre). In an E2F1-null background (Rb(F/F);K14creER(TM); and E2F1(-/-) mice), the phenotype due to acute Rb1 loss is not ameliorated by E2F1 loss, but rather exacerbated, indicating that pRb functions in epidermis do not rely solely on E2F1. On the other hand, Rb(F/F);K14creER(TM);E2F1(-/-) mice develop spontaneous epidermal tumours of hair follicle origin with high incidence. These tumours, which retain a functional p19(arf)/p53 axis, also show aberrant activation of β-catenin/Wnt pathway. Gene expression studies revealed that these tumours display relevant similarities with specific human tumours. These data demonstrate that the Rb/E2F1 axis exerts essential functions not only in maintaining epidermal homoeostasis, but also in suppressing tumour development in epidermis, and that the disruption of this pathway may induce tumour progression through specific alteration of developmental programs.

Purpose: We have previously demonstrated a hyperplastic phenotype when Rb expression was disrupted within the intestinal epithelium. These findings mimic resection-induced adaptation suggesting a possible mechanistic role for Rb during adaptation. The purpose of the present study was to elucidate a mechanism for how Rb deficiency induces intestinal hyperplasia., Methods: Enterocytes isolated from intestine-specific Rb knockout mice (Rb-IKO) underwent a microarray to elucidate their gene expression profile. IGF2 expression was significantly elevated, which was subsequently confirmed by RT-PCR and in situ mRNA hybridization. Mice with deficient expression of IGF2 or its receptor IGF1R were therefore crossed with Rb-IKO mice to determine the significance of IGF2 in mediating the Rb-IKO intestinal phenotype., Results: Expression of IGF2 was significantly elevated in villus enterocytes of Rb-IKO mice. The mucosal hyperplasia in Rb-IKO mice was reversed when either IGF2 or IGF1R expression was genetically disrupted in Rb-IKO mice., Conclusion: IGF-2 expression is significantly elevated in villus enterocytes and is required for the hyperplastic intestinal mucosal phenotype of Rb-IKO mice. The trophic effects of IGF2 require intact IGF1R signaling within the intestinal epithelium. These findings reveal novel regulatory roles for Rb in expanding intestinal mucosal surface area., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer deaths among women in the United States, but its pathogenesis is poorly understood. Some epithelial cancers are known to occur in transitional zones between two types of epithelium, whereas others have been shown to originate in epithelial tissue stem cells. The stem cell niche of the ovarian surface epithelium (OSE), which is ruptured and regenerates during ovulation, has not yet been defined unequivocally. Here we identify the hilum region of the mouse ovary, the transitional (or junction) area between the OSE, mesothelium and tubal (oviductal) epithelium, as a previously unrecognized stem cell niche of the OSE. We find that cells of the hilum OSE are cycling slowly and express stem and/or progenitor cell markers ALDH1, LGR5, LEF1, CD133 and CK6B. These cells display long-term stem cell properties ex vivo and in vivo, as shown by our serial sphere generation and long-term lineage-tracing assays. Importantly, the hilum cells show increased transformation potential after inactivation of tumour suppressor genes Trp53 and Rb1, whose pathways are altered frequently in the most aggressive and common type of human EOC, high-grade serous adenocarcinoma. Our study supports experimentally the idea that susceptibility of transitional zones to malignant transformation may be explained by the presence of stem cell niches in those areas. Identification of a stem cell niche for the OSE may have important implications for understanding EOC pathogenesis.

The retinoblastoma protein (Rb) family members are essential regulators of cell cycle progression, principally through regulation of the E2f transcription factors. Growing evidence indicates that abnormal cell cycle signals can participate in neuronal death. In this regard, the role of Rb (p105) itself has been controversial. Germline Rb deletion leads to massive neuronal loss, but initial reports argue that death is non-cell autonomous. To more definitively resolve this question, we generated acute murine knock-out models of Rb in terminally differentiated neurons in vitro and in vivo. Surprisingly, we report that acute inactivation of Rb in postmitotic neurons results in ectopic cell cycle protein expression and neuronal loss without concurrent induction of classical E2f-mediated apoptotic genes, such as Apaf1 or Puma. These results suggest that terminally differentiated neurons require Rb for continuous cell cycle repression and survival.

The retinoblastoma gene, RB1, is frequently inactivated in a subset of tumors, including retinoblastoma and osteosarcoma (OS). One characteristic of OS, as well as other tumors in which RB1 is frequently inactivated, is the lack of N-cadherin-mediated cell-cell adhesions. The frequent inactivation of RB1 and parallel loss of N-cadherin expression in OS prompted us to ask whether these observations are directly related to each other. In this study, we observed reduced N-cadherin expression in RB1(-/-) calvarial osteoblasts. In addition, RB1(-/-) cell lines had increased migration potential compared to their RB1(+/+) counterparts. These properties of RB1(-/-) cell lines correlated with an adipogenic potential lacking in RB1(+/+) cell lines, suggesting that each property is present in an immature progenitor cell. The isolation of a cell population with low surface expression of N-cadherin and enhanced adipogenic ability supports this view. Interestingly, the acute loss of pRb does not affect N-cadherin expression or migration or confer adipogenic potential to immortalized RB1(+/+) calvarial cells, suggesting that these traits are not a direct consequence of pRb loss; rather, pRb loss leads to the expansion and immortalization of an immature progenitor pool characterized by these properties.

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 β-galactosidase [SA-β-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, χ(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-β-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-β-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.