ABSTRACT
Adenoid cystic carcinoma (ACC) and basal cell adenoma (BCA) share many histological characteristics and often need a differential diagnosis in clinical pathology. Recently, we found homeobox protein engrailed-1 (EN1) was a potential diagnostic marker for ACC in an organoids library of salivary gland tumors (SGTs). Here we aim to confirm EN1 as a differential diagnostic marker for ACC, and further investigate the regulatory mechanism and biological function of EN1 in tumor progression. The transcriptional analysis, quantitative polymerase chain reaction, Western blot and immunohistochemistry staining were performed and revealed that EN1 was specifically and highly expressed in ACC, and accurately differentiated ACC from BCA. Furthermore, TGFß signaling pathway was found associated with ACC, and the regulation of EN1 through TGFß was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFß-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFß1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFß1 in ACC was significantly associated with the clinical stage. In summary, our study verified EN1 is a good diagnostic marker to differentiate ACC from BCA. TGFß-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.
Subject(s)
Adenoma , Carcinoma, Adenoid Cystic , Salivary Gland Neoplasms , Humans , Adenoma/pathology , Biomarkers, Tumor/genetics , Carcinoma, Adenoid Cystic/pathology , Homeodomain Proteins , Salivary Gland Neoplasms/pathology , Transforming Growth Factor betaABSTRACT
BACKGROUND: The objective is to estimate the importance of the decrease of smoking habits in Sweden for the occurrence of lung cancer. METHODS: The change in smoking habits in the general population was retrieved from surveys and on taxation of sale of cigarettes. We used data from the Swedish Cancer Register on incidence of lung cancer between 1970 and 2021, stratified for sex, age and cell type, and compared the occurrence overtime in ages between 40 and 84 years. RESULTS: The sale of cigarettes peaked in 1980 to 1800 cigarettes per person and decreased to 600 per person in 2021. The change in incidence rates of squamous cell cancer and other cell types varied over time, sex, and age in a pattern that partly seems to be explained by change in the prevalence of daily smokers. The incidence of adenocarcinoma was similar in men and women 1970-2021 and increased, e.g. for women and men 75-79 years of age from around 20 cases in early 1970s to around 120 cases per 100 000 person-years in the 2020s. CONCLUSIONS: Our data indicate that the risk of lung cancer several years after smoking cessation is less favourable than previously studies have indicated. There is a similar increase in the incidence of adenocarcinoma in men and women which is hard to explain only with changing smoking habits. The change from non-filter to filter cigarettes in the 1960s-1970s may be a contributing factor.
Subject(s)
Lung Neoplasms , Smoking , Humans , Sweden/epidemiology , Lung Neoplasms/epidemiology , Male , Female , Aged , Middle Aged , Adult , Aged, 80 and over , Incidence , Smoking/epidemiology , Registries , Prevalence , Smoking Cessation/statistics & numerical dataABSTRACT
Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants-Fusobacterium nucleatum and Enterococcus faecalis-as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/ß-Catenin, TGF-ß, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/ß-Catenin and NF-κB signaling pathways: ß-Catenin (p = 0.0068 *), LRP-5 (p = 0.0059 **), and LRP-6 (p = 0.0329 *), as well as NF-kB (p = 0.0034 **) and TRAF6 (p = 0.0285 *). These results suggest that oral bacteria can up- and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET.
