Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.

Receptor-activated transcription factors and beyond: multiple modes of Smad2/3-dependent transmission of TGF-ß signaling.

Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine that is widely distributed throughout the body. Its receptor proteins, TGF-ß type I and type II receptors, are also ubiquitously expressed. Therefore, the regulation of various signaling outputs in a context-dependent manner is a critical issue in this field. Smad proteins were originally identified as signal-activated transcription factors similar to signal transducer and activator of transcription proteins. Smads are activated by serine phosphorylation mediated by intrinsic receptor dual specificity kinases of the TGF-ß family, indicating that Smads are receptor-restricted effector molecules downstream of ligands of the TGF-ß family. Smad proteins have other functions in addition to transcriptional regulation, including post-transcriptional regulation of micro-RNA processing, pre-mRNA splicing, and m6A methylation. Recent technical advances have identified a novel landscape of Smad-dependent signal transduction, including regulation of mitochondrial function without involving regulation of gene expression. Therefore, Smad proteins are receptor-activated transcription factors and also act as intracellular signaling modulators with multiple modes of function. In this review, we discuss the role of Smad proteins as receptor-activated transcription factors and beyond. We also describe the functional differences between Smad2 and Smad3, two receptor-activated Smad proteins downstream of TGF-ß, activin, myostatin, growth and differentiation factor (GDF) 11, and Nodal.

The Snail signaling branch downstream of the TGF-ß/Smad3 pathway mediates Rho activation and subsequent stress fiber formation.

Cancer cells acquire malignant phenotypes through an epithelial-mesenchymal transition, which is induced by environmental factors or extracellular signaling molecules, including transforming growth factor-ß (TGF-ß). Among epithelial-mesenchymal transition-associated cell responses, cell morphological changes and cell motility are closely associated with remodeling of the actin stress fibers. Here, we examined the TGF-ß signaling pathways leading to these cell responses. Through knockdown experiments in A549 lung adenocarcinoma cells, we found that Smad3-mediated induction of Snail, but not that of Slug, is indispensable for morphological changes, stress fiber formation, and enhanced motility in cells stimulated with TGF-ß. Ectopic expression of Snail in SMAD3-knockout cells rescued the defect in morphological changes and stress fiber formation by TGF-ß, indicating that the role of Smad3 in these responses is to upregulate Snail expression. Mechanistically, Snail is required for TGF-ß-induced upregulation of Wnt5b, which in turn activates RhoA and subsequent stress fiber formation in cooperation with phosphoinositide 3-kinase. However, ectopic expression of Snail in SMAD3-knockout cells failed to rescue the defect in cell motility enhancement by TGF-ß, indicating that activation of the Smad3/Snail/Wnt5b axis is indispensable but not sufficient for enhancing cell motility; a Smad3-dependent but Snail-independent pathway to activate Rac1 is additionally required. Therefore, the Smad3-dependent pathway leading to enhanced cell motility has two branches: a Snail-dependent branch to activate RhoA and a Snail-independent branch to activate Rac1. Coordinated activation of these branches, together with activation of non-Smad signaling pathways, mediates enhanced cell motility induced by TGF-ß.

Indole-derived compound SIS3 targets a subset of activated Smad complexes.

Smad2 and Smad3 are receptor-regulated Smad proteins that transmit signals from cytokines belonging to the transforming growth factor (TGF)-ß family, which are vital for adult tissue homeostasis. The overactivation of such proteins often engenders the development of pathological conditions. Smad3 reportedly mediates TGF-ß-induced fibrosis. Although various potential Smad3-specific inhibitors are being developed, their specificity and action mechanisms remain largely unknown. This study aimed to establish a biochemical platform to monitor Smad2- or Smad3-dependent TGF-ß signaling using SMAD2, SMAD3 and SMAD2/3 knockout cell lines alongside TGF-ß-dependent luciferase reporters and Smad mutant proteins. Using this platform, SIS3, an indole-derived compound widely used as a specific Smad3 inhibitor, was observed to preferentially suppress a subset of activated Smad complexes. However, its inhibition did not favor Smad3 signaling over Smad2 signaling. These findings indicate that SIS3 can be employed as a probe to examine the heterogeneous nature of Smad signaling that induces gene expression. However, its use as a Smad3-specific inhibitor should be avoided.

