Rb Target Research Articles

FRMD8 targets both CDK4 activation and RB degradation to suppress colon cancer growth

Cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (RB) are both important cell-cycle regulators that function in different scenarios. Here, we report that FERM domain-containing 8 (FRMD8) inhibits CDK4 activation and stabilizes RB, thereby causing cell-cycle arrest and inhibiting colorectal cancer (CRC) cell growth. FRMD8 interacts separately with CDK7 and CDK4, and it disrupts the interaction of CDK7 with CDK4, subsequently inhibiting CDK4 activation. FRMD8 competes with MDM2 to bind RB and attenuates MDM2-mediated RB degradation. Frmd8 deficiency in mice accelerates azoxymethane/dextran-sodium-sulfate-induced colorectal adenoma formation. The FRMD8 promoter is hypermethylated, and low expression of FRMD8 predicts poor prognosis in CRC patients. Further, we identify an LKCHE-containing FRMD8 peptide that blocks MDM2 binding to RB and stabilizes RB. Combined application of the CDK4 inhibitor and FRMD8 peptide leads to marked suppression of CRC cell growth. Therefore, using an LKCHE-containing peptide to interfere with the MDM2-RB interaction may have therapeutic value in CDK4/6 inhibitor-resistant patients.

Discovery of biomarkers related to abnormal lipid metabolism in liver and serum and intervention mechanism of ginsenoside Rb_1 in hyperlipidemia rats based on non-targeted metabolomics

Through the non-targeted metabolomics study of endogenous substances in the liver and serum of hyperlipidemia rats, the biomarkers related to abnormal lipid metabolism in hyperlipidemia rats were found, and the target of ginsenoside Rb_1 in improving hyperlipidemia was explored and its mechanism was elucidated. The content of serum biochemical indexes of rats in each group was detected by the automatic biochemical analyzer. The metabolite profiles of liver tissues and serum of rats were analyzed by HPLC-MS. Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to compare and analyze the metabolic data in the normal group, the hyperlipidemia group, and the ginsenoside Rb_1 group, and screen potential biomar-kers. The related metabolic pathways were further constructed by KEGG database analysis. The results showed that hyperlipemia induced dyslipidemia in rats, which was alleviated by ginsenoside Rb_1. The non-targeted metabolomics results showed that there were 297 differential metabolites in the liver tissues of hyperlipidemia rats, 294 differential metabolites in the serum samples, and 560 diffe-rential metabolites in the hyperlipidemia rats treated by ginsenoside Rb_1. Perillic acid and N-ornithyl-L-taurine were common metabolites in the liver and serum samples, which could be used as potential biomarkers for ginsenoside Rb_1 in the improvement of hyperlipidemia. As revealed by pathway enrichment in the liver and serum, ginsenoside Rb_1 could participate in the metabolic pathway of choline in both the liver and serum. In addition, ginsenoside Rb_1 also participated in the ABC transporter, alanine, aspartic acid, and glutamate metabolism, protein digestion and absorption, β-alanine metabolism, taurine and hypotaurine metabolism, caffeine metabolism, valine, leucine, and isoleucine biosynthesis, arachidonic acid metabolism, and methionine and cysteine metabolism to improve dyslipidemia in rats.

The oncogenic roles of JC polyomavirus in cancer.

