Notch1 Knockdown Research Articles

Hypoxia reduces the osteogenic differentiation of peripheral blood mesenchymal stem cells by upregulating Notch-1 expression

ABSTRACTPurpose: Mesenchymal stem cells (MSCs) seeded on biocompatible scaffolds have therapeutic potential for bone defect repair. However, MSCs can be affected by hypoxia and nutritional deficiency due to a lack of blood vessels in the scaffolds. Here, we explored the effects of hypoxia on MSC differentiation to clarify these mechanisms.Methods: Peripheral blood mesenchymal stem cells (PBMSCs) were cultured in small individual chambers with oxygen concentrations of 1%, 9%, and 21%. Cell proliferation was evaluated by Cell Counting Kit 8 assays, and cell survival was determined using live/dead assays. Scratch assays were performed to evaluate cell migration. Ca2+ deposition/mineralization experiments, reverse transcription quantitative real-time polymerase chain reaction, and Western blotting were performed to assess the osteogenic differentiation of cells. Notch1 expression was downregulated by lentivirus-transfected PBMSCs to observe the effects of Notch1 knockdown on osteogenic gene and protein expression.Results: PBMSCs exposed to hypoxia (1% O2) demonstrated accelerated proliferation, increased migration, and reduced survival in the absence of serum. Although 9% oxygen promoted osteogenic differentiation, the osteogenic differentiation of PBMSCs was significantly reduced by 1% O2, and this effect was associated with increased Notch1 expression. Reducing Notch1 expression using small interfering RNA significantly restored the osteogenic differentiation of PBMSCs.Conclusions: Hypoxia accelerated proliferation, increased migration, and reduced PBMSC differentiation into osteoblasts by increasing Notch1 expression. These findings may contribute to the development of appropriate cell culture or in vivo transplantation conditions to maintain the full osteogenic potential of PBMSCs.

NOTCH signaling is activated in and contributes to resistance in enzalutamide-resistant prostate cancer cells

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is a Food and Drug Administration-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period, tumors will develop drug resistance. In this study using RNA-Seq and bioinformatics analyses, we observed that NOTCH signaling is a deregulated pathway in enzalutamide-resistant cells. NOTCH2 and c-MYC gene expression positively correlated with AR expression in samples from patient with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide resistance. Cleaved NOTCH1, HES1 (Hes family BHLH transcription factor 1), and c-MYC protein expression levels are elevated in two enzalutamide-resistant cell lines, MR49F and C4-2R, indicating NOTCH signaling activation. Moreover, inhibition of the overexpressed ADAM metallopeptidase domain 10 (ADAM10) in the resistant cells induces an exclusive reduction in cleaved NOTCH1 expression. Furthermore, exposure of enzalutamide-resistant cells to both PF-03084014 and enzalutamide increased cell death, decreased colony formation ability, and resensitized cells to enzalutamide. Knockdown of NOTCH1 in C4-2R increased enzalutamide sensitivity by decreasing cell proliferation and increasing cleaved PARP expression. In a 22RV1 xenograft model, PF-03084014 and enzalutamide decreased tumor growth through reducing cell proliferation and increasing apoptosis. These results indicate that NOTCH1 signaling may contribute to enzalutamide resistance in prostate cancer, and inhibition of NOTCH signaling can resensitize resistant cells to enzalutamide.

