NOTCH3 limits the epithelial-mesenchymal transition and predicts a favorable clinical outcome in esophageal cancer.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human squamous cell carcinomas and is characterized by chemotherapy resistance and poor prognosis associated with the epithelial-mesenchymal transition (EMT). A subset of ESCC displays loss-of-function mutations in genes encoding Notch receptor family members, including NOTCH3. Although Notch signaling regulates EMT in ESCC cells, the role of NOTCH3 in EMT and chemotherapy resistance remains elusive. This study aimed to examine the role of NOTCH3 in EMT and chemotherapy resistance, and determine whether NOTCH3 expression can be used to predict the response to chemotherapy. METHODS: In vitro and in vivo assays were conducted to clarify the contribution of NOTCH3 to chemotherapy resistance. Using specimens from 120 ESCC patients treated with neoadjuvant chemotherapy, we compared the expression levels of NOTCH3 and genes involved in EMT according to the degree of chemotherapy sensitivity. RESULTS: In ESCC cells, chemotherapy resistance was associated with NOTCH3 downregulation and concurrent activation of EMT. RNA interference to silence NOTCH3 resulted in induction of the EMT marker Vimentin (VIM), leading to chemotherapy resistance in ESCC cells. Conversely, ectopic expression of the activated form of NOTCH3 suppressed EMT and sensitized cells to chemotherapy. Results of chromatin immunoprecipitation assays suggested that NOTCH3 may repress transcription of the VIM. CONCLUSIONS: Our findings suggest that NOTCH3 may control chemotherapy sensitivity by regulating EMT. NOTCH3 may serve as a novel biomarker to predict better clinical outcomes in ESCC patients.

Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells.

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

ER stress and Rho kinase activation underlie the vasculopathy of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.

NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma.

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRß+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRßlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.