JC polyomavirus (JCPyV) belongs to the human polyomavirus family. Based on alternative splicing, the early region encodes the large and small T antigens, while the late region encodes the capsid structural proteins (VP1, VP2, and VP3) and the agnoprotein. The regulatory transcription factors for JCPyV include Sp1, TCF-4, DDX1, YB-1, LCP-1, Purα, GF-1, and NF-1. JCPyV enters tonsillar tissue through the intake of raw sewage, inhalation of air droplets, or parent-to-child transmission. It persists quiescently in lymphoid and renal tissues during latency. Both TGF-β1 and TNF-α stimulates JCPyV multiplication, while interferon-γ suppresses the process. The distinct distribution of caspid receptors (α-2, 6-linked sialic acid, non-sialylated glycosaminoglycans, and serotonin) determines the infection capabilities of JCPyV virions, and JCPyV entry is mediated by clathrin-mediated endocytosis. In permissive cells, JCPyV undergoes lytic proliferation and causes progressive multifocal leukoencephalopathy, while its DNA is inserted into genomic DNA and leads to carcinogenesis in non-permissive cells. T antigen targets p53, β-catenin, IRS, Rb, TGF-β1, PI3K/Akt and AMPK signal pathways in cancer cells. Intracranial injection of T antigen into animals results in neural tumors, and transgenic mice develop neural tumors, lens tumor, breast cancer, gastric, Vater’s, colorectal and pancreatic cancers, insulinoma, and hepatocellular carcinoma. Additionally, JCPyV DNA and its encoded products can be detected in the brain tissues of PML patients and brain, oral, esophageal, gastric, colorectal, breast, cervical, pancreatic, and hepatocellular cancer tissues. Therefore, JCPyV might represent an etiological risk factor for carcinogenesis and should be evaluated for early prevention, diagnosis, and treatment of cancers.

Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.

Identification of Epigenetically Modified Hub Genes and Altered Pathways Associated With Retinoblastoma.

Retinoblastoma (Rb) is the most common childhood malignancy initiated by biallelic mutation in RB1 gene and driven by various epigenetic events including DNA methylation and microRNA dysregulation. Hence, understanding the key genes that are critically modulated by epigenetic modifications in RB1 −/− cells is very important to identify prominent biomarkers and therapeutic targets of Rb. In this study, we for the first time have integrated various Rb microarray NCBI-GEO datasets including DNA Methylation (GSE57362), miRNA (GSE7072) and mRNA (GSE110811) to comprehensively investigate the epigenetic consequences of RB loss in retinoblastoma tumors and identify genes with the potential to serve as early diagnostic markers and therapeutic targets for Rb. Interestingly, the GEO2R and co-expression network analysis have identified three genes namely E2F3, ESR1, and UNC5D that are significantly deregulated by modified DNA methylation, mRNA and microRNA expression in Rb tumors. Due to their recognition in all epigenetic, transcriptomic, and miRNA datasets, we have termed these genes as “common genes”. The results of our integrative bioinformatics analysis were validated in vitro by studying the gene and protein expression of these common genes in Y79, WERI-Rb-1, Rb cell lines and non-tumorigenic retinal pigment epithelial cell line (hTERT-RPE). The expression of E2F3 and UNC5D were up-regulated and that of ESR1 was down-regulated in Rb tumor cells when compared to that in non-tumorigenic hTERT-RPE cells. More importantly, UNC5D, a potent tumor suppressor gene in most cancers is significantly up-regulated in Y79 and Weri Rb1 cells, which, in turn, questions its anti-cancer properties. Together, our study shows that E2F3, ESR1, and UNC5D may be crucially involved in Rb tumorigenesis and possess the potential to act as early diagnostic biomarkers and therapeutic targets of Rb.

Age-related neurodegeneration and cognitive impairments of NRMT1 knockout mice are preceded by misregulation of RB and abnormal neural stem cell development

