Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009. © 2009 Wiley‐Liss, Inc.     

Correlation between HIV‐1 seropositivity and prevalence of a γ‐secretase polymorphism in two distinct ethnic populations

Susceptibility for human immunodeficiency virus type 1 (HIV‐1) infection may be influenced by host genetics. Recent findings with a Wistar rat model raised the possibility that the γ‐secretase pathway may be associated with an individual's susceptibility to infection. A functional single‐nucleotide polymorphism (SNP) in the γ‐secretase component APH1B (Phe217Leu; rs1047552) was therefore analyzed for association with HIV‐1 infection. The SNP showed a tendency for association with HIV‐1 infection in a Xhosa indigenous South African Bantu study (P = 0.087), and associated significantly in a Caucasian Dutch study (P = 0.049). Together, the results suggest a role for the γ‐secretase pathway in susceptibility to HIV‐1 infection. J. Med. Virol. 81:1847–1851, 2009. © 2009 Wiley‐Liss, Inc.     

Calsenilin Contributes to Neuronal Cell Death in Ischemic Stroke

Calsenilin is a calcium sensor protein that interacts with presenilin and increases calcium‐triggered neuronal apoptosis, and γ‐secretase activity. Notch is a cell surface receptor that regulates cell‐fate decisions and synaptic plasticity in brain. The aim of the present study was to characterize the role of calsenilin as a regulator of the γ‐secretase cleavage of Notch in ischemic stroke. Here, we determined the modulation of expression level and cellular distribution of calsenilin in neurons subjected to ischemic‐like conditions. The levels of calsenilin and presenilin were increased in primary neurons after oxygen and glucose deprivation. Furthermore, calsenilin was found to enhance the γ‐secretase cleavage of Notch and to contribute to cell death under ischemia‐like conditions. The inhibition of γ‐secretase activity and a presenilin deficiency were both found to protect against calsenilin‐mediated ischemic neuronal death. The expression of calsenilin was found to be increased in brain following experimental ischemic stroke. These findings establish a specific molecular mechanism by which the induction of calsenilin enhances Notch activation in ischemic stroke, and identify calsenilin as an upstream of the γ‐secretase cleavage of Notch.     

Inhibition of BMP activity protects epithelial barrier function in lung injury

Epithelial injury is a central finding in pulmonary disease and is accompanied by disruption of epithelial barrier function, leading to pulmonary oedema and inflammation. Injured epithelial cells lose their properties and gain mesenchymal characteristics, a phenotypic switch that contributes to lung remodelling after injury. Here we studied bone morphogenetic protein (BMP) signalling and, in particular, the role of BMP2 and the BMP modulator BMPER in injured lung epithelium. Increased BMP activity, reflected by up‐regulation of the Smad1/5–Id1 axis, is detected after injury of lung epithelium in vitro and in vivo. Two members of the BMP family, BMP2 and BMPER, have opposing effects. BMP2 is up‐regulated after epithelial injury and causes epithelial dysfunction and hyperpermeability, mediated by the Smad1/5–Id1‐dependent down‐regulation of E‐cadherin. In contrast, BMPER expression is decreased following injury, which in turn impairs epithelial integrity, characterized by reduction of E‐cadherin and epithelial leakage in vitro and in vivo. High levels of BMPER antagonized BMP2‐Smad5–Id1 signalling and prevented BMP2‐mediated decrease of E‐cadherin and hyperpermeability, suggesting that BMPER restores epithelial homeostasis. Supporting this notion, pharmacological inhibition of BMP signalling by LDN193189 prevented reduction of E‐cadherin and disruption of epithelial barrier function. Inhibition of excessive BMP activation could be a new approach to restore epithelial integrity and prevent disruption of epithelial barrier function after lung injury. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype

Zardawi S J, Zardawi I, McNeil C M, Millar E K A, McLeod D, Morey A L, Crea P, Murphy N C, Pinese M, Lopez‐Knowles E, Oakes S R, Ormandy C J, Qiu M R, Hamilton A, Spillane A, Soon Lee C, Sutherland R L, Musgrove E A & O’Toole S A
(2010) Histopathology 56, 286–296 High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype Aims: Activation of Notch signalling results in hyperplasia and tumorigenesis in murine mammary epithelium. However, there is little information regarding the expression of Notch1 in premalignant lesions and early breast cancer. We investigated expression of Notch1 in breast cancer development and its association with molecular subtypes. Methods and results: Immunohistochemical expression of Notch1 was determined in a murine model of mammary carcinogenesis and in breast tissue from two cohorts of breast cancer patients, the first (n = 222) comprising a histological progression series and the second an outcome series of 228 patients with operable invasive ductal carcinoma. Enhanced expression of Notch1 protein was an early event in both murine and human breast cancer development with progressive increases in expression with the development of hyperplasia and malignancy. High Notch1 was not prognostic in the outcome cohort. There was, however, a highly significant association of high Notch1 protein with the HER‐2 molecular subtype of breast cancer (P = 0.008). Conclusions: These data demonstrate that aberrant Notch regulation is an early event in mammary carcinogenesis and is associated with the HER‐2 molecular subtype of breast cancer, and suggest the Notch signalling pathway may be a potential therapeutic target worthy of further investigation.     

