Heterogeneous expression and role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in small cell lung cancer.

The present study investigated the expression and role of ROR2 in small cell lung cancer (SCLC). To examine the expression of ROR2, 27 surgically resected SCLC tissue samples were immunostained for ROR2. Sixteen tissue samples were positive and some showed intratumor heterogeneity in staining intensity. The heterogeneity of ROR2 expression was also observed in tumor tissues from a PDX model of SCLC, in which there were cells with high ROR2 expression (ROR2high cells) and without its expression (ROR2low cells). These cells were subjected to a RNA sequence analysis. GSEA was performed and the results obtained revealed the enrichment of molecules such as G2M checkpoint, mitotic spindle, and E2F targets in ROR2high cells. The rate of EdU incorporation was significantly higher in ROR2high cells than ROR2low cells from the PDX model and the SCLC cell lines. Cell proliferation was suppressed in ROR2 KO SBC3 cells in vitro and in vivo. Comparisons of down-regulated differentially expressed genes in ROR2 KO SBC3 cells with up-regulated DEG in ROR2high cells from the PDX model revealed 135 common genes. After a Metascape analysis of these genes, we focused on Aurora kinases. In SCLC cell lines, the knockdown of ROR2 suppressed Aurora kinases. Therefore, ROR2 appears to regulate the cell cycle through Aurora kinases. The present results reveal a role for ROR2 in SCLC and afford a candidate system (ROR2-Aurora kinase) accompanying tumor heterogeneity in SCLC.

Pulmonary Neuroendocrine Cells and Small Cell Lung Carcinoma: Immunohistochemical Study Focusing on Mechanisms of Neuroendocrine Differentiation.

Neuroendocrine (NE) differentiation has been histochemically detected in normal and cancer tissues and cells. Immunohistochemical analyses have provided a more detailed understanding of NE biology and pathology. Pulmonary NE cells are a rare lung epithelial type, and small cell carcinoma of the lung (SCLC) is a high-grade NE tumor. Pulmonary NE and SCLC cells share common mechanisms for NE differentiation. Neural or NE cell lineage-specific transcription factors, such as achaete-scute homologue 1 (Ascl1) and insulinoma-associated protein 1 (INSM1), are crucial for the development of pulmonary NE cells, and NE differentiation is influenced by the balance between Ascl1 and the suppressive neural transcription factor, hairy-enhancer of split 1, a representative target molecule of the Notch signaling pathway. In this review, we discuss the importance of Ascl1 and INSM1 in identifying pulmonary NE and SCLC cells and introduce Ascl1-related molecules detected by comparative RNA-sequence analyses. The molecular classification of SCLC based on the expression of lineage-specific transcription or co-transcription factors, including ASCL1, NEUROD1, POU2F3, and YAP1, was recently proposed. We attempted to characterize these 4 SCLC subtypes using integrated immunohistochemical studies, which will provide insights into the molecular characteristics of these subtypes and clarify the inter- and intratumor heterogeneities of SCLC.

The role of YAP1 in small cell lung cancer.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ, also known as WWTR1) are core downstream effectors of the Hippo pathway, which is involved in diverse biological processes. The oncogenic effects of YAP and TAZ in non-small cell lung cancer (NSCLC) have recently been reported; however, their roles in SCLC remain unclear. Immunohistochemistry (IHC) on lung cancer tissues and Western blotting (WB) on lung cancer cell lines were performed to examine the expression of YAP1. Genome editing using CRISPR/Cas9 was then used to knockout the YAP1 gene in the H69AR cell line. An RNA-sequence analysis, gene ontology (GO) analysis, WB, cell counting assay, invasion assays, and xenograft studies were conducted on these cells to investigate the biological roles of YAP1. IHC revealed that insulinoma-associated protein 1 was expressed in most cases (28 out of 32 cases), while only four cases expressed YAP1. The knockout of YAP1 in H69AR cells, a chemically induced SCLC-Y subtype, reduced cell proliferation and invasion capacity and restored drug sensitivity. Xenograft assays revealed that the knockout of YAP1 suppressed cell proliferation. Tumor tissues showed the expression of neuroendocrine markers and a low Ki-67 index. In SCLC, YAP1 plays an important role in biological functions, such as cell proliferation, EMT, drug sensitivity, and neuroendocrine differentiation.