N-terminal methylation is an important posttranslational modification that regulates protein/DNA interactions and plays a role in many cellular processes, including DNA damage repair, mitosis, and transcriptional regulation. Our generation of a constitutive knockout mouse for the N-terminal methyltransferase NRMT1 demonstrated its loss results in severe developmental abnormalities and premature aging phenotypes. As premature aging is often accompanied by neurodegeneration, we more specifically examined how NRMT1 loss affects neural pathology and cognitive behaviors. Here we find that Nrmt1−/− mice exhibit postnatal enlargement of the lateral ventricles, age-dependent striatal and hippocampal neurodegeneration, memory impairments, and hyperactivity. These morphological and behavior abnormalities are preceded by alterations in neural stem cell (NSC) development. Early expansion and differentiation of the quiescent NSC pool in Nrmt1−/− mice is followed by its subsequent depletion and many of the resulting neurons remain in the cell cycle and ultimately undergo apoptosis. These cell cycle phenotypes are reminiscent to those seen with loss of the NRMT1 target retinoblastoma protein (RB). Accordingly, we find misregulation of RB phosphorylation and degradation in Nrmt1−/− mice, and significant de-repression of RB target genes involved in cell cycle. We also identify novel de-repression of Noxa, an RB target gene that promotes apoptosis. These data identify Nα-methylation as a novel regulatory modification of RB transcriptional repression during neurogenesis and indicate that NRMT1 and RB work together to promote NSC quiescence and prevent neuronal apoptosis.

Diverging targets mediate the pathological role of miR-199a-5p and miR-199a-3p by promoting cardiac hypertrophy and fibrosis

MicroRNA-199a-5p (miR-199a-5p) and -3p are enriched in the myocardium, but it is unknown whether miR-199a-5p and -3p are co-expressed in cardiac remodeling and what roles they have in cardiac hypertrophy and fibrosis. We show that miR-199a-5p and -3p are co-upregulated in the mouse and human myocardium with cardiac remodeling and in Ang-II-treated neonatal mouse ventricular cardiomyocytes (NMVCs) and cardiac fibroblasts (CFs). miR-199a-5p and -3p could aggravate cardiac hypertrophy and fibrosis in vivo and in vitro. PPAR gamma coactivator 1 alpha (Ppargc1a) and sirtuin 1 (Sirt1) were identified as target genes to mediate miR-199a-5p in promoting both cardiac hypertrophy and fibrosis. However, miR-199a-3p aggravated cardiac hypertrophy and fibrosis through targeting RB transcriptional corepressor 1 (Rb1) and Smad1, respectively. Serum response factor and nuclear factor κB p65 participated in the upregulation of miR-199a-5p and -3p in Ang-II-treated NMVCs and mouse CFs, and could be conversely elevated by miR-199a-5p and -3p. Together, Ppargc1a and Sirt1, Rb1 and Smad1 mediated the pathological effect of miR-199a-5p and -3p by promoting cardiac hypertrophy and fibrosis, respectively. This study suggests a possible new strategy for cardiac remodeling therapy by inhibiting miR-199a-5p and -3p.

Abstract 5295: Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance

Abstract The Retinoblastoma (Rb) tumor suppressor protein plays a role in the control of proliferation, apoptosis, differentiation, invasion and the maintenance of genome stability. Its activity is regulated by phosphorylation on several serine/threonine sites, which is thought to inactivate its tumor suppressive function. Cyclin dependent kinases (CDKs) are responsible for most of the Rb phosphorylation that occurs in cells. It is well established that alterations in the Rb pathway (consisting of CDK4/6, D-type cyclins, the CDK inhibitor p16INK4a, and Rb itself) that lead to excessive phosphorylation of Rb have been observed in almost all cancer types. Alterations that lead to increased CDK activity toward Rb are more common than mutations to the Rb gene itself, a finding that prompted investigations into the development of clinical treatments that inhibit the phosphorylation of Rb by CDKs. The CDK4/6 inhibitors palbociclib and abemaciclib were recently clinically approved for use in breast cancer. However, acquired resistance to CDK4/6 inhibition has been shown to limit clinical efficiency. Targeting Rb phosphorylation by CDK4/6 inhibition has been shown to activate alternate pro-survival pathways such as the AKT pathway. In our studies we developed a method of siRNA-mediated activation of Rb phosphatase that targets Rb phosphorylation in attempt to circumvent the development of resistance. Phosphatase Nuclear Targeting Subunit (PNUTS) associates with and regulates PP1 activity toward Rb. In several cancer cell types, such as HCT116 colon cancer, MIA PaCa-2 pancreatic cancer and MCF7 breast cancer, PNUTS depletion causes dephosphorylation of Rb without AKT activation. Furthermore, in Panc1 pancreatic cancer cells, activation of AKT is observed after 1-hour palbociclib (500nM) treatment, whereas PNUTS depletion does not induce AKT. In MD-MBA-231 breast cancer cells, 1-hour treatment of palbociclib or abemaciclib (20nM) induces the AKT pathway as well as the STAT pathways, however PNUTS depletion in these cells does not induce AKT activation. It has been shown that CDK4/6 inhibitors activate AKT by causing loss of Rb interaction with SIN1, a component the mTORC2 complex. Our studies reveal that PNUTS depletion causes dephosphorylation of Rb, without disrupting Rb-mediated inhibition of mTORC2. Thus, the stimulation of Rb phosphatase activity may target Rb phosphorylation without the development of resistance. Citation Format: Kathleen H. DiSalvo, Brian C. Velez, Rebecca L. Kravtsov, Nancy A. Krucher. Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5295.