Loss of miR‐101 expression promotes Wnt/β‐catenin signalling pathway activation and malignancy in colon cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer‐related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR‐101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR‐101 re‐expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR‐101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR‐101 expression and important CRC pro‐malignant features, such as inflammation, activation of the Wnt/β‐catenin signalling pathway and epithelial–mesenchymal transition (EMT). We then propose that up‐regulated miR‐101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR‐101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dichotomy of Notch signalling in regulating tumour immune surveillance

Notch signalling is an evolutionarily conserved multifaceted pathway that controls diverse cellular processes. Its role in regulating development and tissue homeostasis is well established. Aberrant activation of the Notch pathway has been implicated in the initiation and progression of many types of cancers. However, although in some cancers Notch signalling acts as a tumour‐promoter, in others it is reported to suppress tumour growth and progression. Accumulating evidence suggests the involvement of both the innate and adaptive immune system in the development of various tumours. Currently, extensive studies on investigating the effects of Notch signalling in tumour immune surveillance are being carried out. Interestingly, recent literature shows how the changing expression of Notch genes in different T cell subsets like CD4 and CD8 helps in controlling anti‐tumour immune responses. In this review, we discuss in depth the roles of Notch signalling molecules and different immune cells in the context of the tumour microenvironment. We also outline how current knowledge can be exploited to develop novel therapies in order to control the propagation of cancer stem cells.     

TUSC3 promotes colorectal cancer progression and epithelial–mesenchymal transition (EMT) through WNT/β‐catenin and MAPK signalling

Colorectal cancer (CRC) is one of the most common malignancies and is the second leading cause of cancer death in humans. Tumour suppressor candidate 3 (TUSC3) plays an important role in embryogenesis and metabolism. Deletion of TUSC3 often causes non‐syndromic mental retardation. Even though TUSC3 deregulation is frequently observed in epithelial cancers, the function of TUSC3 in CRC has remained unknown. In this study, we observed greater expression of TUSC3 at the mRNA and protein level in clinical colorectal tumour samples compared with paired normal tissues. Gain‐ and loss‐of‐function analyses were performed to evaluate the functional significance of TUSC3 in CRC initiation and progression. Immunoblotting, immunofluorescence, and co‐immunoprecipitation analyses were used to identify potential pathways with which TUSC3 might be involved. Overexpression of TUSC3 in CRC cells induced epithelial–mesenchymal transition (EMT) in CRC cells, accompanied by down‐regulation of the epithelial marker, E‐cadherin, and up‐regulation of the mesenchymal marker, vimentin. Increased proliferation, migration, and invasion, as well as accelerated xenograft tumour growth, were observed in TUSC3‐overexpressing CRC cells, while opposite effects were achieved in TUSC3‐silenced cells. In conclusion, our study demonstrated the oncogenic role of TUSC3 in CRC and showed that TUSC3 may be responsible for alternations in the proliferation ability, aggressiveness, and invasive/metastatic potential of CRC through regulating the MAPK, PI3K/Akt, and Wnt/β‐catenin signalling pathways. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Lentivirus‐mediated somatic recombination and development of a novel mouse model for sporadic colorectal cancer

In this study, we have developed a novel mouse model for sporadic colorectal cancer (CRC) by utilizing APC conditional knockout (Apc^CKO) mouse and lentivirus encoding Cre recombinase and a reporter gene (EGFP or LacZ). Lentiviral transduction of colonic crypt stem cells allowed for the long‐term expression of reporter gene as well as excision of floxed Apc alleles, which resulted in tumor development. Tumors represented adenoma stages along with the nuclear accumulation of β‐catenin. Loss of E‐cadherin at the cellular junctions and strong expression of Vimentin suggested the sign of active epithelial‐mesenchymal transition. Moreover, nuclear staining of Ki67 inside epithelial cells of aberrant crypts demonstrated their higher proliferative nature. Erratic downstream signaling of activated Wnt/β‐catenin, AKT/mTOR, and Notch pathways provided strong evidence towards the higher proliferative index of epithelial cells inside the aberrant crypts. These results do recapitulate the findings of previous APC mutant mouse models. Our model represents sporadic CRC more precisely as (i) tumors result from somatic mutations but not from germline; (ii) tumors develop in colon not in small intestine; (iii) few tumors develop at the distal end of colons. Additionally, our model allows for the long‐term expression of the gene(s), which get integrated into the host cell genome and provides an ability to track the tumor growth. © 2016 Wiley Periodicals, Inc.     