ZMYM3 May Promote Cell Proliferation in Small Cell Lung Carcinoma.

Zinc finger, myeloproliferative, and mental retardation-type containing 3 (ZMYM3) is a highly conserved protein among vertebrates. Although it promotes DNA repair and moderate histone acetylation, the other functions of ZMYM3 remain unclear. We herein examined the physiological functions of ZMYM3 in human lung cancer using a ZMYM3-knockdown small cell lung cancer (SCLC) cell line. ZMYM3-knockdown SCLC cells grew slowly and the Ki-67 labeling index was lower in ZMYM3-knockdown cells than in mock cells. The subcutaneous tumors that formed after xenotransplantation into immunodeficient mice were slightly smaller in the ZMYM3-knockdown group than in the mock group. Furthermore, public RNA-sequencing data analyses showed similar RNA profiles between ZMYM3 and some cell proliferation markers. These results indicate that ZMYM3 promotes cell proliferation in human lung carcinomas, particularly SCLC.

INSM1 immunostaining in solid papillary carcinoma of the breast.

Solid papillary carcinoma (SPC) is a histological subtype of breast carcinomas. At least 50% of SPC show neuroendocrine differentiation. Insulinoma-associated protein 1 (INSM1) is a transcription factor now employed as a useful neuroendocrine marker. It is suppressed by the Notch signaling pathway in other neuroendocrine tumors. However, the usefulness of INSM1 as a neuroendocrine marker and the relationships between INSM1 and NOTCH receptors in SPC of the breast currently remain unclear. To clarify the usefulness of INSM1 as a neuroendocrine marker and the relationships between INSM1 and NOTCH receptors in SPC, we performed immunohistochemistry using 19 tissue specimens of SPC of the breast. We complementarily analyzed public RNA sequencing data on breast carcinomas. Immunohistochemical examinations revealed that the staining intensity of INSM1 was significantly higher in the neuroendocrine group than in the non-neuroendocrine group. Positive correlations were observed between INSM1 and synaptophysin (SYP), or chromogranin-A (CHGA). In all cases, NOTCH 2 and 3 were positive, while NOTCH 1 and 4 were negative. According to public RNA data analyses, there were positive correlations between INSM1 and SYP, or CHGA, and negative correlations between INSM1 and NOTCH1-3. INSM1 is useful as a diagnostic marker for SPC with neuroendocrine differentiation in the breast.

Integrated Immunohistochemical Study on Small-Cell Carcinoma of the Lung Focusing on Transcription and Co-Transcription Factors.

Small-cell lung cancer (SCLC) is an aggressive malignant cancer that is classified into four subtypes based on the expression of the following key transcription and co-transcription factors: ASCL1, NEUROD1, YAP1, and POU2F3. The protein expression levels of these key molecules may be important for the formation of SCLC characteristics in a molecular subtype-specific manner. We expect that immunohistochemistry (IHC) of these molecules may facilitate the diagnosis of the specific SCLC molecular subtype and aid in the appropriate selection of individualized treatments. We attempted IHC of the four key factors and 26 candidate SCLC target molecules selected from the gene expression omnibus datasets of 47 SCLC samples, which were grouped based on positive or negative results for the four key molecules. We examined differences in the expression levels of the candidate targets and key molecules. ASCL1 showed the highest positive rate in SCLC samples, and significant differences were observed in the expression levels of some target molecules between the ASCL1-positive and ASCL1-negative groups. Furthermore, the four key molecules were coordinately and simultaneously expressed in SCLC cells. An IHC study of ASCL1-positive samples showed many candidate SCLC target molecules, and IHC could become an essential method for determining SCLC molecular subtypes.

Distinct transcriptional programs of SOX2 in different types of small cell lung cancers.