Subject(s)
Dental Papilla , Mouth , Osteogenesis , Stem Cells , Adult , Bacteria , Cell Differentiation/physiology , Cytokines , Dental Papilla/metabolism , Humans , Mouth/microbiology , NF-kappa B/metabolism , Osteogenesis/genetics , Protein Array Analysis/methods , RNA, Messenger , Stem Cells/metabolism , Transcriptome , Wnt Signaling Pathway , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
BACKGROUND: Transforming growth factor ß (TGFß) functions as a double-edged sword in prostate cancer tumorigenesis. In initial stages of the disease, TGFß acts as a growth inhibitor upon tumor cells, whereas it in later stages of disease rather promotes invasion and metastatic potential. One well-known cellular source of TGFß in the bone metastatic site is the bone-forming osteoblasts. Here we have studied the effects by osteoblast-derived factors on metastatic potential in several human prostate cancer cell lines. METHODS: Effects on metastatic potential in prostate cancer cells by osteoblast-derived factors were studied in vitro using several methods, including Transwell migration and evaluation of formation of pro-migratory protrusions. Confocal microscopy was used to evaluate possible changes in differentiation state in tumor cells by analysis of markers for epithelial-to-mesenchymal transition (EMT). The Matrigel-on-top 3D culture method was used for further assessment of metastatic characteristics in tumor cells by analysis of formation of filopodium-like protrusions (FLPs). RESULTS: Osteoblast-derived factors increased migration of PC-3U cells, an effect less prominent in cells overexpressing a mutated type I TGFß receptor (TßRI) preventing non-canonical TRAF6-dependent TGFß signaling. Osteoblast-derived factors also increased the formation of long protrusions and loss of cell-cell contacts in PC-3U cells, suggesting induction of a more aggressive phenotype. In addition, treatment with TGFß or osteoblast-derived factors of PC-3U cells in Matrigel-on-top 3D cultures promoted formation of FLPs, previously shown to be essential for metastatic establishment. CONCLUSIONS: These findings suggests that factors secreted from osteoblasts, including TGFß, can induce several cellular traits involved in metastatic potential of PC-3U cells, further strengthening the role for bone cells to promote metastatic tumor cell behavior.
Subject(s)
Osteoblasts/metabolism , Prostatic Neoplasms/pathology , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Cell Line, Tumor , Cell Movement/physiology , Cell Shape/physiology , Culture Media, Conditioned , Epithelial-Mesenchymal Transition , Humans , Male , Prostatic Neoplasms/metabolismABSTRACT
Cancer cells produce high levels of TGFß, a multipotent cytokine. Binding of TGFß to its cell surface receptors, the transmembrane serine/threonine kinases TßRII and TßRI, causes phosphorylation and activation of intracellular latent Smad transcription factors. Nuclear Smads act in concert with specific transcription factors to reprogram epithelial cells to become invasive mesenchymal cells. TGFß also propagates non-canonical signals, so it is crucial to have a better understanding of the underlying molecular mechanisms which favor this pathway. Here we highlight our recent discovery that TGFß promotes the proteolytic cleavage of TßRI in cancer cells, resulting in the liberation and nuclear translocation of its intracellular domain, acting as co-regulator to transcribe pro-invasive genes. This newly identified oncogenic TGFß pathway resembles the Notch signaling pathway. We discuss our findings in relation to Notch and provide a short overview of other growth factors that transduce signals via nuclear translocation of their cell surface receptors.
Subject(s)
Cell Membrane/metabolism , Cell Transformation, Neoplastic/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Cell Nucleus/metabolism , Disease Progression , Humans , Neoplasms/metabolism , Neoplasms/pathology , Proteolysis , Receptor, Transforming Growth Factor-beta Type I , Receptors, Cytoplasmic and Nuclear/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
The transcription factor nuclear factor κB (NF-κB) plays a central role in regulating inflammation in response to several external signals. The TGFß-associated kinase 1 (TAK1) is an upstream regulator of NF-κB signaling. In TGFß-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-κB activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1ß. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-α and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGFß, which in all cases resulted in NF-κB activation. Expression of a K34R-mutant TAK1 led to a reduced NF-κB activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-α-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-κB signaling downstream of several receptors.
Subject(s)
MAP Kinase Kinase Kinases/metabolism , Transcription Factor RelA/metabolism , Ubiquitination , Active Transport, Cell Nucleus/drug effects , Amino Acid Substitution , Animals , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Down-Regulation/drug effects , Humans , Inflammation/metabolism , Interleukin-6/metabolism , Lipopolysaccharides/pharmacology , Lysine/metabolism , MAP Kinase Kinase Kinases/chemistry , MAP Kinase Kinase Kinases/genetics , Mice , Mutation , Receptors, Interleukin-1/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/drug effects , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitination/drug effectsABSTRACT
Introduction: Bacterial persistence is considered one of the main causal factors for regenerative endodontic treatment (RET) failure in immature permanent teeth. This interference is claimed to be caused by the interaction of bacteria that reside in the root canal with the stem cells that are one of the essentials for RET. The aim of the study was to investigate whether prolonged exposure of stem cells from the apical papilla (SCAP) to bacterial remnants of Fusobacterium nucleatum, Actinomyces gerensceriae, Slackia exigua, Enterococcus faecalis, Peptostreptococcaceae yurii, commonly found in infected traumatized root canals, and the probiotic bacteria Lactobacillus gasseri and Limosilactobacillus reuteri, can alter SCAP's inflammatory response and mineralization potential. Methods: To assess the effect of bacterial remnants on SCAP, we used UV-C-inactivated bacteria (as cell wall-associated virulence factors) and bacterial DNA. Histochemical staining using Osteoimage Mineralization Assay and Alizarin Red analysis was performed to study SCAP mineralization, while inflammatory and osteo/odontogenic-related responses of SCAPs were assessed with Multiplex ELISA. Results: We showed that mineralization promotion was greater with UV C-inactivated bacteria compared to bacterial DNA. Immunofluorescence analysis detected that the early mineralization marker alkaline phosphatase (ALP) was increased by the level of E. coli lipopolysaccharide (LPS) positive control in the case of UV-C-inactivated bacteria; meanwhile, DNA treatment decreased the level of ALP compared to the positive control. SCAP's secretome assessed with Multiplex ELISA showed the upregulation of pro-inflammatory factors IL-6, IL-8, GM-CSF, IL-1b, neurotrophic factor BDNF, and angiogenic factor VEGF, induced by UV-C-killed bacteria. Discussion: The results suggest that long term stimulation (for 21 days) of SCAP with UV-C-inactivated bacteria stimulate their mineralization and inflammatory response, while DNA influence has no such effect, which opens up new ideas about the nature of RET failure.