Smad2Δexon3 and Smad3 have distinct properties in signal transmission leading to TGF-ß-induced cell motility.

In mammalian cells, Smad2 and Smad3, two receptor-regulated Smad proteins, play crucial roles in the signal transmission of transforming growth factor-ß (TGF-ß) and are involved in various cell regulatory processes, including epithelial-mesenchymal transition-associated cell responses, that is, cell morphological changes, E-cadherin downregulation, stress fiber formation, and cell motility enhancement. Smad2 contains an additional exon encoding 30 amino acid residues compared with Smad3, leading to distinct Smad2 and Smad3 functional properties. Intriguingly, Smad2 also has an alternatively spliced isoform termed Smad2Δexon3 (also known as Smad2ß) lacking the additional exon and behaving similarly to Smad3. However, Smad2Δexon3 and Smad3 signaling properties have not yet been compared in detail. In this study, we reveal that Smad2Δexon3 rescues multiple TGF-ß-induced in vitro cellular responses that would become defective upon SMAD3 KO but does not rescue cell motility enhancement. Using Smad2Δexon3/Smad3 chimeric proteins, we identified that residues Arg-104 and Asn-210 in Smad3, which are not conserved in Smad2Δexon3, are key for TGF-ß-enhanced cell motility. Moreover, we discovered that Smad2Δexon3 fails to rescue the enhanced cell motility as it does not mediate TGF-ß signals to downregulate transcription of ARHGAP24, a GTPase-activating protein that targets Rac1. This study reports for the first time distinct signaling properties of Smad2Δexon3 and Smad3.

Ets family proteins regulate the EMT transcription factors Snail and ZEB in cancer cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during epithelial tumor progression. Snail and ZEB1/2 (ZEB1 and ZEB2), known as EMT transcription factors, are key regulators of this transition. ZEB1/2 are positively correlated with EMT phenotypes and the aggressiveness of cancers. On the contrary, Snail is also correlated with the aggressiveness of cancers, but is not correlated with the expression of EMT marker proteins. Snail is induced by transforming growth factor-ß (TGF-ß), a well-known inducer of EMT, in various cancer cells. Interestingly, Snail induction by TGF-ß is markedly enhanced by active Ras signals. Thus, cancer cells harboring an active Ras mutation exhibit a drastic induction of Snail by TGF-ß alone. Here, we found that members of the E26 transformation-specific (Ets) transcription factor family, Ets1 and Ets2, contribute to the upregulation of both Snail and ZEB1/2. Snail induction by TGF-ß and active Ras is dramatically inhibited using siRNAs against both Ets1 and Ets2 together, but not on their own; in addition, siRNAs against both Ets1 and Ets2 also downregulate the constitutive expression of Snail and ZEB1/2 in cancer cells. Examination of several alternatively spliced variants of Ets1 revealed that p54-Ets1, which includes exon VII, but not p42-Ets1, which excludes exon VII, regulates the expression of the EMT transcription factors, suggesting that Ets1 is a crucial molecule for regulating Snail and ZEB1/2, and thus cancer progression is mediated through post-translational modification of the exon VII domain.

Simple digital system for the stabilization of holographic recordings of a slow grating in photorefractive materials.

We report on a digital system for the stabilization of an interference pattern of light fringes. This system uses a Raspberry Pi computer to operate a continuously stabilized setup and, because of the particular features of this stabilization setup, it is possible to record slow gratings in photorefractive materials. Our system proved to be effective, less expensive, and easy to operate, compared to the frequently employed setup with a lock-in amplifier, as it does not require specific equipment and/or specialized personnel.

Group V Secretory Phospholipase A2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line.

