Numb Expression Research Articles

Hsa_circ_0058124 promotes papillary thyroid cancer tumorigenesis and invasiveness through the NOTCH3/GATAD2A axis

BackgroundDespite a good and overall prognosis, papillary thyroid cancer (PTC) can still affect the quality of life of many patients, and can even be life-threatening due to its invasiveness and metastasis. Emerging studies demonstrate that circular RNAs (circRNAs) participate in the regulation of various cancers. However, the circRNA profile in invasive PTC is still not well understood.MethodsCompeting endogenous RNA (ceRNA) microarrays were performed to determine circRNAs contributed to the tumorigenesis and invasiveness of PTC. Bioinformatics methods were used to narrow down the candidate circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) assays revealed a significant upregulation of hsa_circ_0058124 in PTC tissue and a close correlation with a poor prognosis for PTC patients. RNA fluorescence in situ hybridization and Cell fractionation assay were used to investigate the subcellular location of hsa_circ_0058124. Then, we examined the functions of hsa_circ_0058124 in PTC by cell proliferation, cell cycle, apoptosis, migration and invasion assay. Mechanistically, RNA sequencing and GSEA analysis were applied to predict the downstream pathway of hsa_circ_0058124. Dual-luciferase report assays were used to explore the potential miRNA sponge role of hsa_circ_0058124. Western blotting, cell proliferation, cell cycle, cell apoptosis, migration and invasion, and mouse xenograft assay were used to validate the effects of hsa_circ_0058124/NOTCH3/GATAD2A axis on PTC progression.ResultsIn the current study, a novel hsa_circ_0058124 on 2q35 was identified and explored in PTC. Hsa_circ_0058124 is associated with the malignant features and poor outcomes of PTC patients. Hsa_circ_0058124 acts as an oncogenic driver that promotes PTC cell proliferation, tumorigenicity, tumor invasion, and metastasis, which functions as a competing endogenous RNA to modulate miRNA-218-5p and its target gene NUMB expression, and consequently with repression of the NOTCH3/GATAD2A signaling axis in vitro and in vivo.ConclusionsThis study unveils a novel biomarker panel consisting of the hsa_circ_0058124/NOTCH3/GATAD2A axis which is critical for PTC tumorigenesis and invasiveness and may represent a novel therapeutic target for intervening in PTC progression.

Inhibition of the Numb/Notch signaling pathway increases radiation sensitivity in human nasopharyngeal carcinoma cells.

Radiation therapy is the primary treatment for primary nasopharyngeal carcinoma (NPC). The aim of this study is to identify the effect of the Numb/Notch signaling pathway on radiation sensitivity in human NPC cells. NPC tissues and normal nasopharyngeal tissues were collected. To evaluate the regulatory effects of the Numb/Notch signaling pathway, NPC cells were subjected to radiotherapy and various doses of the Numb/Notch signaling pathway inhibitor gamma secretase inhibitor (GSI). Next, the expression of Notch and Numb proteins was determined in NPC tissues and normal nasopharyngeal tissues, and the correlation of Notch and Numb protein expression with the clinicopathological features of NPC tissues was analyzed. Then, the effect of radiotherapy on NPC cell survival rate, survival fraction, apoptosis rate, proliferation, migration, and invasion as well as Numb/Notch signaling pathway-related molecules was detected. The results demonstrated that the Numb/Notch signaling pathway was activated in NPC tissues. Following treatment with radiotherapy and GSI, the Numb/Notch signaling pathway was inhibited. In addition, the NPC cell survival rate, survival fraction, cell proliferation, migration, and invasion were decreased, whereas the colony number and apoptosis rate were increased. Following radiotherapy and GSI treatment, Numb expression was increased, whereas Notch1, Hes1, Jagged1, and c-Myc expression was decreased. However, the greatest difference was noted upon treatment with radiotherapy +15 μM GSI. The results reported in this study suggest that a high dose of the inhibitor of the Numb/Notch signaling pathway GSI increased the radiation sensitivity in human NPC cells.