Arsenic Trioxide Suppresses Tumor Growth through Antiangiogenesis via Notch Signaling Blockade in Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is a highly malignant type of lung cancer with no effective second-line chemotherapy drugs. Arsenic trioxide (As2O3) was reported to exert antiangiogenesis activities against lung cancer and induce poor development of vessel structures, similar to the effect observed following the blockade of Notch signaling. However, there are no direct evidences on the inhibitory effects of As2O3 on tumor growth and angiogenesis via blockade of Notch signaling in SCLC. Here, we found that As2O3 significantly inhibited the tumor growth and angiogenesis in SCLC and reduced the microvessel density. As2O3 disturbed the morphological development of tumor vessels and downregulated the protein levels of delta-like canonical Notch ligand 4 (Dll4), Notch1, and Hes1 in vivo. DAPT, a Notch signaling inhibitor, exerted similar effects in SCLC. We found that both As2O3 treatment and Notch1 expression knockdown resulted in the interruption of tube formation by human umbilical vein endothelial cells (HUVECs) on Matrigel. As2O3 had no effects on Dll4 level in HUVECs but significantly inhibited the expression of Notch1 and its downstream gene Hes1 regardless of Dll4 overexpression or Notch1 knockdown. These findings suggest that the antitumor activity of As2O3 in SCLC was mediated via its antiangiogenic effect through the blockade of Notch signaling, probably owing to Notch1 targeting.

Identification of a novel miR-206-Notch3 pathway regulating mouse myoblasts proliferation

MicroRNAs (miRNAs) are endogenous short non-coding RNAs and exert their function by targeting mRNAs of genes. miRNA-206 (miR-206) is exclusively expressed in adult skeletal muscles and plays an important role in myogenesis. However, the regulatory mechanisms of miR-206 in myoblasts proliferation and differentiation are still limited. In this study, we validated that Notch3 is a direct target gene of mouse miR-206 using dual-luciferase reporter assay and miR-206-overexpressed experiments. Furthermore, we demonstrated that the negative effect of overexpression of miR-206 and knockdown of Notch3 on C2C12 cells proliferation, and found that the effect was produced by controlling the transition of G0/G1 and S phase in C2C12 cell cycle. In summary, our results provide direct evidences that miR-206 regulates skeletal muscle cells proliferation and cell cycle arrest partly by targeting the Notch3 gene, which strengthens our understanding of regulatory mechanisms on miR-206 in skeletal muscle growth and development.

Arecoline N-oxide regulates oral squamous cell carcinoma development through NOTCH1 and FAT1 expressions.

Areca nut has been evaluated as a group I carcinogen to humans. However, the exact compounds of areca nut causing oral cancer remain unproven. Previous findings from our lab revealed that arecoline N-oxide (ANO), a metabolite of arecoline, exhibits an oral fibrotic effect in immune-deficient NOD/SCID mice. The aim of this study is to investigate the oral potentially malignant disorders (OPMD) inductive activity between areca-alkaloid arecoline and its metabolite ANO in C57BL/6 mice. Our findings show that ANO showed higher activity in inducing hyperplasia with leukoplakia and collagen deposition in C57BL/6 mice compared with the arecoline treated groups. Importantly, immunohistochemical studies showed significant upregulation of NOTCH1, HES1, FAT1, PCNA, and Ki67 expressions in the pathological hyperplastic part. In addition, in vitro studies showed that upregulation of NOTCH1 and FAT1 expressions in ANO treated HGF-1 and DOK cell models. We found that NOTCH1 regulates TP53 expression from NOTCH1 knockdown oral cancer cells. The DNA damage was significantly increased after arecoline and ANO treatment. Further, we found that arecoline-induced H2AX expression was regulated by FMO3. Altogether, our findings show that ANO exhibited higher toxicity in OPMD activity and play a significant role in the induction of areca nut mediated oral tumorigenesis.