AIMS: It was suggested that group V secretory phospholipase A2 (sPLA2-V) existed in the nucleus. This study examined whether nuclear sPLA2-V plays a role in endocytosis of acetylated low-density lipoprotein (AcLDL) in monocyte/macrophage-like cell line RAW264.7 cells. METHODS: RAW264.7 cells were transfected with shRNA vector targeting sPLA2-V (sPLA2-V-knockdown [KD] cells) or empty vector (sPLA2-V-wild-type [WT] cells). AcLDL endocytosis was assessed by incubation with 125I-AcLDL or AcLDL conjugated with pHrodo. Actin polymerization was assessed by flow cytometry using Alexa Fluor 546-phalloidin. RESULTS: In immunofluorescence microscopic studies, sPLA2-V was detected in the nucleus. ChIP-Seq and ChIP-qPCR analyses showed binding of sPLA2-V to the promoter region of the phosphoglycerate kinase 1 (Pgk1) gene. In the promoter assay, sPLA2-V-KD cells had lower promoter activity of the Pgk1 gene than sPLA2-V-WT cells, and this decrease could be reversed by transfection with a vector encoding sPLA2-V-H48Q that lacks enzymatic activity. Compared with sPLA2-V-WT cells, sPLA2-V-KD cells had decreased PGK1 protein expression, beclin 1 (Beclin1) phosphorylation at S30, and class III PI3-kinase activity that could also be restored by transfection with sPLA2-V-H48Q. sPLA2-V-KD cells had impaired actin polymerization and endocytosis, which was reversed by introduction of sPLA2-V-H48Q or PGK1 overexpression. In sPLA2-V-WT cells, siRNA-mediated depletion of PGK1 suppressed Beclin1 phosphorylation and impaired actin polymerization and intracellular trafficking of pHrodo-conjugated AcLDL. CONCLUSIONS: Nuclear sPLA2-V binds to the Pgk1 gene promoter region and increases its transcriptional activity. sPLA2-V regulates AcLDL endocytosis through PGK1-Beclin1 in a manner that is independent of its enzymatic activity in RAW264.7 cells.

MAB21L4 regulates the TGF-ß-induced expression of target genes in epidermal keratinocytes.

Smad proteins transduce signals downstream of transforming growth factor-ß (TGF-ß) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-ß and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-ß induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-ß was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-ß. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-ß-induced MAB21L4 up-regulates the gene expression induced by TGF-ß, possibly through the inhibition of c-Ski via physical interaction in the cytosol.

EHF suppresses cancer progression by inhibiting ETS1-mediated ZEB expression.

ETS homologous factor (EHF) belongs to the epithelium-specific subfamily of the E26 transformation-specific (ETS) transcription factor family. Currently, little is known about EHF's function in cancer. We previously reported that ETS1 induces expression of the ZEB family proteins ZEB1/δEF1 and ZEB2/SIP1, which are key regulators of the epithelial-mesenchymal transition (EMT), by activating the ZEB1 promoters. We have found that EHF gene produces two transcript variants, namely a long form variant that includes exon 1 (EHF-LF) and a short form variant that excludes exon 1 (EHF-SF). Only EHF-SF abrogates ETS1-mediated activation of the ZEB1 promoter by promoting degradation of ETS1 proteins, thereby inhibiting the EMT phenotypes of cancer cells. Most importantly, we identified a novel point mutation within the conserved ETS domain of EHF, and found that EHF mutations abolish its original function while causing the EHF protein to act as a potential dominant negative, thereby enhancing metastasis in vivo. Therefore, we suggest that EHF acts as an anti-EMT factor by inhibiting the expression of ZEBs, and that EHF mutations exacerbate cancer progression.

TGF-ß-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Transforming growth factor-ß (TGF-ß) signaling promotes cancer progression. In particular, the epithelial-mesenchymal transition (EMT) induced by TGF-ß is considered crucial to the malignant phenotype of cancer cells. Here, we report that the EMT-associated cellular responses induced by TGF-ß are mediated by distinct signaling pathways that diverge at Smad3. By expressing chimeric Smad1/Smad3 proteins in SMAD3 knockout A549 cells, we found that the ß4 region in the Smad3 MH1 domain is essential for TGF-ß-induced cell motility, but is not essential for other EMT-associated responses including epithelial marker downregulation. TGF-ß was previously reported to enhance cell motility by activating Rac1 via phosphoinositide 3-kinase. Intriguingly, TGF-ß-dependent signaling mediated by Smad3's ß4 region causes the downregulation of multiple mRNAs that encode GTPase activating proteins that target Rac1 (ARHGAPs), thereby attenuating Rac1 inactivation. Therefore, two independent pathways downstream of TGF-ß type I receptor contribute cooperatively to sustained Rac1 activation, thereby leading to enhanced cell motility.

Modulation of TNFR 1-triggered two opposing signals for inflammation and apoptosis via RIPK 1 disruption by geldanamycin in rheumatoid arthritis.