Abstract 859: Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines

Abstract Background: Notch pathway has been involved in cell differentiation, proliferation, apoptosis, angiogenesis and drug resistance, as well as in epithelial-to-mesenchymal transition. Notch pathway activation leads to a proteolytic cleavage, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and activates target genes, such as HES1 (interacting with Notch for maintenance of stem cells and progenitor phenotype). Our team has demonstrated the involvement of PlGF/VEGFR1/Notch4 axis in the angiogenesis of hepatocellular carcinoma, but what about the role of Notch in other cancer types? The aim of this work is to characterize the basal activation and the role of Notch4 in a panel of human cancer cell lines. Materials and Methods: We characterized a panel of 8 pancreatic (PDAC), 8 head and neck (H&N), 5 colorectal (CRC), 5 cholangiocarcinoma (CK), and 10 hepatocellular (HCC) human carcinoma cell lines for intracellular domain of the notch protein 4 (NICD4), HES1, E-cadherin, and Vimentin expression by Western Blot. In each tumor type, cell lines with high and low Notch4 activation were selected to assess basal cell proliferation and migration, using MTT and wound-healing assay, respectively. In high versus low Notch4 cell lines, we assessed NUMB expression (involved in NICD proteasomal degradation) and the effect of a Notch inhibitor, PF- 03084014. Results: Notch4 activation assessed by NICD4 expression was observed in 6 out of 8 (6/8) PDAC, 7/8 H&N, 2/5 CRC, 4/5 CK, and 5/10 HCC cell lines. In PDAC cells, Notch4 activation was correlated with high Vimentin expression. In PDAC-, H&N-, CRC-, and CK-low NICD4 cells, we observed an overexpression of NUMB, suggesting a correlation between low Notch4 activation and proteasomal degradation. In most tumor types, Notch4 activation was associated with an increased proliferation rate and basal migration. PF-03084014 had pronounced antiproliferative effects in high NICD4 cells in comparison to low NICD4 cells, except for HCC. In H&N cells, PF-03084014 displayed no effect on Notch4 activation. We also observed a decrease in HES1 expression (significant in high NICD4 cells) and an increased in AKT phosphorylation (significant in low NICD4 cells). We will further analyze the effect of PF-03084014 on cell signaling and migration in other tumor types to display the results at the conference. Conclusions: In this study, we demonstrated a correlation between high Notch4 basal activation and increased proliferation/migration in a panel of human cancer cell lines, as well as higher sensitivity to the Notch inhibitor PF-03084014. In highly sensitive H&N cell lines, PF-03084014 inhibited Notch signaling pathway, whereas we observed an activation of AKT survival pathway in the low-sensitive ones. Since Notch inhibition is an interesting topic for antitumor therapy, this study could help to select tumor types that could be good candidate for Notch inhibition in the clinic. Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Sandrine Faivre, Annemilaï Tijeras-Raballand. Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines [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 859.

Abstract 1164: Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines

Abstract TCGA genomic study in papillary thyroid carcinoma (PTC) samples highlights two groups of tumors: BRAF-like and RAS-like, whose genotype might influence its biological behavior. The mechanisms behind progressive loss of thyroid differentiation, observed mainly in the most aggressive histotype anaplastic thyroid carcinoma (ATC), could be associated with the reactivation of embryonic signaling and acquisition of stem cell-like phenotype. In this study we focused on Notch and Nodal embryonic signaling pathways, which are crucial for stemness maintenance in embryonic cells and has also been linked to cancer cell stemness. By assessing the expression of Notch and Nodal pathway components in BRAF- and RAS-like cell lines, we sought to investigate if the reactivation of these pathways is involved in dedifferentiation mechanisms observed in ATC. For that purpose, RNA and protein were extracted from three PTC (TPC-1, BCPAP and K1), six ATC (KTC2, SW1736, C643, Hth7, Hth74 and Hth83) and non-tumoral (Nthy-ori 3-1) cell lines. For gene expression analysis, cDNA generate from mRNA was amplified by qPCR using specific primers for NOTCH1, NOTCH4, HES1, NUMB, CER1 and LEFTY1/2. For protein expression analysis, SDS-PAGE-fractionated proteins were transferred onto nitrocellulose membrane, incubated with anti-Nodal antibody and visualized by chemoluminescence. NOTCH1 was downregulated in BRAF-like PTC cell lines, while slightly overexpressed in RET/PTC-containing TPC-1. On the other hand, NOTCH1 was overexpressed in Hth83 and C643 ATC lines, both HRAS-mutated, and also in BRAF-mutated SW1736. NOTCH4 was overexpressed in PI3K-mutated K1 and all ATC cell lines. Transcription repressor HES1, which prevents cell cycle arrest by inhibiting p27, was highly expressed in all cell lines compared to Nthy. Levels of NUMB expression were elevated in all ATC cell lines with no difference between BRAF- and RAS-mutated, but were inversely related to those of HES1, as expected for a Notch pathway inhibitor. CER1 and LEFTY1/2, which encode Nodal inhibitors, were highly expressed in ATC cell lines (4/6 and 5/6, respectively) but with no apparent distinction between BRAF- and RAS-mutated cells. At the same time, Nodal protein levels are only upregulated in TP53-mutated BCPAP and C643, suggesting that alterations in MAPK components are not sufficient to promote Nodal overexpression. Our results show re-expression of Notch embryonic signaling components in both PTC and ATC cell lines with either BRAF or RAS oncogenes. Moreover, additional genetic alterations such as in PI3K and TP53 might also interfere with the expression of both Notch and Nodal components. We therefore highlight the importance of tumor genotype, which might determine the modulation of embryonic-related genes and therefore contribute to the undifferentiated phenotype in thyroid cancer. Financial support: FAPESP and CNPq. Citation Format: Paola M. Dantonio, Cesar Seigi Fuziwara, Kelly C. Saito, Edna T. Kimura. Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines [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 1164.

HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells

ABSTRACTGlioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2–3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth.We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-β and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway.Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.

RETRACTED ARTICLE: Numb inhibits epithelial-mesenchymal transition via RBP-Jκ-dependent Notch1/PTEN/FAK signaling pathway in tongue cancer

BackgroundOral cancer has been estimated as the sixth most frequent solid cancer all over the world, in which tongue squamous cell carcinoma (TSCC) is the most common type of oral cancers. However, the mechanism of TSCC metastasizing to lymph node and distant sites has not been completely understood.MethodsIn this study, RT-qPCR method was used to detect the mRNA level of Numb, PTEN and Notch1 genes, as well as EMT-associated genes. Western blot assay was utilized to detect protein level of these genes. In addition, we determined cell proliferation by MTT assay and employed transwell invasion assay and wound healing assay to probe the abilities of invasion and migration, respectively. To investigate the role of PTEN, its inhibitor VO-Ohpic trihydrate was used to treat SCC-4 and CAL27 cells.ResultsWe found that Numb expression was downregulated in SCC-9 and CAL-27 cells compared to NHOK cells. Instead, Notch1 level in SCC-9 and CAL-27 cells were higher than that in NHOK cells. Furthermore, the results showed that Numb overexpression significantly suppressed proliferation, migration and invasion of SCC-9 and CAL-27 cells via regulating Notch1 signaling and EMT-related genes expression. By contrast, we observed that RBP-Jκ knockdown had an inhibitory role in proliferation, migration and invasion of SCC-9 and CAL-27 cells. In cells with Numb overexpression or RBP-Jκ knockdown, p-FAK and EMT-related genes were remarkably regulated.ConclusionsOur findings provide new mechanism of understanding the metastasis of TSCC and help develop therapeutic strategies for treating tongue cancer.

Numb inhibits cell proliferation, invasion, and epithelial-mesenchymal transition through PAK1/β-catenin signaling pathway in ovarian cancer.

ObjectiveThe present study aimed to investigate the expression of Numb in ovarian cancer tissues and to assess the effect of Numb on cell proliferation, invasion, and EMT in ovarian cancer.MethodsReal-time PCR and Western blotting were used to detect the mRNA and protein expression of Numb, PAK1, β-catenin, and epithelial–mesenchymal transition (EMT)-related proteins. MTT was employed to check the effect of Numb on proliferation of ovarian cancer cells. Transwell assay was performed to examine the functions of Numb and PAK1 on migration and invasion of ovarian cancer cells.ResultsThe Numb expression was significantly downregulated while PAK1 and β-catenin were significantly upregulated in both ovarian cancer tissues and cell lines. Silencing of Numb promoted cell proliferation, migration, invasion, and EMT in ovarian cancer cell lines while overexpressed Numb reversed the above effects. Moreover, the EMT process induced by the inhibition of Numb was regulated through Numb-mediated PAK1/β-catenin signaling pathway.ConclusionNumb was downregulated and associated with cell proliferation, invasion, and EMT in ovarian cancer through regulating PAK1/β-catenin signaling, providing a novel potential biomarker and potential therapeutic target for ovarian cancer.

NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian cancer cells by inhibiting cell proliferation and epithelial-mesenchymal transition.

BackgroundNUMB is an inhibitory regulator of NOTCH signaling, which is critical for the induction of epithelial-mesenchymal transition (EMT). Loss of NUMB expression is correlated with the genesis and development of multiple tumors. Recent studies reported that NUMB expression was upregulated in human ovarian cancer. However, the role of NUMB in ovarian cancer is still unclear. Here, we invested the effect of NUMB knockdown on the proliferation and EMT in ovarian cancer cells and explored the role of NUMB in the effect of cisplatin.MethodsTwo ovarian cancer cells (OVCAR-3 and SK-OV-3) were used in the experiments. The proliferation and apoptosis of ovarian cancer cells was examined using methyl thiazolyl tetrazolium (MTT) test and flow cytometry assays. The invasion and migration of ovarian cancer cells were examined using Transwell assays. The expression of EMT markers were examined using Simple Western analysis.ResultsNUMB knockdown inhibited cell proliferation, invasion, and migration in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell growth inhibiting and apoptosis in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell invasion in SK-OV-3 cells. NUMB knockdown also decreased the expression of N-cadherin and Vimentin in SK-OV-3 cells.ConclusionsNUMB acted as an oncogene in ovarian cancer and NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian carcinoma cells by inhibiting cell proliferation and EMT.

Serine/arginine protein-specific kinase 2 promotes the development and progression of pancreatic cancer by downregulating Numb and p53.

Serine/arginine protein-specific kinase 2 (SRPK2) plays a vital role in the progression of a range of different malignancies, including pancreatic cancer. However, the mechanisms are poorly understood. Previous studies have shown that in hepatocellular carcinoma, SRPK2 knockdown leads to the upregulation of the cell fate determining protein Numb, and in pancreatic cancer cells, Numb knockdown prevents ubiquitin-mediated degradation of p53. In this study, we investigated the relationship between SRPK2, Numb and p53 in the development of pancreatic cancer with or without chemical agent treatment in vitro. SRPK2 expression was upregulated in pancreatic cancer tissues and associated with decreased overall survival in pancreatic cancer patients, indicating that expression of this protein can be used as a marker of unfavourable prognosis. Expression of SRPK2 was positively associated with tumour T stage and Union for International Cancer Control (UICC) stage, and negatively associated with Numb expression in serial tissue sections. In pancreatic cancer cells, SRPK2 downregulation or overexpression led to modulation of Numb and wild-type p53 protein expression in response to oxaliplatin treatment. Furthermore, these three endogenous proteins could be coimmunoprecipitated as a triple complex. Numb or p53 knockdown reversed the upregulation of p53 that was induced by silencing SRPK2. SRPK2 overexpression promoted cell invasion and migration, and decreased chemosensitivity of cancer cells to gemcitabine or oxaliplatin treatment. Conversely, SRPK2 silencing decreased cell invasion and migration and increased chemosensitivity; these effects were reversed by silencing p53 in oxaliplatin-treated pancreatic cancer cells. Our data suggest that SRPK2 negatively regulates p53 by downregulating Numb under chemical agent treatment. Thus, SRPK2 promotes the development and progression of pancreatic cancer in a p53-dependent manner.

NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian cancer cells by inhibiting cell proliferation and epithelial-mesenchymal transition

Background: NUMB is an inhibitory regulator of NOTCH signaling, which is critical for the induction of epithelial-mesenchymal transition (EMT). Loss of NUMB expression is correlated with the genesis and development of multiple tumors. Recent studies reported that NUMB expression was upregulated in human ovarian cancer. However, the role of NUMB in ovarian cancer is still unclear. Here, we invested the effect of NUMB knockdown on the proliferation and EMT in ovarian cancer cells and explored the role of NUMB in the effect of cisplatin. Methods: Two ovarian cancer cells (OVCAR-3 and SK-OV-3) were used in the experiments. The proliferation and apoptosis of ovarian cancer cells was examined using methyl thiazolyl tetrazolium (MTT) test and flow cytometry assays. The invasion and migration of ovarian cancer cells were examined using Transwell assays. The expression of EMT markers were examined using Simple Western analysis. Results: NUMB knockdown inhibited cell proliferation, invasion, and migration in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell growth inhibiting and apoptosis in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell invasion in SK-OV-3 cells. NUMB knockdown also decreased the expression of N-cadherin and Vimentin in SK-OV-3 cells. Conclusions: NUMB acted as an oncogene in ovarian cancer and NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian carcinoma cells by inhibiting cell proliferation and EMT.