Overexpression of Notch3 is associated with metastasis and poor prognosis in osteosarcoma patients

BackgroundNotch signaling abnormalities are associated with the development of various tumors, including hematopoietic and epithelium-derived tumors. However, the role of Notch signaling in tumors originating from mesenchymal cells is unclear. The effect of Notch3 expression on the prognosis of osteosarcoma and its role and mechanism in osteosarcoma cells have never been reported.Materials and methodsIn this study, we performed a clinicopathological analysis of 70 cases of osteosarcoma, with primary focus on survival. Osteosarcoma cell lines MTH and U2OS were used. After knockdown of Notch3 by lentiviral transfection and siRNA, the cell cycle, cell viability, and wound healing capacity were assessed. Subsequently, the Transwell assay was performed, and the expression levels of hairy and enhancer of split-1 (Hes1) and matrix metalloproteinase 7 (MMP7) were detected by RT-PCR and Western blot assay. The expression of MMP7 was also detected after knockdown of Hes1. Animal experiments were performed by injecting the cell lines MTH of Notch3 knockdown into mice tail veins and comparing the development of lung metastasis with the control group.ResultsComparison of survival curves showed that Notch3 expression significantly impacts patient survival. Additionally, multivariate analysis revealed that Notch3 is an independent prognostic factor for osteosarcoma. In in vivo experiments, osteosarcoma-associated pulmonary metastasis in nude mice was reduced after Notch3 silencing. The expression of downstream effector molecule, Hes1, and that of the invasion and metastasis-associated proteolytic enzyme, MMP7, were reduced, and MMP7 was further decreased by Hes1 knockdown in in vitro experiments.ConclusionNotch3 is a prognostic factor for osteosarcoma and might regulate its invasion and metastasis through the downstream target gene Hes1 and effector MMP7.

Epigenetic knockdown of Notch1 inhibits hepatitis B virus X protein‑induced hepatocarcinogenesis of L02/HBx cells.

The present study aimed to investigate the effects of Notch1 on the development of hepatitis B virus X protein (HBx)‑induced hepatocarcinogenesis. The L02/HBx cells were transfected with a short hairpin RNA (shRNA) specially targeting Notch1 (Notch1‑shRNA). The mRNA and protein expression levels of Notch1 signaling pathway‑related molecules (Notch1, Hes1 and NICD) were detected after knockdown of Notch1. The effects of Notch1 knockdown on the proliferation was analyzed by Cell Counting Kit‑8 assay, and cell cycle and apoptosis of L02/HBx cells invitro were investigated by flow cytometry. The invivo tumor xenograft model was established by subcutaneously injection of mice with Notch1‑shRNA or sh‑NC transfected cells. The effects of Notch1 knockdown on tumor progression invivo were then explored by H&E staining and immunohistochemistry. The results showed that knockdown of Notch1 inhibited the activation of the Notch1 signaling pathway. In addition, decreased viability and colony formation ability of L02/HBx cells were detected along with downregulated protein expression levels of Ki‑67 and PCNA (proliferating cell nuclear antigen). In addition, knockdown of Notch1 led to L02/HBx cell cycle arrest at G0/G1 phase by decreasing the expression of cyclin D1, CDK4, E2F1 and increasing the expression of p21 and retinoblastoma gene (Rb). Moreover, knockdown of Notch1 promoted the apoptosis of L02/HBx cells by activation of caspase‑3 and caspase‑9. Invivo experiments demonstrated that knockdown of Notch1 inhibited the tumorigenicity of L02/HBx cells. Our findings revealed that inhibition of the Notch1 signaling pathway may inhibit the development of HBx‑induced hepatocellular carcinoma. Notch1 may serve as a promising therapeutic target for HCC.

Co-Expression Analysis Reveals Mechanisms Underlying the Varied Roles of NOTCH1 in NSCLC

