Delayed manipulation of regeneration within injured peripheral axons.

While several new translational strategies to enhance regrowth of peripheral axons have been identified, combined approaches with different targets are rare. Moreover, few have been studied after a significant delay when growth programs are already well established and regeneration-related protein expression has waned. Here we study two approaches, Rb1 (Retinoblastoma 1) knockdown that targets overall neuron plasticity, and near nerve insulin acting as a growth factor. Both are validated to boost regrowth only at the outset of regeneration. We show that local delivery of Rb1 siRNA alone, with electroporation to an area of prior sciatic nerve injury generated knockdown of Rb1 mRNA in ipsilateral lumbar dorsal root ganglia. While mice treated with Rb1-targeted siRNA, compared with scrambled control siRNA, starting 2 weeks after the onset of regeneration, had only limited behavioural or electrophysiological benefits, they had enhanced reinnervation of epidermal axons. We next confirmed that intrinsic Rb1 knockdown combined with exogenous insulin had dramatic synergistic impacts on the growth patterns of adult sensory neurons studied in vitro, prompting analysis of a combined approach in vivo. Using an identical delayed post-injury protocol, we noted that added insulin not only augmented epidermal reinnervation rendered by Rb1 knockdown alone but also improved indices of mechanical sensation and motor axon recovery. The findings illustrate that peripheral neurons that are well into attempted regrowth retain their responsiveness to both intrinsic and exogenous approaches that improve their recovery. We also identify a novel local approach to manipulate gene expression and outcome in regrowing axons.

Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 -/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 -/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 -/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 -/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 -/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

Trametinib downregulates survivin expression in RB1-positive KRAS-mutant lung adenocarcinoma cells.

High expression levels of survivin in KRAS-mutant lung adenocarcinomas are linked with unfavorable patient outcomes, suggesting that survivin is a promising target for tumor treatment. We found that trametinib, a MEK inhibitor, downregulates survivin expression in the RB1-positive KRAS-mutant lung adenocarcinoma cell lines H358 and H441. In these cell lines, trametinib treatment induced p21 expression and dephosphorylated RB1, leading to sustained suppression of survivin. Knockdown of p21 or RB1 restored survivin expression in trametinib-treated cells, at least partially, which supports the contribution of these molecules to trametinib-mediated survivin suppression. In RB1-negative KRAS-mutant lung adenocarcinoma H2009 cells, survivin downregulation by trametinib was only slight and transient, and trametinib-resistant (TR) cells developed within 1 month of treatment. H2009 TR cells depended much more on survivin for survival than its parental cells, as evidenced by apoptosis induction when survivin was depleted. These findings collectively suggest that trametinib is effective for the treatment of RB1-positive KRAS-mutant lung adenocarcinomas through sustained survivin suppression, but not for RB1-negative lung adenocarcinomas. Thus, the RB1 status could be a biomarker for trametinib application in KRAS-mutant lung adenocarcinomas.

Gamma-Tocotrienol prevents cell cycle arrest in aged human fibroblast cells through p16INK4a pathway

Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16INK4a pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 μM of palm γ-tocotrienol for 24 hours. Silencing of p16INK4a was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16INK4a were determined by western blot technique. The finding of this study showed that p16INK4a mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (AU)

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γ-Tocotrienol prevents cell cycle arrest in aged human fibroblast cells through p16INK4a pathway.

Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16INK4a pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 µM of palm γ-tocotrienol for 24 hours. Silencing of p16INK4a was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16INK4a were determined by western blot technique. The finding of this study showed that p16INK4a mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions.

miR-590 promotes cell proliferation and invasion in T-cell acute lymphoblastic leukaemia by inhibiting RB1.

MicroRNAs play important roles in the pathogenesis of cancers by inhibiting gene expression at posttranscriptional level. Here, we identified that miR-590 and its predicted target gene RB1 are differentially expressed in T-cell acute lymphoblastic leukaemia (T-ALL). The correlation between miR-590 and RB1 was further confirmed in 395 T-ALL patients. In T-ALL cell lines, miR-590 promoted the cell proliferation by increasing G1/S transition. Moreover, migration and invasion assay showed that miR-590 promotes the migration and invasion of T-ALL cells by increasing E-cadherin and inhibiting MMP-9. Luciferase assays confirmed that miR-590 directly binds to the 3'untranslated region of RB1, and western blotting showed that miR-590 suppresses the expression of RB1 at the protein levels. This study indicated that miR-590 inhibits RB1 and promotes proliferation and invasion of T-ALL cells. Thus, miR-590 may represent a potential therapeutic target for T-ALL intervention.