Opposing Regulation of Cancer Properties via KRT19-Mediated Differential Modulation of Wnt/β-Catenin/Notch Signaling in Breast and Colon Cancers.

Although Keratin 19 (KRT19) has been reported as a tumor cell marker and found to interact with other proteins that modulate cancer properties, its role in cancer prognosis remains to be fully elucidated. We found that KRT19 expression was increased in both colon and breast cancer, but that knockdown of KRT19 showed opposing effects on cancer properties. In colon cancer, KRT19 knockdown resulted in suppression of cancer via downregulation of Wnt/Notch signaling without altering NUMB transcription. In breast cancer, KRT19 knockdown led to an increase in cancer properties because of attenuated Wnt and enhanced Notch signaling. In colon cancer, KRT19 interacted with β-catenin but not with RAC1, allowing the LEF/TCF transcription factor to bind primarily to the LEF1 and TCF7 promoter regions, whereas in breast cancer, KRT19 interacted with the β-catenin/RAC1 complex and led to apparent upregulation of NUMB expression and NUMB-mediated suppression of Notch signaling. These results reveal a novel differential role of KRT19 in carcinogenesis, due to differential modulation of Wnt/β-catenin/Notch signaling crosstalk through various interactions of KRT19 with only β-catenin or with the β-catenin/RAC1 complex, which might have implications for clinical cancer research.

Telomere Damage Maintains Hematopoietic Stem Cells (HSCs) in an Activated Metabolic State, Which Compromises Their Self-Renewal Capability

DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown.Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit+Sca+Lin-CD34-flk2-CD150+CD48-CD41-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability.To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERTER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERTER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p<10-3) that was associated with a decreased number of the lymphoid-biased MPP4 cells, and an increased number of both megakaryocyte-biased MPP2 and myeloid-biased MPP3 cells. HSC exhaustion and increased myeloid-to lymphoid output were reminiscent of stressed hematopoiesis and premature aging. G5/G6 HSCs exhibited a significant accumulation of telomere dysfunction-induced foci (p<10-5) but did not display increased levels of apoptosis in steady-state conditions.HSC exhaustion could result from apoptosis and/or senescence induced by telomere damage in HSCs entering the cell cycle or from an altered balance between self-renewal and differentiation. To distinguish between these two possibilities, we first investigated the effect of inducing young G5/G6 HSCs out of a homeostatic quiescent state. By tracking the real-time changes in the expression level of annexin V on HSCs induced to differentiate towards the myeloid lineage, we found that apoptosis was not the primary fate of G5/G6 HSCs upon entry into the cell cycle. Similarly, in vivo treatment with poly I:C induced the G5/G6 HSCs to enter into the cell cycle at the same rate as that of the G0 mice without inducing apoptosis. Transcriptomic analysis of poly I:C-treated G0 and G5/G6 HSCs, compared with vehicle-treated controls, revealed a significant enrichment of genes involved in the regulation of the cell cycle and platelet production, which is consistent with previous findings showing that megakaryocyte differentiation of HSCs occurs in response to poly I:C to replenish platelets that are lost during inflammatory insult. Importantly, we did not observe any significant change in gene expression between G0 and G5/G6 HSCs isolated from poly I:C-treated mice, which confirmed that telomeric damage did not limit HSCs' proliferation potential by activating programs of senescence or apoptosis.Next, we evaluated the capability of single HSCs isolated from G0 or G5/G6 mice to either self-renew or differentiate. An evaluation of Numb inheritance and expression in G0 and G5/G6 HSCs (n=133 and n=113, respectively) induced to proliferate in vitro showed that G5/G6 HSCs had a 2-fold lower frequency of symmetric self-renewal division (p<10-3) and a concomitant 2-fold higher frequency of symmetric commitment (p<10-4). Accordingly, PB analysis revealed that the CD45.2-derived reconstitution was severely compromised in mice competitively transplanted with G5/G6 HSCs (0.26% vs 77%; p<10-3). Single cell RNA-seq analysis of G0 and G5/G6 HSCs followed by the differential analysis of the clusters showed that 40% of G5/G6 HSCs were in an activated metabolic state associated with hyperactive OXPHOS and ROS signaling pathways, which are directly involved in HSC functional decline. Finally, we reactivated telomerase to investigate the possibility of restoring normal HSC function upon elimination of damage. Single cell RNA-seq and functional studies are ongoing to evaluate whether HSCs' activated metabolic state and compromised self-renewal capability are reversible processes.This study challenges the concept that telomeric damage limits HSC's proliferative potential and offers unparalleled opportunities for unraveling regenerative strategies to ameliorate their decline. DisclosuresColla:Abbvie: Research Funding.