IntroductionNotch receptor family dysregulation can be tumor promoting or suppressing depending on cellular context. Our studies shed light on the mechanistic differences that are responsible for NOTCH1’s opposing roles in lung adenocarcinoma and lung squamous cell carcinoma. MethodsWe integrated transcriptional patient-derived datasets with gene co-expression analyses to elucidate mechanisms behind NOTCH1 function in subsets of NSCLC. Differential co-expression was examined using hierarchical clustering and principal component analysis. Enrichment analysis was used to examine pathways associated with the underlying transcriptional networks. These pathways were validated in vitro and in vivo. Endogenously epitope-tagged NOTCH1 was used to identify novel interacting proteins. ResultsNOTCH1 co-expressed genes in lung adenocarcinoma and squamous carcinoma were distinct and associated with either angiogenesis and immune system pathways or cell cycle control and mitosis pathways, respectively. Tissue culture and xenograft studies of lung adenocarcinoma and lung squamous models with NOTCH1 knockdown showed growth differences and opposing effects on these pathways. Differential NOTCH1 interacting proteins were identified as potential mediators of these differences. ConclusionsRecognition of the opposing role of NOTCH1 in lung cancer, downstream pathways, and interacting proteins in each context may help direct the development of rational NOTCH1 pathway-dependent targeted therapies for specific tumor subsets of NSCLC.

MicroRNA‑495 targets Notch1 to prohibit cell proliferation and invasion in oral squamous cell carcinoma.

MicroRNAs (miRNAs) are associated with the initiation and progression of oral squamous cell carcinoma (OSCC) by regulating a variety of cancer‑associated behaviors. Fully understanding the regulatory mechanism of miRNAs in the pathogenesis of OSCC may provide novel promising approaches for the identification of prognostic biomarkers and therapeutic targets for this particular malignancy. In the present study, reverse transcription‑quantitative polymerase chain reaction analysis was performed to detect miRNA (miR)‑495 expression in OSCC tissues and cell lines. The effects of miR‑495 on the proliferation and invasion of OSCC cells were determined using Cell Counting Kit‑8 and Matrigel invasion assays, respectively. The mechanisms underlying the action of miR‑495 in OSCC cells were also investigated. Results from the present study revealed that miR‑495 expression was downregulated in OSCC tissues and cell lines compare with in adjacent normal tissues and human oral keratinocytes, respectively. Exogenous expression of miR‑495 restricted cell proliferation and invasion of OSCC cells invitro. Notch1 was identified as a direct functional target of miR‑495 in OSCC. Furthermore, Notch1 knockdown exhibited inhibitory effects, similar to those induced by miR‑495 overexpression in OSCC cells. Restoration of Notch1 expression rescued the suppressive effects of miR‑495 on OSCC cell proliferation and invasion. These findings suggested an important role for miR‑495 in the regulation of OSCC cell growth and metastasis, at least partly by directly targeting Notch1. In addition, the findings of the present study revealed the potential of miR‑495 as a novel therapeutic target for the treatment of patients with OSCC.

Regulation of differentiation of MEG01 to megakaryocytes and platelet-like particles by Valproic acid through Notch3 mediated actin polymerization

Valproic acid (VPA) is one of the HDAC inhibitors used for the treatment of neurological disorders and hematological malignancies. Its role in self-renewal and proliferation of hematopoietic stem cells (HSCs) is well studied, but little is known about its involvement in regulating megakaryopoiesis and thrombopoiesis. In this study, we evaluated the role of VPA in megakaryopoiesis by using MEG-01, a megakaryoblast cell line. Our results show that VPA treatment differentiates MEG-01 cells to megakaryocytes (MK) and platelet-like particles. It was confirmed by augmented expression of MK and PLT-specific markers, higher ploidy, and PLT functionality. We assessed the molecular events underlying megakaryopoiesis. In the present study, we found an upregulation of Notch3 and its downstream target PDGFR-β upon VPA treatment. The direct role of Notch3 in megakaryopoiesis has not yet been studied. PDGFR-β is known to control actin organization during vascular smooth muscle cell differentiation. The actin cytoskeleton plays important role during proplatelet and PLT formation. We found an upregulation of Rac/Cdc42 GTPase and its downstream effectors that are the key players during actin polymerization events. We speculate that VPA induces PLT formation through Notch-3 signaling that in turn modulates actin polymerization that is one of the crucial steps necessary for thrombopoiesis. These studies were also confirmed with knockdown of Notch3 in MEG01 by using ShRNA approach as well as with apheresis-derived CD34+ cells. Altogether, these findings provide an evidence for a novel role of Notch3 in regulating platelet formation.