SOX2 is recognized as an oncogene in human small cell lung cancer (SCLC), which is an aggressive neuroendocrine (NE) tumor. However, the role of SOX2 in SCLC is not completely understood, and strategies to selectively target SOX2 in SCLC cells remain elusive. Here, we show, using next-generation sequencing, that SOX2 expressed in the ASCL1-high SCLC (SCLC-A) subtype cell line is dependent on ASCL1, which is a lineage-specific transcriptional factor, and is involved in NE differentiation and tumorigenesis. ASCL1 recruits SOX2, which promotes INSM1 and WNT11 expression. Immunohistochemical studies revealed that SCLC tissue samples expressed SOX2, ASCL1, and INSM1 in 18 out of the 30 cases (60%). Contrary to the ASCL1-SOX2 signaling axis controlling SCLC biology in the SCLC-A subtype, SOX2 targets distinct genes such as those related to the Hippo pathway in the ASCL1-negative, YAP1-high SCLC (SCLC-Y) subtype. Although SOX2 knockdown experiments suppressed NE differentiation and cell proliferation in the SCLC-A subtype, they did not sufficiently impair the growth of the SCLC-Y subtype cell lines in vitro and ex vivo. The present results support the importance of the ASCL1-SOX2 axis as a main subtype of SCLC, and suggest the therapeutic potential of targeting the ASCL1-SOX2 axis.

Kiss1R Identification and Biodistribution Analysis Employing a Western Ligand Blot and Ligand-Derivative Stain with a FITC-Kisspeptin Derivative.

It is not always easy to establish specific antibodies against receptors. Most receptors are hydrophobic and have complicated three-dimensional structures, making them difficult to use as immunogens. Thus, we developed receptor detection methods with a fluorescein-labeled ligand as an antibody alternative, which we referred to as a western ligand blot (WLB) and ligand derivative stain (LDS). Kisspeptin receptor (Kiss1R) was detected by its ligand. Kiss1R expression was confirmed in eight human cell lines by the WLB and in four pathological tissues by the LDS. Next, Kiss1R was stained by LDS in organs, revealing Kiss1R expression by [67 Ga]Ga-DOTA-kisspeptin 10 accumulation. As a result, Kiss1R-expressing cells in each organ could be stained with fluorescein-labeled kisspeptin 14 instead of an antibody and observed by light microscopy. The combination of the WLB and LDS allows identification of receptors in tissues, which can be readily applied to target receptor detection by a synthetic ligand derivative.

Significance of achaete-scute complex homologue 1 (ASCL1) in pulmonary neuroendocrine carcinomas; RNA sequence analyses using small cell lung cancer cells and Ascl1-induced pulmonary neuroendocrine carcinoma cells.

ASCL1 is one of the master transcription factors of small cell lung carcinoma (SCLC). To investigate the significance of ASCL1 in pulmonary neuroendocrine carcinoma, we performed 2 comparative RNA-seq studies between H69 (ASCL1-positive, classical type SCLC) and H69AR (ASCL1-negative, variant type SCLC) and between ASCL1-transfected A549 adenocarcinoma cell lines (A549(ASCL1+) cell lines) and A549(control) cell lines. RNA-seq analyses revealed that 940 genes were significantly different between the H69 and H69AR cell lines, and 728 between the A549(ASCL1+) and A549(control) cell lines. In total, 120 common genes between these analyses were selected as candidate ASCL1-related genes, and included genes with various cellular functions, such as neural development, secretion, growth, and morphology. Their expression degrees in three classical and two variant SCLC cell lines, two A549(ASCL1+) and two A549(control) cell lines were subjected to quantitative PCR analyses. Since the candidate ASCL1-related genes were strongly expressed in the classical SCLC and A549(ASCL1+) cell lines and more weakly expressed in the variant SCLC and A549(control) cell lines, the ASCL1-related 7 molecules INSM1, ISL1, SYT4, KCTD16, SEZ6, MS4A8, and COBL were further selected. These molecules suggested diverse functions for A549(ASCL1+): INSM1 and ISL1 are transcription factors associated with neuroendocrine differentiation, while SYT4, KTCD16, and SEZ6 may be related to neurosecretory functions and MS4A8 and COBL to cell growth and morphology. An immunohistochemistry of these seven molecules was performed on lung carcinoma tissues and the xenotransplanted tumors of A549(ASCL1+), and they were preferentially and positively stained in ASCL1-postive tumor tissues.