Subject(s)
Escherichia coli , Osteogenesis , DNA, Bacterial , Escherichia coli/genetics , Cell Differentiation/genetics , Stem Cells/physiology , Cells, Cultured , Cell ProliferationABSTRACT
The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabA's binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.
ABSTRACT
Transforming growth factor-beta (TGFß) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFß signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TßRI and TßRII, respectively). The activated TßR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFß also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFß-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFß-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFß are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFß-induced responses by non-Smad signaling pathways.
Subject(s)
Signal Transduction/physiology , Smad Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Epithelial-Mesenchymal Transition/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP Phosphohydrolases/metabolism , Integrins/metabolism , MAP Kinase Kinase Kinases/metabolism , Receptors, Transforming Growth Factor beta/metabolism , TNF Receptor-Associated Factor 6/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Transforming growth factor ß (TGF-ß) is a multifunctional cytokine regulating homeostasis and immune responses in adult animals and humans. Aberrant and overactive TGF-ß signaling promotes cancer initiation and fibrosis through epithelial-mesenchymal transition (EMT), as well as the invasion and metastatic growth of cancer cells. TGF-ß is a key factor that is active during hypoxic conditions in cancer and is thereby capable of contributing to angiogenesis in various types of cancer. Another potent role of TGF-ß is suppressing immune responses in cancer patients. The strong tumor-promoting effects of TGF-ß and its profibrotic effects make it a focus for the development of novel therapeutic strategies against cancer and fibrosis as well as an attractive drug target in combination with immune regulatory checkpoint inhibitors. TGF-ß belongs to a family of cytokines that exert their function through signaling via serine/threonine kinase transmembrane receptors to intracellular Smad proteins via the canonical pathway and in combination with co-regulators such as the adaptor protein and E3 ubiquitin ligases TNF receptor-associated factor 4 (TRAF4) and TNF receptor-associated factor 6 (TRAF6) to promote non-canonical pathways. Finally, the outcome of gene transcription initiated by TGF-ß is context-dependent and controlled by signals exerted by other growth factors such as EGF and Wnt. Here, we discuss the synergistic cooperation between TGF-ß and hypoxia in development, fibrosis and cancer.
Subject(s)
Neoplasms , Transforming Growth Factor beta , Animals , Fibrosis , Humans , Hypoxia , Neoplasms/metabolism , Smad Proteins/metabolism , TNF Receptor-Associated Factor 4/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Transforming growth factor-ß (TGF-ß) signaling has important roles during embryonic development and in tissue homeostasis. TGF-ß ligands exert cellular effects by binding to type I (TßRI) and type II (TßRII) receptors and inducing both SMAD-dependent and SMAD-independent intracellular signaling pathways, the latter of which includes the activation of the tyrosine kinase Src. We investigated the mechanism by which TGF-ß stimulation activates Src in human and mouse cells. Before TGF-ß stimulation, inactive Src was complexed with TßRII. Upon TGF-ß1 stimulation, TßRII associated with and phosphorylated TßRI at Tyr182. Binding of Src to TßRI involved the interaction of the Src SH2 domain with phosphorylated Tyr182 and the interaction of the Src SH3 domain with a proline-rich region in TßRI and led to the activation of Src kinase activity and Src autophosphorylation. TGF-ß1-induced Src activation required the kinase activities of TßRII and Src but not that of TßRI. Activated Src also phosphorylated TßRI on several tyrosine residues, which may stabilize the binding of Src to the receptor. Src activation was required for the ability of TGF-ß to induce fibronectin production and migration in human breast carcinoma cells and to induce α-smooth muscle actin and actin reorganization in mouse fibroblasts. Thus, TGF-ß induces Src activation by stimulating a direct interaction with TßRI that depends on tyrosine phosphorylation of TßRI by TßRII.