Notch1 serves as a prognostic factor and regulates metastasis via regulating EGFR expression in hypopharyngeal squamous cell carcinoma.

ObjectiveHypopharyngeal squamous cell carcinoma (HSCC) remains one of the most lethal malignancies in head and neck. Notch1 has been validated to play prominent roles in the occurrence and development of various types of cancer. The aim of this study was to explore the function and underlying mechanism of Notch1 in HSCC.Patients and methodsSeventy-one cancer tissue samples and adjacent noncancerous formalin-fixed paraffin embedded tissue specimens were analyzed by immunohistochemistry. As Notch1 is overexpressed in HSCC, we further questioned whether there was a relationship between Notch1 and the clinicopathological characteristics. After confirming the successful knockdown of Notch1 by siRNA, the migration and invasion after gene knockdown were investigated by Transwell chambers. We then tried to identify YBX1 and EGFR expression using real-time PCR (RT-PCR) and Western blot analyses. To further determine whether the downexpression of EGFR was caused by YBX1 and the overexpression of YBX1 was caused by gene amplification, the expression of EGFR was detected by RT-PCR and Western blot assays.ResultsWe found that the expression of Notch1 and EGFR in HSCC tissues was upregulated compared with those in the adjacent noncancerous tissues. Further clinicopathological characteristics analysis revealed that the expression of Notch1 was positively correlated with distant metastasis (P=0.003) and tumor differentiation (P=0.031). The high expression of Notch1 is an independent prognostic factor for a poor overall survival in patients with HSCC (P=0.015, χ2=10.403). Knocking down of Notch1 significantly inhibits the migration and invasion of FaDu cells in vitro. Mechanistic investigation reveals that Notch1 knockdown is found suppressing the expression of EGFR at transcriptional level. Interestingly, we further found that Notch1 knockdown also decreased the expression of YBX1, which is a transcription factor of EGFR. Moreover, the upregulation of YBX1 reverses the suppression of Notch1 on EGFR. Furthermore, forced overexpression of YBX1 induced the invasion of FaDu cells.ConclusionTaken together, we found a positively cross-linked role of Notch1 signaling in the outcome of HSCC, providing a novel valuable prognostic marker and potential therapeutic target for the treatment of HSCC patients. Notch1 is a core signaling molecule for regulating migration and invasion via interplaying with EGFR in HSCC cells.

Notch3 inhibits epithelial\u2013mesenchymal transition in breast cancer via a novel mechanism, upregulation of GATA-3 expression

Notch3 and GATA binding protein 3 (GATA-3) have been, individually, shown to maintain luminal phenotype and inhibit epithelial–mesenchymal transition (EMT) in breast cancers. In the present study, we report that Notch3 expression positively correlates with that of GATA-3, and both are associated with estrogen receptor-α (ERα) expression in breast cancer cells. We demonstrate in vitro and in vivo that Notch3 suppressed EMT and breast cancer metastasis by activating GATA-3 transcription. Furthermore, Notch3 knockdown downregulated GATA-3 and promoted EMT; while overexpression of Notch3 intracellular domain upregulated GATA-3 and inhibited EMT, leading to a suppression of metastasis in vivo. Moreover, inhibition or overexpression of GATA-3 partially reversed EMT or mesenchymal–epithelial transition induced by Notch3 alterations. In breast cancer patients, high GATA-3 expression is associated with higher Notch3 expression and lower lymph node metastasis, especially for hormone receptor (HR) positive cancers. Herein, we demonstrate a novel mechanism whereby Notch3 inhibit EMT by transcriptionally upregulating GATA-3 expression, at least in part, leading to the suppression of cancer metastasis in breast cancers. Our findings expand our current knowledge on Notch3 and GATA-3's roles in breast cancer metastasis.