OBJECTIVES: To evaluate the ability of geldanamycin to modulate two opposing TNFα/TNFR1-triggered signals for inflammation and cell death. METHODS: The effects of geldanamycin on TNFα-induced proinflammatory cytokine production, apoptosis, NF-κB activation, caspase activation, and necroptosis in a human rheumatoid synovial cell line (MH7A) were evaluated via ELISA/qPCR, flow cytometry, dual-luciferase reporter assay, and western blotting assay, respectively. In addition, therapeutic effects on murine collagen-induced arthritis (CIA) were also evaluated. RESULTS: Geldanamycin disrupted RIPK1 in MH7A, thereby inhibiting TNFα-induced proinflammatory cytokine production and enhancing apoptosis. TNFα-induced NF-κB and MLKL activation was inhibited, whereas caspase 8 activation was enhanced. Recombinant RIPK1 restored the geldanamycin-mediated inhibition of TNFα-induced NF-κB activation. In addition, GM showed more clinical effectiveness than a conventional biologic TNF inhibitor, etanercept, in murine CIA and significantly attenuated synovial hyperplasia, a histopathological hallmark of RA. CONCLUSIONS: GM disrupts RIPK1 and selectively inhibits the TNFR1-triggered NF-κB activation signaling pathway, while enhancing the apoptosis signaling pathway upon TNFα stimulation, thereby redressing the balance between these two opposing signals in a human rheumatoid synovial cell line. Therapeutic targeting RIPK1 may be a novel concept which involves TNF inhibitor acting as a TNFR1-signal modulator and have great potential for a more fundamental, effective, and safer TNF inhibitor. Key Points • Geldanamycin (GM) disrupts RIPK1 and selectively inhibits the TNFR1-triggered NF-κB activation signaling pathway while enhancing the apoptosis signaling pathway upon TNFα stimulation, thereby redressing the balance between these two opposing signals in a human rheumatoid synovial cell line, MH7A. • GM showed more clinical effectiveness than a conventional biologic TNF-inhibitor, etanercept, in murine collagen-induced arthritis (CIA), and significantly attenuated synovial hyperplasia, a histopathological hallmark of RA. • Therapeutic targeting RIPK1 may be a novel concept which involves TNF inhibitor acting as a TNFR1-signal modulator and have great potential for a more fundamental, effective, and safer TNF-inhibitor.

ZEB1 and oncogenic Ras constitute a regulatory switch for stimulus-dependent E-cadherin downregulation.

E-cadherin, an epithelial cell-specific cell adhesion molecule, has both promoting and suppressing effects on tumor invasion and metastasis. It is often downregulated during cancer progression through gene deletion/mutation, transcriptional repression, or epigenetic silencing. We describe a novel regulatory switch to induce stimulus-dependent downregulation of mRNA encoding E-cadherin (CDH1 mRNA) in KRAS-mutated cancer cells. The regulatory switch consists of ZEB1 and oncogenic K-Ras, does not target the promoter region of CDH1, and requires an external cue to temporally downregulate E-cadherin expression. Its repressive effect is maintained as long as the external stimulus continues and is attenuated with cessation of the stimulus. Contextual external cues that turn this regulatory switch on include activation of protein kinase C or fibroblast growth factor signaling. The mode of action is distinct from that of EPCAM repression by ZEB1, which does not require an external cue. Thus, KRAS-mutated cancer cells acquire a novel mode of regulating E-cadherin expression depending on ZEB1, which could contribute to phenotypic plasticity of cancer cells during malignant progression.

Neurotensin receptor 1 signaling promotes pancreatic cancer progression.