Ascl1-induced Wnt11 regulates neuroendocrine differentiation, cell proliferation, and E-cadherin expression in small-cell lung cancer and Wnt11 regulates small-cell lung cancer biology.

The involvement of Wnt signaling in human lung cancer remains unclear. This study investigated the role of Wnt11 in neuroendocrine (NE) differentiation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) in human small-cell lung cancer (SCLC). Immunohistochemical staining of resected specimens showed that Wnt11 was expressed at higher levels in SCLCs than in non-SCLCs; 58.8% of SCLC, 5.2% of adenocarcinoma (ADC), and 23.5% of squamous cell carcinoma tissues stained positive for Wnt11. A positive relationship was observed between Achaete-scute complex homolog 1 (Ascl1) and Wnt11 expression in SCLC cell lines, and this was supported by transcriptome data from SCLC tissue. The expression of Wnt11 and some NE markers increased after the transfection of ASCL1 into the A549 ADC cell line. Knockdown of Ascl1 downregulated Wnt11 expression in SCLC cell lines. Ascl1 regulated Wnt11 expression via lysine H3K27 acetylation at the enhancer region of the WNT11 gene. Wnt11 controlled NE differentiation, cell proliferation, and E-cadherin expression under the regulation of Ascl1 in SCLC cell lines. The phosphorylation of AKT and p38 mitogen-activated protein kinase markedly increased after transfection of WNT11 into the SBC3 SCLC cell line, which suggests that Wnt11 promotes cell proliferation in SCLC cell lines. Ascl1 plays an important role in regulating the Wnt signaling pathway and is one of the driver molecules of Wnt11 in human SCLC. Ascl1 and Wnt11 may employ a cooperative mechanism to control the biology of SCLC. The present results indicate the therapeutic potential of targeting the Ascl1-Wnt11 signaling axis and support the clinical utility of Wnt11 as a biological marker in SCLC.

Significance of Tsukushi in lung cancer.

OBJECTIVES: Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan (SLRP) family, plays multifunctional roles by interacting with signaling molecules during development. However, the role of TSK in cancer remains unknown. The aim of the present study was to investigate the biological significance of TSK in lung cancer. MATERIALS AND METHODS: Immunohistochemistry of lung cancer tissues and reverse transcription polymerase chain reaction (PCR) of lung cancer cell lines were carried out to detect TSK. Then, RNA sequence analysis, Gene Ontology analysis, quantitative real-time PCR, western blotting, cell counting assay, invasion assays, and xenograft studies were done in a human lung adenocarcinoma cell line, H1975 with modification of TSK expression levels, in order to investigate its biological roles, in particular epithelial-mesenchymal transition (EMT) and proliferation. RESULTS: TSK was found to be highly expressed in lung cancer tissues and cell lines. Modification of TSK expression levels in H1975 resulted in changes in molecules related to EMT, including cadherin-1, snail family transcriptional repressor 1, snail family transcriptional repressor 2, and vimentin. The results of cell counting and xenograft assays showed that TSK promotes cell proliferation. CONCLUSIONS: In lung cancer cells, TSK is expressed more highly than the other SLRPs family members, and regulates the EMT and proliferation. Thus, TSK may be a key coordinator of multiple pathways and an important structural element in the lung cancer microenvironment.

Expression of Draxin in Lung Carcinomas.