Suppression of microRNA-495 alleviates high-glucose-induced retinal ganglion cell apoptosis by regulating Notch/PTEN/Akt signaling

High glucose (HG)-induced apoptosis of retinal ganglion cells (RGCs) contributes to the pathogenesis of diabetic retinopathy, which is one of the most common and severe complications of diabetes mellitus. Accumulating evidence has documented that microRNAs (miRNAs) play an important role in the pathogenesis of diabetic retinopathy. However, the role of miRNAs in regulating HG-induced apoptosis of RGCs remains largely unknown. Various studies have suggested that miR-495 is an important regulator of cell apoptosis and survival. In this study, we aimed to investigate whether miR-495 is involved in regulating HG-induced apoptosis of RGCs and reveal its possible relevance in diabetic retinopathy. We found that miR-495 was significantly upregulated in HG-treated RGCs. Downregulation of miR-495 protected RGCs against HG-induced apoptosis, whereas overexpression of miR-495 had the opposite effect. Notably, Notch1 was identified as a target gene of miR-495, as miR-495 negatively regulated Notch1 expression and the Notch signaling pathway. Moreover, downregulation of miR-495 inhibited PTEN expression while promoting Akt activation. However, knockdown of Notch1 significantly abolished the protective effect of miR-495 inhibition against HG-induced apoptosis. Overall, our study suggests that downregulation of miR-495 alleviates HG-induced apoptosis of RGCs by targeting Notch1 to regulate PTEN/Akt signaling, which provides novel insights into understanding the pathogenesis of HG-induced apoptosis of RGCs.

PO-279 Interplay between Indoleamine 2,3-dioxigenase 1 and Notch1 in radiation response of cervical stem cancer cells

IntroductionCervical cancer is the fourth common cancer in female around the world. Cancer stem cells (CSCs) are responsible for the initiation, maintenance, drug resistance and metastasis of tumours and are considered as an effective therapeutic target. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyses tryptophan metabolising to kynurenine and plays a role in immunosuppressive microenvironment within cancers. Notch1 expression participates in radioresistance of cancer cells. The purpose of this study is to explore the relationship between Notch1 and IDO1 expression in radiation response of cervical CSCs.Material and methodsCervical CSCs were enriched by tumorsphere cultivation from HeLa or SiHa cervical cancer cell lines. The expression of IDO1 and Notch1 was determined by western blot or real-time RT-PCR. The IDO1 activity in cell lysates was determined by measurement of kynurenine fluorescence converted from tryptophan. Radiation response of cervical cancer cells were determined by clonogenic assay. The binding of Notch intracellular domain (NICD) on IDO1 promoter was determined by chromatin immunoprecipitation (ChIP) assay. shRNA mediated RNA interference was used for knockdown of specific genes.Results and discussionsWe first found that the expression of IDO1, as well as NICD, was increased in cervical tumorspheres by western blot analysis. We also found that the IDO1 activity in total cell lysates was significantly increased in cervical tumorspheres than the parental cells. Using Jurkat T cells as a model, the growth rate of T cells was significantly decreased in conditional medium from tumorsphere cultivation when compared to those from parental adherent culture. In addition, IDO1 expression was suppressed in HeLa or SiHa cervical cancer cells with knockdown of Notch1. The binding of NICD on IDO1 promoter was inhibited by Notch1 inhibitor. The radiosensitivity of cervical cancer cells was increased by the treatment of IDO1 inhibitor but was decreased after treatment of kynurenine. Furthermore, the expression of NICD protein, as well as Notch1 or BMI1 mRNA, was decreased in cervical tumorspheres with IDO1 knockdown. We also observed that Inhibition of AhR or ARNT, the signalling molecules of kynurenine, suppressed CSC activity of cervical cancer cells.ConclusionOur data indicated that there is a reciprocal regulation mechanism of Notch1 and IDO1 expression in cervical CSCs. These results will provide new insights in the development of the combination of immunotherapy and radiotherapy in against cervical cancer.

