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Targeting Notch signaling as a novel therapy for retinoblastoma.

ABSTRACT: Retinoblastoma is the most common intraocular malignancy of childhood. Notch plays a key role in retinal cells from which retinoblastomas arise, and we therefore studied the role of Notch signaling in promoting retinoblastoma proliferation. Moderate or strong nuclear expression of Hes1 was found in 10 of 11 human retinoblastoma samples analyzed immunohistochemically, supporting a role for Notch in retinoblastoma growth. Notch pathway components were present in WERI Rb1 and Y79 retinoblastoma lines, with Jag2 and DLL4 more highly expressed than other ligands, and Notch1 and Notch2 more abundant than Notch3. The cleaved/active form of Notch1 was detectable in both lines. Inhibition of the pathway, achieved using a ?-secretase inhibitor (GSI) or by downregulating Jag2, DLL4 or CBF1 using short hairpin RNA, potently reduced growth, proliferation and clonogenicity in both lines. Upregulation of CXCR4 and CXCR7 and downregulation of PI3KC2? were identified by microarray upon Jag2 suppression. The functional importance of PI3KC2? was confirmed using shRNA. Synergy was found by combining GSI with Melphalan at their IC50. These findings indicate that Notch pathway is active in WERI Rb1 and Y79, and in most human retinoblastoma samples, and suggest that Notch antagonists may represent a new approach to more effectively treat retinoblastoma.

SUBMITTER: Asnaghi L

PROVIDER: S-EPMC5342532 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Targeting Notch signaling as a novel therapy for retinoblastoma.

Asnaghi Laura L Tripathy Arushi A Yang Qian Q Kaur Harpreet H Hanaford Allison A Yu Wayne W Eberhart Charles G CG

Oncotarget 20161001 43

Retinoblastoma is the most common intraocular malignancy of childhood. Notch plays a key role in retinal cells from which retinoblastomas arise, and we therefore studied the role of Notch signaling in promoting retinoblastoma proliferation. Moderate or strong nuclear expression of Hes1 was found in 10 of 11 human retinoblastoma samples analyzed immunohistochemically, supporting a role for Notch in retinoblastoma growth. Notch pathway components were present in WERI Rb1 and Y79 retinoblastoma lin ...[more]

PMID: 27661116

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Project description:Retinoblastoma (RB) is one of the most common forms of childhood intraocular cancer. While the occurrence of RB is traditionally associated with dysregulation of the RB1 gene, efforts have been made to assess the role of several other pathways that may result in RB. The Notch signaling pathway has been identified as one of the sentinel pathways in retinal development and has been indicated to serve as a tumor suppressor. However, epigenetic modifications of the Notch signaling pathway, and their consequences on tumor establishment and progression, have received little attention. The present study attempted to elucidate the microRNA (miR)-mediated dysregulation of the Notch signaling pathway and its implications on tumor initiation. Upon recruitment of patients with RB (age, 4-25 months), the levels of miR-34b-5p were determined in tumor and adjacent healthy tissues. Simultaneously, the serum levels of miR-34b-5p were measured in tumor and healthy samples using reverse transcriptase-quantitative PCR (RT-qPCR). Binding of miR-34b-5p to Notch1 and Notch2 were confirmed bioinformatically. In vitro studies were performed in Y79 and Weri-Rb-1 RB cell lines. The cell lines were transfected with miR-34b-5p constructs and miR-34b-5p overexpression was confirmed using RT-qPCR. The impact of miR-34b-5p overexpression on cell growth and cancer stemness markers (Sox-2, Nanog, and CD133) was examined. The expression levels of Notch1 and Notch2 were evaluated in the presence of miR-34b-5p. The rescue of cell growth and cancer stemness phenotypes was evaluated by co-transfection of miR-34b-5p with Notch1 or Notch2. The results of the present study indicated that the expression levels of miR-34b-5p were reduced in patient tissues and serum samples compared with those in healthy tissues and samples. Notch1 and Notch2 expression level was negatively correlated with the expression level of miR-34b-5p. Overexpression of miR-34b-5p resulted in reduced cell proliferation, migration, invasion and cancer stemness compared with the control group. Further in vivo experiments confirmed the inhibitory effects of miR-34b-5p on RB cell proliferation. Upon co-transfection of miR-34b-5p with Notch1 or Notch2, these phenotypes were rescued with reversal of cell growth and tumor sphere formation. Collectively, the results indicated that miR-34b-5p functions as a tumor suppressor in RB via regulating the Notch signaling pathway. Therefore, miR-34b-5p may be explored for its utility as a therapeutic target in RB.

Project description:RATIONALE:The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. OBJECTIVE:The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. METHODS AND RESULTS:Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)-deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. CONCLUSIONS:These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

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Targeting Notch signaling as a novel therapy for retinoblastoma.

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Targeting Notch signaling as a novel therapy for retinoblastoma.

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