Pancreatic cancer is one of the cancers with the poorest prognosis, with a 5-year survival rate of approximately 5-10%. Thus, it is urgent to identify molecular targets for the treatment of pancreatic cancer. Using serial transplantations in a mouse pancreatic orthotopic inoculation model, we previously produced highly malignant pancreatic cancer sublines with increased tumor-forming abilities in vivo. Here, we used these sublines to screen molecular targets for the treatment of pancreatic cancer. Among the genes with increased expression levels in the sublines, we focused on those encoding cell surface receptors that may be involved in the interactions between cancer cells and the tumor microenvironment. Based on our previous RNA-sequence analysis, we found increased expression levels of neurotensin (NTS) receptor 1 (NTSR1) in highly malignant pancreatic cancer sublines. Furthermore, re-analysis of clinical databases revealed that the expression level of NTSR1 was increased in advanced pancreatic cancer and that high NTSR1 levels were correlated with a poor prognosis. Overexpression of NTSR1 in human pancreatic cancer cells Panc-1 and SUIT-2 accelerated their tumorigenic and metastatic abilities in vivo. In addition, RNA-sequence analysis showed that MAPK and NF-κB signaling pathways were activated upon NTS stimulation in highly malignant cancer sublines and also revealed many new target genes for NTS in pancreatic cancer cells. NTS stimulation increased the expression of MMP-9 and other pro-inflammatory cytokines and chemokines in pancreatic cancer cells. Moreover, the treatment with SR48692, a selective NTSR1 antagonist, suppressed the activation of the MAPK and NF-κB signaling pathways and induction of target genes in pancreatic cancer cells in vitro, while the administration of SR48692 attenuated the tumorigenicity of pancreatic cancer cells in vivo. These findings suggest that NTSR1 may be a prognostic marker and a molecular target for pancreatic cancer treatment.

Structural basis for inhibitory effects of Smad7 on TGF-ß family signaling.

Smad6 and Smad7 are classified as inhibitory Smads (I-Smads). They are crucial in the fine-tuning of signals by cytokines of the transforming growth factor-ß (TGF-ß) family. They are negative feedback regulators and principally target the activated type I receptors as well as the activated Smad complexes, but with distinct specificities. Smad7 inhibits Smad signaling from all seven type I receptors of the TGF-ß family, whereas Smad6 preferentially inhibits Smad signaling from the bone morphogenetic protein (BMP) type I receptors, BMPR1A and BMPR1B. The target specificities are attributed to the C-terminal MH2 domain. Notably, Smad7 utilizes two alternative molecular surfaces for its inhibitory function against type I receptors. One is a basic groove composed of the first α-helix and the L3 loop, a structure that is shared with Smad6 and receptor-regulated Smads (R-Smads). The other is a three-finger-like structure (consisting of residues 331-361, 379-387, and the L3 loop) that is unique to Smad7. The underlying structural basis remains to be elucidated in detail. Here, we report the crystal structure of the MH2 domain of mouse Smad7 at 1.9 Å resolution. The three-finger-like structure is stabilized by a network of hydrogen bonds between residues 331-361 and 379-387, thus forming a molecular surface unique to Smad7. Furthermore, we discuss how Smad7 antagonizes the activated Smad complexes composed of R-Smad and Smad4, a common partner Smad.

Dissociation of the AhR/ARNT complex by TGF-ß/Smad signaling represses CYP1A1 gene expression and inhibits benze[a]pyrene-mediated cytotoxicity.

Cytochrome P450 1A1 (CYP1A1) catalyzes the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and is transcriptionally regulated by the aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex upon exposure to PAHs. Accordingly, inhibition of CYP1A1 expression reduces production of carcinogens from PAHs. Although transcription of the CYP1A1 gene is known to be repressed by transforming growth factor-ß (TGF-ß), how TGF-ß signaling is involved in the suppression of CYP1A1 gene expression has yet to be clarified. In this study, using mammalian cell lines, along with shRNA-mediated gene silencing, CRISPR/Cas9-based genome editing, and reporter gene and quantitative RT-PCR assays, we found that TGF-ß signaling dissociates the B[a]P-mediated AhR/ARNT heteromeric complex. Among the examined Smads, Smad family member 3 (Smad3) strongly interacted with both AhR and ARNT via its MH2 domain. Moreover, hypoxia-inducible factor 1α (HIF-1α), which is stabilized upon TGF-ß stimulation, also inhibited AhR/ARNT complex formation in the presence of B[a]P. Thus, TGF-ß signaling negatively regulated the transcription of the CYP1A1 gene in at least two different ways. Of note, TGF-ß abrogated DNA damage in B[a]P-exposed cells. We therefore conclude that TGF-ß may protect cells against carcinogenesis because it inhibits CYP1A1-mediated metabolic activation of PAHs as part of its anti-tumorigenic activities.