Numb Isoforms Deregulation in Medulloblastoma and Role of p66 Isoform in Cancer and Neural Stem Cells.

Numb is an intracellular protein with multiple functions. The two prevalent isoforms, Numb p66 and Numb p72, are regulators of differentiation and proliferation in neuronal development. Additionally, Numb functions as cell fate determinant of stem cells and cancer stem cells and its abnormal expression has been described in several types of cancer. Involvement of deregulated Numb expression has been described in the malignant childhood brain tumor medulloblastoma, while Numb isoforms in these tumors and in cancer stem-like cells derived from them, have not been studied to date. Here we show that medulloblastoma stem-like cells and cerebellar neuronal stem cells (NSCs) express Numb p66 where its expression tampers stemness features. Furthermore, medulloblastoma samples evaluated in this study express decreased levels of Numb p66 while overexpressed Numb p72 compared with normal tissues. Our results uncover different roles for the two major Numb isoforms examined in medulloblastoma and a critical role for Numb p66 in regulating stem-like cells and NSCs maintenance.

Effect of experimentally induced hypothyroidism during gestation period on activity dependent neurotrophic factor (ADNF) in newborn rat brain tissue.

Purpose The aim of the study was to evaluate the effects of prenatal hypothyroidism on neonatal rats by the way of activity-dependent neuroprotective factor (ADNF) expression. Methods Twenty-one Wistar albino neonatal rats were divided into two subgroups; a control group and neonatal rats with experimental maternal hypothyroidism. Hypothyroidism was induced by using propylthiouracil (PTU). Neonatal rats obtained PTU from breast milk continuously for 1 week after birth. The rats from the control group were fed only normal feed and water. After birth, body weight and blood thyroid hormone levels were tested. Glial fibrillary acidic protein (GFAP), Slug, Numb, Notch-1 and ADNF antibodies were used for immunohistochemical analysis. Real-time polymerase chain reaction (RT-PCR) and Western blotting analyses were used to evaluate ADNF gene expression levels from 1-week-old rat's brain. Results There was no difference between the two groups for birth weights. The thyroxine (T4) level from the experimental group was <0.4 ng/mL, and it was 0.8 ng/mL for the control group. It was shown that, the results from the experimental group samples had significantly lower ADNF mRNA levels than control group (p < 0.05). The increase from GFAP and Numb expression and decrease from Slug expression were shown in the experimental group. Local differences were identified for ADNF and a decrease was shown in both sides of brain. There was no difference for Notch-1 expression for both groups. Conclusion In this study, decreasing ADNF expression might contribute to developing neurological problems in congenital hypothyroidism.

Numb Depletion Promotes Drp1-Mediated Mitochondrial Fission and Exacerbates Mitochondrial Fragmentation and Dysfunction in Acute Kidney Injury.