Proteogenomic Analysis of Human Colon Cancer Reveals New Therapeutic Opportunities

We performed the first proteogenomic study on aprospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis ofpaired tumor and normal adjacent tissues produceda catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colontumors and suggests a rationalefor targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 Tcell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors,suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpointblockade. Proteogenomics presents new avenuesfor biological discoveries and therapeutic development.

A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family.

The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO’s capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity.

Comparative Assessment of the Effects of Soil Carbon and Nitrogen Amendment on Subsurface Soil pH, Eh and Sulfate Content of Acid Sulfate Soils

The harmful impacts of acid sulfate soils (ASS) on various ecosystems are diverse and management of the impacts needs ecosystem-specific strategies. The importance of subsurface soil carbon and nitrogen for amelioration of sulfuric soil and curtailing of sulfidic soil oxidation as an option for ASS management was investigated in this study. The results showed the mechanisms involved in amelioration of sulfuric soil and curtailing of sulfidic soil oxidation were dependent on amendment and the microbial ecology the resources were capable of supporting microbial metabolism. Glucose as a single nutrient source capable of engaging a few soil microbes was ineffective in ameliorating the sulfuric soil, compared to complex organic matter, which is a source of multiple nutrients and is capable of engaging a diversity of soil microbes. Addition of complex organic matter effectively ameliorated the sulfuric soil and prevented sulfidic soil oxidation. The alkalization processes was limited by resource availability and the processes ceased as resources got depleted. Polynucleotide analysis of at least one group of sulfate reducing bacteria (SRB, Desulfovibrio) showed that the concentrations of these polynucleotides is high when availability of resources is high and decreases as the resource is limited over time. A three-fold increase in RNA and a ten-fold increase in DNA concentrations of the target SRB were measured in soil amended with organic matter and incubated for four weeks; after six weeks, they decreased.

Abstract 5483: Investigating the role of the retinoblastoma protein in induction and maintenance of oncogene-induced cellular senescence in tumor suppression