Guidance molecules, such as Netrin-1, and their receptors have important roles in controlling axon pathfinding, modulate biological activities of various cancer cells, and may be a useful target for cancer therapy. Dorsal repulsive axon guidance protein (Draxin) is a novel guidance molecule that binds not only common guidance molecule receptors with Netrin-1, but also directly binds the EGF domain of Netrin-1 through a 22-amino-acid peptide (22aa). By immunostaining, Draxin was positively expressed in small cell carcinoma, adenocarcinoma (ADC), and squamous cell carcinoma of the lung. In addition, western blot analysis revealed that Draxin was expressed in all histological types of lung cancer cell lines examined. Knockdown of Draxin in an ADC cell line H358 resulted in altered expression of molecules associated with proliferation and apoptosis. The Ki-67 labeling index of Draxin-knockdown ADC cells was increased compared to that of control ADC cells. In H358 cells, treatment of 22aa induced phosphorylation of histone H3, but did not change apoptosis-associated enzymes. These data suggest that Draxin might be involved in cell proliferation and apoptosis in lung adenocarcinoma cells.

Pathobiology of Notch2 in lung cancer.

Notch signalling has been reported to be involved in initiation, progression, and suppression in various types of cancer. The pathological significance of Notch1 has been well studied in lung cancer, but that of Notch2 remains unclear. An immunohistochemical study was performed to clarify the expression of NOTCH2 in non-neoplastic lung tissues and lung cancers in comparison with Clara (Club) cell 10 kDa protein (CC10), and western blotting analysis was performed to detect NOTCH2 in human cancer cell lines. A Notch2 gene knockdown experiment was carried out to reveal the function of Notch2. Transient transfection of the intracellular domain of the Notch2 (N2ICD) gene was used. In addition, the relationship of the expressions of Notch1, 2, and 3 was studied. Immunohistochemical study of lung tissues revealed that NOTCH2 was detected in bronchiolar epithelial cells and was often colocalised with CC10, and that adenocarcinoma tissues were more positively stained than those of squamous cell carcinoma and small cell carcinoma. In human lung cancer cell lines, expression of NOTCH2 was similar to that of NOTCH1, and preferentially detected in non-small cell lung carcinoma (NSCLC) cell lines. Knockdown of the Notch2 gene in NSCLC cell lines showed no remarkable changes in expression of molecules associated with cell differentiation, proliferation, apoptosis, and motility, and the seemingly unvalued effects of Notch2 gene knockdown could be masked by concomitant Notch1 activation, as indicated by an increase in the intracellular domain of NOTCH1. Additionally, transient transfection of the N2ICD gene induced CC10 expression in an adenocarcinoma cell line. The present study revealed that Notch2 is important in Club cell differentiation in normal lungs and adenocarcinoma. Notch2 is regulated mutually with Notch1, and the balance of the expression of Notch receptors could determine the biological behaviours of lung cancer cells.

Correlation between histone acetylation and expression of Notch1 in human lung carcinoma and its possible role in combined small-cell lung carcinoma.

Combined small-cell lung carcinoma (cSCLC) is composed of small-cell lung carcinoma (SCLC) admixed with non-small-cell lung carcinoma (NSCLC). Evaluating the molecular differences between SCLC and NSCLC could lead to a better understanding of the pathogenesis of such neoplasms. Therefore, in this study, we investigated the correlation between histone acetylation and Notch1 expression in lung carcinoma. Using chromatin immunoprecipitation (ChIP) assay, we measured the level of acetylated histone H3 around the promoter region of Notch1 in SCLC and NSCLC cells. We then treated SCLC cells with trichostatin A (TSA) and characterized the level of histone H3 acetylation at Notch1. In addition, TSA-treated cells were injected into immune-compromised mice, for analysis of the ex vivo tumor xenograft phenotype. The level of acetylated histone H3 surrounding the Notch1 promoter was lower in lung cancer cells not expressing Notch1. Tumors originated from TSA-treated SCLC cells occasionally formed an epithelial-like glandular arrangement of cells; with Notch1 expression and decreased expression of neuroendocrine (NE) markers. Histone deacetylation around the promoter region of Notch1 inhibits Notch1 protein expression in SCLC and the restoration of Notch1 expression in SCLC leads to the concurrent appearance of epithelial-like areas within the SCLC, which could provide a possible mechanism for histogenesis of cSCLC.

Significance of Stat3 Signaling in Epithelial Cell Differentiation of Fetal Mouse Lungs.