Notch in the kidney: development and disease

Notch signalling is a highly conserved cell-cell communication mechanism that regulates development, tissue homeostasis, and repair. Within the kidney, Notch has an important function in orchestrating kidney development. Recent studies indicate that Notch plays a key role in establishing proximal epithelial fate during nephron segmentation as well as the differentiation of principal cells in the renal collecting system. Notch signalling is markedly reduced in the adult kidney; however, increased Notch signalling has been noted in both acute and chronic kidney injury. Increased glomerular epithelial Notch signalling has been associated with albuminuria and glomerulosclerosis, while tubular epithelial Notch activation caused fibrosis development most likely inducing an improper epithelial repair pathway. Recent studies thereby indicate that Notch is a key regulator of kidney development, repair, and injury.     

Arid1a inactivation in an Apc‐ and Pten‐defective mouse ovarian cancer model enhances epithelial differentiation and prolongs survival

Inactivation of the ARID1A tumour suppressor gene is frequent in ovarian endometrioid (OEC) and clear cell (OCCC) carcinomas, often in conjunction with mutations activating the PI3K–AKT and/or canonical Wnt signalling pathways. Prior work has shown that conditional bi‐allelic inactivation of the Apc and Pten tumour suppressor genes in the mouse ovarian surface epithelium (OSE) promotes outgrowth of tumours that reflect the biological behaviour and gene expression profiles of human OECs harbouring comparable Wnt and PI3K–AKT pathway defects, although the mouse tumours are more poorly differentiated than their human tumour counterparts. We found that conditional inactivation of one or both Arid1a alleles in OSE concurrently with Apc and Pten inactivation unexpectedly prolonged the survival of tumour‐bearing mice and promoted striking epithelial differentiation of the cancer cells, resulting in morphological features akin to those in human OECs. Enhanced epithelial differentiation was linked to reduced expression of the mesenchymal markers N‐cadherin and vimentin, and increased expression of the epithelial markers Crb3 and E‐cadherin. Global gene expression profiling showed enrichment for genes associated with mesenchymal–epithelial transition in the Arid1a‐deficient tumours. We also found that an activating (E545K) Pik3ca mutation, unlike Pten inactivation or Pik3ca H1047R mutation, cannot cooperate with Arid1a loss to promote ovarian cancer development in the mouse. Our results indicate that the Arid1a tumour suppressor gene has a key role in regulating OEC differentiation, and paradoxically the mouse cancers with more initiating tumour suppressor gene defects had a less aggressive phenotype than cancers arising from fewer gene alterations. Microarray data have been deposited in NCBI's Gene Expression Omnibus (GSE67695). Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dengue virus up‐regulates expression of notch ligands Dll1 and Dll4 through interferon‐β signalling pathway

The Notch signalling pathway is involved in multiple cellular processes and has been recently indicated to modulate the host immune response. However, the role of the Notch pathway in dengue virus (DENV) infection remains unknown. Our study has screened the expression profile of Notch receptors, ligands and target genes in human monocytes, macrophages and dendritic cells in response to DENV infection. The real‐time PCR data showed that Notch ligand Dll1 was significantly induced in DENV‐infected monocytes; and receptor Notch4, ligands Dll1 and Dll4, and target Hes1 were dramatically enhanced in DENV‐infected macrophages and dendritic cells. In macrophages, induction of Dll1 and Dll4 mediated by DENV2 was increased by treatment with interferon‐β (IFN‐β), and was impaired by neutralization of IFN‐β. The DENV‐induced Dll1 and Dll4 expression level was decreased by silencing key innate immune molecules, including Toll‐like receptor 3 (TLR3), MyD88, RIG‐I and IPS‐I. In IFN‐receptor‐depleted macrophages, the Dll1 and Dll4 induction was significantly alleviated. Functionally, activation of Notch signalling by Dll1 in CD4⁺ T cells enhanced the expression of a T helper type 1 (Th1) cytokine IFN‐γ, while Notch activation in macrophages had no direct effect on replication of DENV. Our data revealed that the expressions of Notch ligands in antigen‐presenting cells were differentially induced by DENV via innate immune signalling, which is important for Th1/Th2 differentiation during adaptive immune response.     