Abstract 545: NOTCH1 co-expression analysis reveals novel insights underlying its opposing effects as an oncogenic and tumor suppressor in lung cancer

Abstract Background: Aberrations in the family of Notch receptors (1, 2, 3, and 4) have been implicated in a range of solid tumors, including lung cancer. NOTCH1 biology is complex where Notch plays an oncogenic and tumor suppressor role in lung adenocarcinoma and lung squamous cell carcinoma, respectively. Although the role of Notch in cancer development and progression has received increased appreciation in recent years, there is still a lack of understanding of the mechanisms underlying these opposing activities in lung cancer. Methods: The Cancer Genome Atlas datasets were used to examine gene co-expression patterns of Notch1 in lung adenocarcinoma and lung squamous cell carcinoma. Biological pathways implicated by gene families were assessed using functional annotation tools (DAVID, ToppGene, and IPA). In vitro and in vivo knockdown studies assessed the functional role of Notch1. Results: This novel co-expression analysis supports the hypothesis that NOTCH1 is co-expressed with different genes in lung adenocarcinoma and squamous cell carcinoma. Knockdown of Notch1 in vitro and in vivo support our in silico finding of opposing effects of NOTCH1. Our analysis implicates genes associated with metabolic pathways, immune pathways, angiogenesis and cell cycle that may underlie the differential role of NOTCH1 in lung adenocarcinoma and squamous cell carcinoma. In vitro and in vivo studies support our bioinformatics analysis. Conclusion: These results demonstrate different NOTCH1 gene co-expression patterns in lung adenocarcinoma and squamous cell carcinoma. These findings provide novel insights underlying the context-dependent role of Notch as an oncogene and tumor suppressor in subtypes of lung cancer. Understanding the similarities and differences in co-expression patterns reveal novel insights, suggesting that tumor intrinsic and extrinsic pathways may underlie the dual role of NOTCH1 in lung cancer. Recognition of the mechanisms underlying NOTCH1 opposing roles in cancer may help direct development of Notch-targeted therapies as a monotherapy or in combination with approaches focusing on the tumor microenvironment. Citation Format: Sara L. Sinicropi-Yao, David P. Carbone, Kevin R. Coombes, Joseph M. Amann, David Lopez Lopez. NOTCH1 co-expression analysis reveals novel insights underlying its opposing effects as an oncogenic and tumor suppressor in lung cancer [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 545.

Abstract 157: Targeting cancer stem-like cells in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC), an aggressive, heterogeneous subtype of breast cancer, and immunohistochemically negative for estrogen receptor α (ER-), progesterone receptor (PR-), and lacking amplification of the human epidermal growth factor receptor 2 locus (HER2-). TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlates with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Moreover the role of non-canonical Notch signaling in TNBC remains unknown. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment of NF-κB subunits, IKKα and Notch1 to the cIAP-2 promoter. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. We also demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and mitochondrial respiration. AKT inhibition or NF-κB inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or NF-κB inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. These data suggest that combination treatments affecting Notch, NF-kB or AKT pathways have potential therapeutic importance in targeting CSCs of TNBC. Citation Format: Fokhrul Hossain, Ayse D Ucar Bilyeu, Claudia Sorrentino, Judy Crabtree, Antonio Pannuti, Margarite Matossian, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Targeting cancer stem-like cells in triple negative breast cancer [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 157.

FOXF1 Mediates Endothelial Progenitor Functions and Regulates Vascular Sprouting.