Subject(s)
Receptors, Transforming Growth Factor beta , Transforming Growth Factor beta1 , Humans , Mice , Animals , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Receptor, Transforming Growth Factor-beta Type II , Transforming Growth Factor beta1/metabolism , Protein Serine-Threonine Kinases , Actins , Transforming Growth Factor beta/metabolism , TyrosineABSTRACT
BACKGROUND: Transforming growth factor ß (TGFß) is overexpressed in several advanced cancer types and promotes tumor progression. We have reported that the intracellular domain (ICD) of TGFß receptor (TßR) I is cleaved by proteolytic enzymes in cancer cells, and then translocated to the nucleus in a manner dependent on the endosomal adaptor proteins APPL1/2, driving an invasiveness program. How cancer cells evade TGFß-induced growth inhibition is unclear. METHODS: We performed microarray analysis to search for genes regulated by APPL1/2 proteins in castration-resistant prostate cancer (CRPC) cells. We investigated the role of TßRI and TRAF6 in mitosis in cancer cell lines cultured in 10% FBS in the absence of exogenous TGFß. The molecular mechanism of the ubiquitination of AURKB by TRAF6 in mitosis and the formation of AURKB-TßRI complex in cancer cell lines and tissue microarrays was also studied. FINDINGS: During mitosis and cytokinesis, AURKB-TßRI complexes formed in midbodies in CRPC and KELLY neuroblastoma cells. TRAF6 induced polyubiquitination of AURKB on K85 and K87, protruding on the surface of AURKB to facilitate its activation. AURKB-TßRI complexes in patient's tumor tissue sections correlated with the malignancy of prostate cancer. INTERPRETATION: The AURKB-TßRI complex may become a prognostic biomarker for patients with risk of developing aggressive PC. FUNDING: Swedish Medical Research Council (2019-01598, ML; 2015-02757 and 2020-01291, CHH), the Swedish Cancer Society (20 0964, ML), a regional agreement between Umeå University and Region Västerbotten (ALF; RV-939377, -967041, -970057, ML). The European Research Council (787472, CHH). KAW 2019.0345, and the Kempe Foundation SMK-1866; ML. National Microscopy Infrastructure (NMI VR-RFI 2016-00968).
Subject(s)
Aurora Kinase B/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Prostatic Neoplasms, Castration-Resistant , Receptor, Transforming Growth Factor-beta Type I/metabolism , TNF Receptor-Associated Factor 6 , Cell Line, Tumor , Cytokinesis , Humans , Ligases , Male , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Transforming Growth Factor beta/metabolism , Ubiquitin/metabolismABSTRACT
Renal cell carcinoma (RCC) includes diverse tumour types characterised by various genetic abnormalities. The genetic changes, like mutations, deletions and epigenetic alterations, play a crucial role in the modification of signalling networks, tumour pathogenesis and prognosis. The most prevalent RCC type, clear cell RCC (ccRCC), is asymptomatic in the early stages and has a poorer prognosis compared with the papillary and the chromophobe types RCCs. Generally, ccRCC is refractory to chemotherapy and radiation therapy. Loss of von Hippel-Lindau (VHL) gene and upregulation of hypoxia-inducible factors (HIF), the signature of most sporadic ccRCC, promote multiple growth factors. Hence, VHL/HIF and a variety of pathways, including phosphatase and TEnsin homolog on chromosome 10/phosphatidylinositol-3-kinase (PI3K)/AKT, are closely connected and contribute to the ontogeny of ccRCC. In the recent decade, multiple targeting agents have been developed based on blocking major signalling pathways directly or indirectly involved in ccRCC tumour progression, metastasis, angiogenesis and survival. However, most of these drugs have limitations; either metastatic ccRCC develops resistance to these agents, or despite blocking receptors, tumour cells use alternate signalling pathways. This review compiles the state of knowledge about the PI3K/AKT signalling pathway confined to ccRCC and its cross-talks with VHL/HIF pathway.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Biomarkers, Tumor , Carcinoma, Renal Cell/enzymology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Kidney Neoplasms/enzymology , Phosphatidylinositol 3-Kinase/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Humans , Kidney Neoplasms/drug therapy , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Molecular Targeted Therapy , PTEN Phosphohydrolase/metabolism , Phosphoinositide-3 Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Treatment Outcome , Tumor Hypoxia , Tumor MicroenvironmentABSTRACT