Differential Notch and TGFbeta signaling in primary colorectal tumors and their corresponding metastases

BACKGROUND: Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFbeta signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases. METHODS: Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related to markers of Wnt (beta-catenin), Notch (HES1) and TGFbeta (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed. RESULTS: When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear beta-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear beta-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear beta-catenin. CONCLUSIONS: Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFbeta signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFbeta pathways is reduced in secondary colorectal tumors.     

Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling

Tzelepi V, Karlou M, Wen S, Hoang A, Logothetis C, Troncoso P & Efstathiou E
(2011) Histopathology 58, 1037–1047
Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling Aims: The hedgehog (Hh) signalling pathway has been implicated in the pathogenesis and aggressiveness of prostate cancer through epithelial–mesenchymal interactions. The aim of this study was to elucidate the cell‐type partitioned expression of the Hh pathway biomarkers in the non‐neoplastic and tumour microenvironments and to correlate it with the grade and stage of prostate cancer. Methods and results: Expression of the Hh pathway components (Shh, Smo, Ptch, Gli1) in the microenvironment of non‐neoplastic peripheral zone (n = 119), hormone‐naive primary prostate carcinoma (n = 141) and castrate‐resistant bone marrow metastases (n = 53) was analysed using immunohistochemistry in tissue microarrays and bone marrow sections. Results showed that epithelial Shh, Smo and Ptch expression was up‐regulated, whereas stromal Smo, Ptch, and Gli1 expression was down‐regulated in prostate carcinomas compared to non‐neoplastic peripheral zone tissue. Ptch expression was modulated further in high‐grade and high‐stage primary tumours and in bone marrow metastases. Hh signalling correlated with ki67 and vascular endothelial growth factor (VEGF) but not with CD31 expression. Conclusion: Our results highlight the importance of Hh‐mediated epithelial–mesenchymal interactions in the non‐neoplastic prostate and imply that shifting the balance from paracrine towards autocrine signalling is important in the pathogenesis and progression of prostate carcinoma.     

Efficient molecular subtype classification of high‐grade serous ovarian cancer

High‐grade serous carcinomas (HGSCs) account for approximately 70% of all epithelial ovarian cancers diagnosed. Using microarray gene expression profiling, we previously identified four molecular subtypes of HGSC: C1 (mesenchymal), C2 (immunoreactive), C4 (differentiated), and C5 (proliferative), which correlate with patient survival and have distinct biological features. Here, we describe molecular classification of HGSC based on a limited number of genes to allow cost‐effective and high‐throughput subtype analysis. We determined a minimal signature for accurate classification, including 39 differentially expressed and nine control genes from microarray experiments. Taqman‐based (low‐density arrays and Fluidigm), fluorescent oligonucleotides (Nanostring), and targeted RNA sequencing (Illumina) assays were then compared for their ability to correctly classify fresh and formalin‐fixed, paraffin‐embedded samples. All platforms achieved > 90% classification accuracy with RNA from fresh frozen samples. The Illumina and Nanostring assays were superior with fixed material. We found that the C1, C2, and C4 molecular subtypes were largely consistent across multiple surgical deposits from individual chemo‐naive patients. In contrast, we observed substantial subtype heterogeneity in patients whose primary ovarian sample was classified as C5. The development of an efficient molecular classifier of HGSC should enable further biological characterization of molecular subtypes and the development of targeted clinical trials. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Notch signalling in T-cell lymphoblastic leukaemia/lymphoma and other haematological malignancies

Notch receptors participate in a highly conserved signalling pathway that regulates normal development and tissue homeostasis in a context- and dose-dependent manner. Deregulated Notch signalling has been implicated in many diseases, but the clearest example of a pathogenic role is found in T-cell lymphoblastic leukaemia/lymphoma (T-LL), in which the majority of human and murine tumours have acquired mutations that lead to aberrant increases in Notch1 signalling. Remarkably, it appears that the selective pressure for Notch mutations is virtually unique among cancers to T-LL, presumably reflecting a special context-dependent role for Notch in normal T-cell progenitors. Nevertheless, there are some recent reports suggesting that Notch signalling has subtle, yet important roles in other forms of haematological malignancy as well. Here, we review the role of Notch signalling in various blood cancers, focusing on T-LL with an eye towards targeted therapeutics.