Mitochondrial fragmentation is a crucial mechanism contributing to tubular cell apoptosis during acute kidney injury (AKI). However, the mechanism of modulating mitochondrial dynamics during AKI remains unclear. Numb is a multifunction adaptor protein that is expressed in renal tubules. The aim of the present study was to evaluate the role of Numb in mitochondrial dysfunction during AKI. The expression of Numb was upregulated in both ischemia-reperfusion- and cisplatin-induced AKI. Depletion of Numb from proximal tubules (PT-Nb-KO) exacerbated AKI shown as more severe renal tubular damage and higher serum creatinine than wild-type mice. Numb depletion alone significantly increased mitochondrial fragmentation without altering mitochondrial mass and function, including adenosine triphosphate production, mitochondrial membrane potential, oxygen consumption, and reactive oxygen species production. However, mitochondrial fragmentation and dysfunction were significantly aggravated after cisplatin exposure in PT-Nb-KO mice. Mechanistically, Numb depletion triggered dynamin-related protein 1 (Drp1) recruitment to mitochondria by increasing the phosphorylation of Drp1 at serine 656 residue (human Drp1 ser637). Inhibiting the activity of Rho-associated coiled-coil containing protein kinase (ROCK) by Y-27632 attenuated phosphorylation of Drp1 ser656 and mitochondrial fragmentation in Numb-deficient cells. Administration of mdivi-1, a pharmacological inhibitor of Drp1, restored mitochondrial morphology, attenuated cisplatin-induced tubular injury, and renal dysfunction in PT-Nb-KO mice. Innovation and Conclusion: Our data suggest that Numb depletion promotes mitochondrial fragmentation by promoting the phosphorylation of Drp1 Ser637 and thus exacerbates cisplatin-induced mitochondrial dysfunction and tubular cell apoptosis. These findings add a novel insight into modulating mechanism of mitochondrial dynamics during AKI.

Abstract 5524: Tumor suppressor Numb represses monocarboxylate transporters to reduce the glycolysis in oral carcinoma

Abstract Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide. Our previous studies have shown that Numb pluripotent gene drives suppressor activity against OSCC, and it is co-targeted by multiple oncogenic miRNAs. In this study, we further identified that Numb mRNA expression was down-regulated in a large fraction of OSCC tumors. We established the Numb-knockout (KO) OSCC cell subclones using CRISPR-Cas9 system. We also generated lentiviruses for exogenous expression of Numb 1 and Numb 4 isoforms. The anchorage independent growth potential was found increased in Numb-KO cell subclones. However, such eligibility was repressed after the rescue of the Numb expression by lentiviral infection. In addition, the level of lactate production and the ratio of ECAR/OCR in the Numb-KO cell subclones were higher than parental cells. The knockout of Numb was able to up-regulate the expression of monocarboxylate transporter (MCT) family members MCT1 and MCT4, but not other metabolism genes, to increase the lactate production. Knockdown of both MCT1 and MCT4 significantly decreased the anchorage independent growth and the lactate production of OSCC cells. This study provides new evidences denoting that Numb represses MCTs to attenuate the transformative capability and the glycolysis in oral carcinoma. Citation Format: Chung-Hsien Chou, Chun-Yu Fan Chaing, Shu-Chun Lin, Kuo-Wei Chang, Hsi-Feng Tu. Tumor suppressor Numb represses monocarboxylate transporters to reduce the glycolysis in oral carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5524.

Numb has distinct function in lung adenocarcinoma and squamous cell carcinoma.

Some reports suggest that Numb is a potential tumor suppressor. However, its role in non-small cell lung cancer remains unclear. Non-small cell lung cancer comprises two major histological subtypes, adenocarcinoma and squamous cell carcinoma. To investigate the role of Numb in tumorigenesis of lung adenocarcinoma and squamous cell carcinoma, we firstly performed loss-of-function and gain-of-function assays. Moreover, Numb expression was investigated in surgically resected lung adenocarcinoma and squamous cell carcinoma tissues by immunohistochemistry and correlations with prognosis were analyzed. Numb suppressed the proliferation, migration, and invasion of adenocarcinoma cells and inhibited Notch signaling and epithelial-mesenchymal transition in vitro. Numb overexpression also inhibited subcutaneous adenocarcinoma tumor growth. In contrast, Numb promoted the proliferation, migration, and invasion of squamous cell carcinoma cells, but did not induce any consistent changes in Notch signaling. High Numb expression was associated with favorable prognosis in patients with lung adenocarcinoma, but not in those with squamous cell carcinoma. Collectively, our data demonstrate that Numb plays distinct roles in lung adenocarcinoma and squamous cell carcinoma. In lung adenocarcinoma, Numb impairs tumor growth and inhibits the Notch pathway and epithelial-mesenchymal transition, whereas in lung squamous cell carcinoma it may promote proliferation.