Endothelial colony forming cells (ECFC) or late blood outgrowth endothelial cells (BOEC) have been proposed to contribute to neovascularization in humans. Exploring genes characteristic for the progenitor status of ECFC we have identified the forkhead box transcription factor FOXF1 to be selectively expressed in ECFC compared to mature endothelial cells isolated from the vessel wall. Analyzing the role of FOXF1 by gain- and loss-of-function studies we detected a strong impact of FOXF1 expression on the particularly high sprouting capabilities of endothelial progenitors. This apparently relates to the regulation of expression of several surface receptors. First, FOXF1 overexpression specifically induces the expression of Notch2 receptors and induces sprouting. Vice versa, knock-down of FOXF1 and Notch2 reduces sprouting. In addition, FOXF1 augments the expression of VEGF receptor-2 and of the arterial marker ephrin B2, whereas it downmodulates the venous marker EphB4. In line with these findings on human endothelial progenitors, we further show that knockdown of FOXF1 in the zebrafish model alters, during embryonic development, the regular formation of vasculature by sprouting. Hence, these findings support a crucial role of FOXF1 for endothelial progenitors and connected vascular sprouting as it may be relevant for tissue neovascularization. It further implicates Notch2, VEGF receptor-2, and ephrin B2 as downstream mediators of FOXF1 functions.

Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival.

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer.

Molecular characterization of mesenchymal stem cells in human osteoarthritis cartilage reveals contribution to the OA phenotype

Adult human articular cartilage harbors a population of CD166+ mesenchymal stem cell-like progenitors that become more numerous during osteoarthritis (OA). While their role is not well understood, here we report that they are indeed part of cellular clusters formed in OA cartilage, which is a pathological hallmark of this disease. We hypothesize that these cells, termed OA mesenchymal stem cells (OA-MSCs), contribute to OA pathogenesis. To test this hypothesis, we generated and characterized multiple clonally derived stable/immortalized human OA-MSC cell lines, which exhibited the following properties. Firstly, two mesenchymal stem cell populations exist in human OA cartilage. While both populations are multi-potent, one preferentially undergoes chondrogenesis while the other exhibits higher osteogenesis potential. Secondly, both OA-MSCs exhibit significantly higher expression of hypertrophic OA cartilage markers COL10A1 and RUNX2, compared to OA chondrocytes. Induction of chondrogenesis in OA-MSCs further stimulated COL10A1 expression and MMP-13 release, suggesting that they contribute to OA phenotypes. Finally, knocking down RUNX2 is insufficient to inhibit COL10A1 in OA-MSCs and also requires simultaneous knockdown of NOTCH1 thereby suggesting altered gene regulation in OA stem cells in comparison to chondrocytes. Overall, our findings suggest that OA-MSCs may drive pathogenesis of cartilage degeneration and should therefore be a novel cell target for OA therapy.

Notch3 is involved in adipogenesis of human adipose-derived stromal/stem cells

Human adipose-derived stromal/stem cells (hASCs) have tremendous therapeutic potential and the ability to offer insight into human development and disease. Here we subject human ASCs to siRNA-mediated knockdown of Notch3 cultured under both self-renewing and adipogenic differentiation conditions. Self-renewal was monitored by assessing viability and proliferation rates through staining and alamarBlue assays, respectively. Adipogenesis was measured through Oil-Red O staining, western blot, and quantitative real-time RT-PCR that determined expression levels of multipotency and adipogenic markers over time. Notch3 was expressed in self-renewing hASCs but knockdown, as validated by qRT-PCR and western blot, showed no impact on cell viability, as measured through live-dead staining, or cell proliferation rates, as measured through alamarBlue assays. However, although Notch3 expression was observed to increase during adipogenesis, in the absence of Notch3 there was a significant increase in hASC adipogenesis as demonstrated through an increased number of lipid vesicles, and increased expression of adipogenic markers ppar-γ, adiponectin, fabp4, and plin2. Although Notch3 is only one of four Notch receptors expressed on the surface of hASCs, this receptor appears important for proper regulation of adipogenic differentiation, possibly serving as a negative regulator to prevent inappropriate adipogenesis or promote other lineage commitments of ASCs.