Abstract Oncogene-induced cellular senescence is a tumor suppressor response resulting in irreversible cell cycle exit, and is dependent on the p53 and RB pathways. Loss of p53 has recently been shown to allow re-entry of senescent cells into the cell cycle. The role of the RB1 protein in maintenance of the senescent state is less well studied. We investigated whether RB1 is necessary for the induction as well as the maintenance of oncogene-induced senescence, and we also evaluated whether RB1 activation is sufficient to induce and/or maintain senesce in the absence of a functional p53 pathway. Inactivation of RB1 prior to onset of senescence led to failure of senescence induction, with impaired cell cycle exit and failure to induce senescence markers. On the other hand, loss of RB1 after cells had already entered oncogene-induced senescence was able to reverse some features of the senescence phenotype, but did not result in successful cell division and accumulation. Interestingly, in the absence of p53, constitutively active RB1 was sufficient for cells to undergo oncogene-induced senescence, and effectively prevented colony formation. Finally, while p53 inactivation was sufficient for senescent cells to re-enter the cell cycle, constitutive activation of RB1 prevented cell cycle re-entry, even in the absence of p53. Our findings show that RB1 is necessary for both the induction and maintenance of oncogene-induced senescence, and that active RB is sufficient for senescence induction as well as its maintenance, both in the presence and absence of p53. These results have direct implications on targeting RB activation as a senescence-inducing approach in both p53 wild type and p53-defective premalignant and malignant tumors. Note: This abstract was not presented at the meeting. Citation Format: Mohamad Harajly, Hasan Zalzali, Farah Ghamloush, Raya H. Saab. Investigating the role of the retinoblastoma protein in induction and maintenance of oncogene-induced cellular senescence in tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5483. doi:10.1158/1538-7445.AM2017-5483

Abstract P6-09-53: Targeting RB loss for the precision treatment of triple negative breast cancer

Abstract This abstract was withdrawn by the authors.

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.

X-Ray Mass Attenuation Coefficient of Medicinal Plant Using Different Energies 32.890KeV to 13.596KeV

To study the X-ray Mass attenuation coefficients of different materials and medicinal plants is necessary for accurate X-ray fluorescent analysis. The present measurement X-ray mass attenuation coefficients held in to two types of medicinal plants which are collected from Gulbarga and Bidar districts, using different X-ray energy range 32.890 KeV to 13.596 KeV. The Measurement performed using NaI (TI) detector which coupled to MCA and a source of 241Am is used to get X-rays in the energies of Ba, Ag, Mo, and Rb target in to different thickness (40 mg to 100 mg) pellet samples of Eucalyptus Obliqua and Mangifera indica medicinal plants. These two medicinal plants are used as medicines in the Indian herbal systems. The theoretical values are compared with the experimental values obtained using WinXCOM, which is personal computer software package. In this present study the mass attenuation coefficient of x-ray increases with decreasing energies.

Serine 220 phosphorylation of the Merkel cell polyomavirus large T antigen crucially supports growth of Merkel cell carcinoma cells.

Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT(S220A) demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches.

MicroRNA-188 suppresses G1/S transition by targeting multiple cyclin/CDK complexes.

BackgroundAccelerated cell cycle progression is the common feature of most cancers. MiRNAs can act as oncogenes or tumor suppressors by directly modulating cell cycle machinery. It has been shown that miR-188 is upregulated in UVB-irradiated mouse skin and human nasopharyngeal carcinoma CNE cells under hypoxic stress. However, little is known about the function of miR-188 in cell proliferation and growth control.ResultsOverexpression of miR-188 inhibits cell proliferation, tumor colony formation and G1/S cell cycle transition in human nasopharyngeal carcinoma CNE cells. Using bioinformatics approach, we identify a series of genes regulating G1/S transition as putative miR-188 targets. MiR-188 inhibits both mRNA and protein expression of CCND1, CCND3, CCNE1, CCNA2, CDK4 and CDK2, suppresses Rb phosphorylation and downregulates E2F transcriptional activity. The expression level of miR-188 also inversely correlates with the expression of miR-188 targets in human nasopharyngeal carcinoma (NPC) tissues. Moreover, studies in xenograft mouse model reveal that miR-188 is capable of inhibiting tumor initiation and progression by suppressing target genes expression and Rb phosphorylation.ConclusionsThis study demonstrates that miR-188 exerts anticancer effects, via downregulation of multiple G1/S related cyclin/CDKs and Rb/E2F signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0066-6) contains supplementary material, which is available to authorized users.

Investigation of key miRNAs and target genes in bladder cancer using miRNA profiling and bioinformatic tools.

Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs' regulatory networks. In this study, we aimed to construct potential networks of bladder-cancer-related miRNAs and their known target genes using miRNA expression profiling and bioinformatics tools and to investigate potential key molecules that might play roles in bladder cancer regulatory networks. Global miRNA expression profiles were obtained using microarray followed by RT-qPCR validation using two randomly selected miRNAs. Known targets of deregulated miRNAs were utilized using DIANA-TarBase database v6.0. The incorporation of deregulated miRNAs and target genes into KEGG pathways were utilized using DIANA-mirPath software. To construct potential miRNA regulatory networks, the overlapping parts of three selected KEGG pathways were visualized by Cytoscape software. We finally gained 19 deregulated miRNAs, including 5 ups- and 14 down regulated in 27 bladder-cancer tissue samples and 8 normal urothelial tissue samples. The enrichment results of deregulated miRNAs and known target genes showed that most pathways were related to cancer or cell signaling pathways. We determined the hub CDK6, BCL2, E2F3, PTEN, MYC, RB, and ERBB3 target genes and hub hsa-let-7c, hsa-miR-195-5p, hsa-miR-141-3p, hsa-miR-26a-5p, hsa-miR-23b-3p, and hsa-miR-125b-5p miRNAs of the constructed networks. These findings provide new insights into the bladder cancer regulatory networks and give us a hypothesis that hsa-let-7c, hsa-miR-195-5p, and hsa-miR-125b-5p, along with CDK4 and CDK6 genes might exist in the same bladder cancer pathway. Particularly, hub miRNAs and genes might be potential biomarkers for bladder cancer clinics.

P16 in HPV-associated cancers

Infections with high-risk human papillomaviruses (HPV) account for approximately 5% of all human cancers. Cervical cancer, in particular, is almost universally caused by “high-risk” HPV infections and these HPVs also cause a large fraction of other anogenital tract cancers as well as oral carcinomas. The viral E6 and E7 proteins are the universal oncogenic drivers of HPV-associated cancers, and persistent E6/E7 expression is necessary for the maintenance of the transformed state. Hence the HPV E6 and E7 proteins should represent excellent therapeutic targets. The HPV E6 and E7 proteins lack intrinsic enzymatic activities and attempts to discover small molecule inhibitors of E6 or E7 containing cellular protein complexes have been futile. HPV E6 and E7 function by associating with host cellular proteins, thereby functionally re-programming cellular signal transduction pathways. The resulting rewiring of cellular circuitry causes “addictions” to certain signaling pathways that are non-essential and/or redundant in normal cells. This in turn generates specific cellular vulnerabilities that may be more readily druggable. The article (McLaughlin-Drubin ME et al., Proc. Natl. Acad. Sci. USA. 2013; 110:16175-16180) identifies a surprising and potentially druggable vulnerability in the retinoblastoma tumor suppressor pathway caused by the high-risk HPV E7 protein. High-risk HPV E7 protein expression triggers a cellular defense response referred to as oncogene induced senescence (OIS), which is mediated by the p16INK4A (p16) tumor suppressor and executed by the retinoblastoma tumor suppressor protein, Rb. In normal cells, p16 expression is epigenetically silenced by polycomb repressive complexes. p16 inhibits cyclin dependent kinase 4 and 6 (CDK4/6). This causes accumulation of hypophosphorylated Rb, which in turn signals cell cycle arrest (Fig. ​(Fig.11 upper panel). E7 protein triggers p16. High-level p16 expression is an excellent biomarker for high-risk HPV-associated lesions and cancers. Cell cycle arrest and senescence in these tumors, however, is subverted by E7 targeting Rb for proteasomal degradation (Fig. ​(Fig.11 lower panel). p16 induction, in the absence of Rb (through Rb degradation by E7) causes a unique and surprising addiction to p16INK4A expression. Even though p16 is a tumor suppressor in most cellular contexts, it is essential for survival of cervical carcinoma lines. The iconoclastic “oncogenic” activity of p16 in HPV E7 expressing cells, like its more familiar tumor suppressor activity, is based on the ability to inhibit CDK4/6. Indeed, a human melanoma derived p16-insensitive CDK4 mutant also causes cell death when expressed in HPV E7-expressing cells. Hence, this oncogenic CDK4 mutant exhibits biological activities that are akin to those of a tumor suppressor in the context of a p16-expressing, Rb defective cell. These results imply that CDK4/6 activity is not tolerated in cells that lack Rb activity. One may hypothesize that there must be CDK4/6 substrates that cause cell death when they are phosphorylated in cells that lack Rb. Tumorigenic activities of the Rb pathway including CDK4/6 and p16 are context dependent. CDK4/CDK6 inhibition may be efficacious only in tumors that retain functional Rb. In contrast, CDK4/CDK6 inhibition in cancers that have suffered Rb mutations is necessary for tumor growth and survival. In such cases it may be therapeutically useful to activate rather than to inhibit CDK4/CDK6 activity. One way CDK4/6 activation may be achieved is by epigenetic silencing of p16 through inhibition of the KDM6B enzyme. Indeed the KDM6-selective small molecule inhibitor GSK-J4 induced cell death specifically in KDM6B/p16-addicted cell lines. This shows that the addiction of HPV-associated lesions and cancer to p16 expression creates a cellular vulnerability to KDM6B inhibition. De-repression of polycomb-silenced genes, such as p16, is a complex, multistep process. Hence, “epigenetic therapies” targeting KDM6B and potentially other enzymes that may also be rate limiting for maintaining the transcriptional competence of the p16 promoter may be efficacious in p16-addicted cancers. Figure 1 The retinoblastoma (Rb) tumor suppressor pathway It has been noted early on that p16 expression is confined to those tumors that contain pRB mutations, and a number of non HPV-associated cancer types, including some breast, prostate, lung and high-grade serous ovarian carcinomas express p16. It will be important to determine whether some of these tumors are similarly addicted to p16 expression and whether they are vulnerable to inhibition of KDM6B and/or other epigenetic enzymes that are necessary for p16 expression.