Melatonin inhibits 17\u03b2-estradiol-induced migration, invasion and epithelial-mesenchymal transition in normal and endometriotic endometrial epithelial cells

BackgroundMelatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear. Here, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic endometrial epithelial cells and explored the pathway that might be involved.MethodsThis hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the expression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by CCK8 assays.ResultsCompared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of Notch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by specific inhibitor blocked 17β-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells.ConclusionsOur data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present in endometriotic endometrium. Melatonin may block 17β-estradiol-induced migration, invasion and EMT in normal and endometriotic epithelial cells by upregulating Numb expression and decreasing the activity of the Notch signaling pathway.

Loss of nuclear NOTCH1, but not its negative regulator NUMB, is an independent predictor of cervical malignancy.

The participation of NOTCH signaling in invasive cervical cancer (ICC) remains controversial since both tumor suppressive and oncogenic properties have been described. Additionally, the role of NUMB, a negative regulator of NOTCH, remains unclear in ICC. We aimed to investigate the role of NOTCH1 and NUMB expression and their localization in cervical intraepithelial neoplasia (CIN) and ICC samples. A total of 144 biopsies were obtained from the Instituto Nacional de Cancerología, México from 2004 to 2017, and were subjected to immunohistochemistry for NOTCH1 and NUMB. We found that nuclear NOTCH1 expression was more frequently found in CIN samples compared with ICC (77.55% vs. 15.79%, p = 0.001). NUMB was almost exclusively found in the nucleus of CIN samples (32.65% vs. 6.32%, p = 0.001). Cytoplasmic expression of NOTCH1 (44.21%) and NUMB (35.79%) was the most frequent localization in ICC. Multivariable-adjusted analysis showed that the loss of nuclear NOTCH1 expression was an independent predictor of malignancy (β = –3.428, 95% confidence interval [95% CI] = –5.127, –1.728, p = 0.001). In contrast, the association between cytoplasmic NUMB expression and cervical cancer was lost after adjusting for nuclear NOTCH1 expression (β = 2.074, 95% [CI] = –0.358, 4.506, P = 0.094). Additionally, patients with cytoplasmic NOTCH1 expression showed a borderline association with longer overall survival (OS) than those with nuclear NOTCH1 expression (P = 0.08). Our data suggest that the loss of nuclear NOTCH1 but not NUMB might be an independent predictor of malignancy in cervical cancer.

Nrf2 antioxidant pathway suppresses Numb-mediated epithelial\u2013mesenchymal transition during pulmonary fibrosis

Epithelial mesenchymal transition (EMT) is a key progression that promotes pulmonary fibrosis (PF). Numb, a phosphotyrosine-binding domain (PTB) protein, is implicated with EMT. Nuclear factor erythroid 2-related factor2 (Nrf2) and its downstream proteins heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) constitute an important pathway of antioxidant defense signal for protecting against PF. It remains elusive whether Nrf2 antioxidant pathway and Numb have a potential relationship in EMT-mediated PF. Here, we observed the effects of Nrf2 pathway and Numb on bleomycin(BLM)-induced PF in Nrf2-knockout (Nrf2−/−) and wild-type (WT) mice. Meanwhile, rat type II alveolar epithelial cells line (RLE-6TN) and human epithelial cells line (A549) were both treated with an Nrf2 activator sulforaphane (SFN), or transfected siRNAs of Nrf2 and Numb to unravel roles of Nrf2 pathway, Numb and the link between them on transforming growth factor β1 (TGF-β1)-induced EMT. We found BLM-induced lung fibrosis were more severe in Nrf2−/− mice compared to WT mice with reduced expressions of HO-1 and NQO1. Numb was enhanced with down-regulated expressions of Nrf2 in BLM groups and further increased in Nrf2−/− groups. In vitro, given exogenous TGF-β1 on RLE-6TN and A549 up-regulated Numb expressions, accompanied with down-regulations of Nrf2 and its target proteins HO-1 and NQO1. Transfected with Nrf2 and Numb siRNAs further aggravated and relieved the progression of EMT, respectively. Inversely, activating Nrf2 pathway by SFN reduced the expression of Numb and EMT-related protein. Moreover, Numb deficiency by siRNA relieved the protection of activating Nrf2 against EMT. In conclusion, activating Nrf2 antioxidant pathway suppresses EMT during PF via inhibiting the abnormal expression of Numb. These findings provide insight into PF pathogenesis and a basis for novel treatment approaches.