To study the significance of signal transducer and activator of transcription (Stat) 3 in lung epithelial development of fetal mice, we examined fetal mouse lungs, focusing on the expression of Clara cell secretory protein (CCSP), Forkhead box protein J1 (Foxj1), calcitonin gene-related peptide (CGRP), phosphorylated Stat3 (Tyr705), and hairy/enhancer of split (Hes) 1, and observed cultured fetal lungs upon treatment with IL-6, a Stat3 activator, or cucurbitacin I, a Stat3 inhibitor. Moreover, the interaction of Stat3 signaling and Hes1 was studied using Hes1 gene-deficient mice. Phosphorylated Stat3 was detected in fetal lungs and, immunohistochemically, phosphorylated Stat3 was found to be co-localized in developing Clara cells, but not in ciliated cells. In the organ culture studies, upon treatment with IL-6, quantitative RT-PCR revealed that CCSP mRNA increased with increasing Stat3 phosphorylation, while cucurbitacin I decreased Hes1, CCSP, Foxj1 and CGRP mRNAs with decreasing Stat3 phosphorylation. In the lungs of Hes1 gene-deficient mice, Stat3 phosphorylation was not markedly different from wild-type mice, the expression of CCSP and CGRP was enhanced, and the treatment of IL-6 or cucurbitacin I induced similar effects on mouse lung epithelial differentiation regardless of Hes1 expression status. Stat3 signaling acts in fetal mouse lung development, and seems to regulate Clara cell differentiation positively. Hes1 could regulate Clara cell differentiation in a manner independent from Stat3 signaling.

Somatostatin receptor staining in FFPE sections using a ligand derivative dye as an alternative to immunostaining.

The confirmation of target expression in tissues is a prerequisite for molecular-targeted therapy. However, difficulties are sometimes associated with the production of appropriate antibodies against receptors. We herein developed a ligand derivative dye for the staining of receptors. The somatostatin receptor (sstr) was selected as the target and FITC-octreotate as the detective agent. We performed a blot analysis to detect sstr in the transfer membrane. The sstr2 recombinant protein or cell lysate from a small cell lung carcinoma cell line (H69) was boiled and loaded onto SDS-PAGE, and the proteins were transferred to a membrane. Even after denaturing processes, FITC-octreotate still bound sstr on the membrane. Furthermore, FITC-octreotate depicted the expression of sstr in formalin-fixed and paraffin-embedded (FFPE) sections, a method that we named ligand derivative staining (LDS). The accuracies of immunostaining and LDS were compared at the points of the detection of sstr using FFPE sections of 30 neuroendocrine tumor specimens. The sensitivity of LDS was 81.8%, while those of immunostaining using anti-sstr2 and sstr5 antibodies were 72.7% and 63.6%, respectively. Thus, LDS appears to be superior to immunostaining. A ligand derivative may be used as a substitute for antibodies, and has the potential to support economical, simple, and accurate detection methods.

Small cell lung cancer, an epithelial to mesenchymal transition (EMT)-like cancer: significance of inactive Notch signaling and expression of achaete-scute complex homologue 1.

Small cell lung cancer (SCLC) is one of the most malignant neoplasms in common human cancers. The tumor is composed of small immature-looking cells with a round or fusiform shape, which possesses weak adhesion features among them, suggesting that SCLC shows the morphological characteristics of epithelial to mesenchymal transition (EMT). SCLC is characterized by high metastatic and recurrent rates, sensitivity to the initial chemotherapy, and easy acquirement of chemoresistance afterwards. These characters may be related to the EMT phenotype of SCLC. Notch signaling is an important signaling pathway, and could have roles in regulating neuroendocrine differentiation, proliferation, cell adhesion, EMT, and chemoresistance. Notch1 is usually absent in SCLC in vivo, but could appear after chemotherapy. Notch1 can enhance cell adhesion by induction of E-cadherin in SCLC, which indicates mesenchymal to epithelial transition. On the other hand, achaete-scute complex homologue 1 (ASCL1), negatively regulated by Notch signaling, is a lineage-specific gene of SCLC, and functions to promote neuroendocrine differentiation as well as EMT. ASCL1-transfected adenocarcinoma cell lines induced neuroendocrine phenotypes and lost epithelial cell features. SCLC is characterized by neuroendocrine differentiation and EMT-like features, which could be produced by inactive Notch signaling and ASCL1 expression. In addition, chemical and radiation treatments can activate Notch signaling, which suppress neuroendocrine differentiation and induces chemoradioresistance, accompanied by secession from EMT. Thus, the status of Notch signaling and ASCL1 expression may determine the cell behaviors of SCLC partly through modifying EMT phenotypes.