The role of micro RNAs let7c, 100 and 218 expression and their target RAS, C-MYC, BUB1, RB, SMARCA5, LAMB3 and Ki-67 in prostate cancer

OBJECTIVE:The aim of this study is to verify the expression of proteins that are controlled by miR-let7c, 100 and 218 using immunohistochemistry in tissue microarray representative of localized and metastasized the lymph nodes and bone prostate cancer.METHODS:To verify the expression of proteins that are controlled by miR-let7c (C-MYC, BUB1, RAS) 100 (SMARCA5, RB) and 218 (LAMB3) and cell proliferation (Ki-67) we used immunohistochemistry and computerized image system ImageJ MacBiophotonics in three tissue microarrays representative of localized prostate cancer and lymph node and bone metastases. miRNA expression was evaluated by qRT-PCR using 60 paraffin blocks to construct the tissue microarray representative of localized disease.RESULTS:RAS expression was increased in localized prostate cancer and bone metastases compared to the lymph nodes (p = 0.017). RB showed an increase in expression from localized prostate cancer to lymph node and bone metastasis (p = 0.036). LAMB3 was highly expressed in localized and lymph node metastases (p<0.001). Cell proliferation evaluated by Ki-67 showed an increase from localized prostate cancer to metastases (p<0.001). We did not found any relationship between C-MYC (p = 0.253), BUB1 (p = 0.649) and SMARCA5 (p = 0.315) protein expression with prognosis or tumor behavior.CONCLUSION:We found that the expression of RAS, RB, LAMB3 and Ki-67 changed in the different stages of prostate cancer. Furthermore, we confirmed the overexpression of the miRNAs let7c, 100 and 218 in localized prostate cancer but failed to show the control of protein expression by the putative controller miRNAs using immunohistochemistry.