Lipidome-based rapid diagnosis with machine learning for detection of TGF-ß signalling activated area in head and neck cancer.

BACKGROUND: Several pro-oncogenic signals, including transforming growth factor beta (TGF-ß) signalling from tumour microenvironment, generate intratumoural phenotypic heterogeneity and result in tumour progression and treatment failure. However, the precise diagnosis for tumour areas containing subclones with cytokine-induced malignant properties remains clinically challenging. METHODS: We established a rapid diagnostic system based on the combination of probe electrospray ionisation-mass spectrometry (PESI-MS) and machine learning without the aid of immunohistological and biochemical procedures to identify tumour areas with heterogeneous TGF-ß signalling status in head and neck squamous cell carcinoma (HNSCC). A total of 240 and 90 mass spectra were obtained from TGF-ß-unstimulated and -stimulated HNSCC cells, respectively, by PESI-MS and were used for the construction of a diagnostic system based on lipidome. RESULTS: This discriminant algorithm achieved 98.79% accuracy in discrimination of TGF-ß1-stimulated cells from untreated cells. In clinical human HNSCC tissues, this approach achieved determination of tumour areas with activated TGF-ß signalling as efficiently as a conventional histopathological assessment using phosphorylated-SMAD2 staining. Furthermore, several altered peaks on mass spectra were identified as phosphatidylcholine species in TGF-ß-stimulated HNSCC cells. CONCLUSIONS: This diagnostic system combined with PESI-MS and machine learning encourages us to clinically diagnose intratumoural phenotypic heterogeneity induced by TGF-ß.

Addiction of mesenchymal phenotypes on the FGF/FGFR axis in oral squamous cell carcinoma cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during epithelial tumor progression. ZEB1/2 are EMT transcription factors that are positively correlated with EMT phenotypes and breast cancer aggressiveness. ZEB1/2 regulate the alternative splicing and hence isoform switching of fibroblast growth factor receptors (FGFRs) by repressing the epithelial splicing regulatory proteins, ESRP1 and ESRP2. Here, we show that the mesenchymal-like phenotypes of oral squamous cell carcinoma (OSCC) cells are dependent on autocrine FGF-FGFR signaling. Mesenchymal-like OSCC cells express low levels of ESRP1/2 and high levels of ZEB1/2, resulting in constitutive expression of the IIIc-isoform of FGFR, FGFR(IIIc). By contrast, epithelial-like OSCC cells showed opposite expression profiles for these proteins and constitutive expression of the IIIb-isoform of FGFR2, FGFR2(IIIb). Importantly, ERK1/2 was constitutively phosphorylated through FGFR1(IIIc), which was activated by factors secreted autonomously by mesenchymal-like OSCC cells and involved in sustained high-level expression of ZEB1. Antagonizing FGFR1 with either inhibitors or siRNAs considerably repressed ZEB1 expression and restored epithelial-like traits. Therefore, autocrine FGF-FGFR(IIIc) signaling appears to be responsible for sustaining ZEB1/2 at high levels and the EMT phenotype in OSCC cells.

Transgenic Analyses in Drosophila Reveal That mCORL1 Is Functionally Distinct from mCORL2 and dCORL.

Uncovering how new members of multigene families acquire new functions is an important topic in evolutionary and developmental genetics. CORL proteins (SKOR in mice, Fussel in humans and fussel in Flybase) are a family of CNS specific proteins related to mammalian Sno/Ski oncogenes. Drosophila CORL (dCORL) participates in TGF-ß and insulin signaling during development and in adult homeostasis but roles for the two mouse CORL proteins (mCORL) are essentially unknown. A series of studies were conducted to test the hypothesis based on previous results that mCORL1 is more similar to dCORL than mCORL2. Neither an updated alignment nor ectopic expression in adult wings were able to distinguish mCORL1 or mCORL2 from dCORL. Transgene experiments employing a dCORL endogenous function in mushroom body neurons showed that mCORL1 is distinct from mCORL2 and dCORL. mCORL1 and mCORL2 are also distinct in biochemical assays of Smad-binding and BMP signaling. Taken together, the data suggests testable new hypotheses for mCORL2 function in mammalian TGF-ß and insulin signaling based on known roles for dCORL. Overall, the study reiterates the value of transgenic methods in Drosophila to provide new information on multigene family evolution and the function of family members in other species.