Evaluation of role of Notch3 signaling pathway in human lung cancer cells.

UNLABELLED: There is still a debate on the extent to which Notch3 signaling is involved in lung carcinogenesis and whether such function is dependent on cancer type or not. PURPOSE: To evaluate Notch3 expression in different types of human lung cancer cells. METHODS: Notch3 was detected in human lung cancer cell lines and in tissues. Then, small interfering RNA (siRNA) was used to down-regulate the expression of Notch3 in H69AR small cell lung carcinoma (SCLC) cells; two non-small cell lung carcinoma (NSCLC) cells; A549 adenocarcinoma (ADC); and H2170 squamous cell carcinoma (SCC). In addition, Notch3 intracellular domain (N3ICD) plasmid was transfected into H1688 human SCLC cells. We observed the effect of deregulating Notch3 signaling on the following cell properties: Notch-related proteins, cell morphology, adhesion, epithelial-mesenchymal transition (EMT), motility, proliferation and neuroendocrine (NE) features of SCLC. RESULTS: Notch3 is mainly expressed in NSCLC, and the expression of Notch1, Hes1 and Jagged1 is affected by Notch3. Notch3 has opposite functions in SCLC and NSCLC, being a tumor suppressor in the former and tumor promoting in the latter, in the context of cell adhesion, EMT and motility. Regarding cell proliferation, we found that inhibiting Notch3 in NSCLC decreases cell proliferation and induces apoptosis in NSCLC. Notch3 has no effect on cell proliferation or NE features of SCLC. CONCLUSION: Notch3 signaling in lung carcinoma is dependent on cell type. In SCLC, Notch3 behaves as a tumor suppressor pathway, while in NSCLC it acts as a tumor-promoting pathway.

Notch1 controls cell chemoresistance in small cell lung carcinoma cells.

BACKGROUND: Small cell lung carcinoma (SCLC) is characterized by a high rate of relapse and failure of chemotherapy because of the emergence of drug resistant cells. Notch signaling controls carcinogenesis in several human malignancies and could be involved in the resistance of cells to several chemotherapeutic agents. Herein, we analyzed the role of Notch1 signaling in the resistance of human SCLC cells to doxorubicin. METHODS: Small interfering ribonucleic acid technology was used to knock down (KD) Notch1 in H69AR and SBC-3 SCLC cells. We detected the effect of inhibiting Notch1 on the expression of drug resistant related molecules: multidrug resistance-associated protein (MRP-1) and anti-apoptotic factor B-cell lymphoma-2, as well as to cell adhesion molecule E-cadherin, which contributes to the adhesion of SCLC cells to the extracellular matrix and confers chemoresistance in a process known as cell adhesion-mediated drug resistance (CAM-DR). We also observed the effect of KD Notch1 on cell survival under high concentrations of doxorubicin treated media. RESULTS: H69AR and SBC-3 cells expressed Notch1 protein and grew as adherent aggregates, which confer resistance to high concentrations of doxorubicin. On inhibiting Notch1, we observed activation of the apoptotic pathway in cells, possibly resulting from the loss of CAM-DR and, thus, SBC-3 cells showed a loss of chemoresistant ability. However, in H69AR cells with KD Notch1, the expression of MRP-1 was increased and, thus, sustained the chemoresistant ability of cells. CONCLUSION: The Notch1 signaling pathway is involved in mediating the drug resistance phenotype of SCLC cells.