The use of stem cells from the apical papilla (SCAPs) has been proposed as a means of promoting root maturation in permanent immature teeth, and plays a significant role in regenerative dental procedures. However, the role of SCAPs may be compromised by microenvironmental factors, such as hypoxic conditions and the presence of bacteria from infected dental root canals. We aim to investigate oral bacterial modulation of SCAP in terms of binding capacity using flow cytometry and imaging, real-time cell proliferation monitoring, and cytokine secretion (IL-6, IL-8, and TGF-ß isoforms) under anaerobic conditions. SCAPs were exposed to key species in dental root canal infection, namely Actinomyces gerensceriae, Slackia exigua, Fusobacterium nucleatum, and Enterococcus faecalis, as well as two probiotic strains, Lactobacillus gasseri strain B6 and Lactobacillus reuteri (DSM 17938). We found that A. gerensceriae, S. exigua, F. nucleatum, and E. faecalis, but not the Lactobacillus probiotic strains bind to SCAPs on anaerobic conditions. Enterococcus faecalis and F. nucleatum exhibited the strongest binding capacity, resulting in significantly reduced SCAP proliferation. Notably, F. nucleatum, but not E. faecalis, induce production of the proinflammatory chemokine IL-8 and IL-10 from SCAPs. Production of TGF-ß1 and TGF-ß2 by SCAPs was dependent on species, cell line, and time, but secretion of TGF-ß3 did not vary significantly over time. In conclusion, SCAP response is compromised when exposed to bacterial stimuli from infected dental root canals in anaerobic conditions. Thus, stem cell-mediated endodontic regenerative studies need to include microenvironmental conditions, such as the presence of microorganisms to promote further advantage in the field.
Subject(s)
Actinobacteria , Stem Cells , Cell Differentiation , Cell ProliferationABSTRACT
Transforming growth factor ß (TGF-ß) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-ß to control TGF-ß signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-ß induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information.
ABSTRACT
HopQ is an outer-membrane protein of Helicobacter pylori that binds to human carcinoembryonic antigen-related cell-adhesion molecules (CEACAMs) with high specificity. We aimed to investigate fluorescence targeting of CEACAM-expressing colorectal tumors in patient-derived orthotopic xenograft (PDOX) models with fluorescently labeled recombinant HopQ (rHopQ). Western blotting, flow cytometry and ELISA were performed to determine the efficiency of rHopQ binding to CEACAMs. rHopQ was conjugated to IR800DyeCW (rHopQ-IR800). Nude mice received orthotopic implantation of colon cancer tumors. Three weeks later, mice were administered 25⯵g or 50⯵g HopQ-IR800 and imaged 24 or 48â¯h later. Intravital images were analyzed for tumor-to-background ratio (TBR). Flow cytometry and ELISA demonstrated binding of HopQ to CEACAM1, 3 and 5. Dose-response intravital imaging in PDOX models demonstrated optimal results 48â¯h after administration of 50⯵g rHopQ-IR800 (TBRâ¯=â¯3.576) in our protocol. Orthotopic models demonstrated clear tumor margins of primary tumors and small regional metastases with a mean TBRâ¯=â¯3.678 (SD⯱â¯1.027). rHopQ showed specific binding to various CEACAMs in PDOX models. rHopQ may be useful for CEACAM-positive tumor and metastasis detection for pre-surgical diagnosis, intra-operative imaging and fluorescence-guided surgery.
ABSTRACT
Prostate cancer disseminates primarily into the adjacent lymph nodes, which is related to a poor outcome. Atypical protein kinase C ζ (PKCζ) is highly expressed in aggressive prostate cancer and correlates with Gleason score, clinical stage, and poor prognosis. Here, we report the molecular mechanisms of PKCζ in lymphatic metastasis during prostate cancer progression. Using zinc-finger nuclease technology or PKCζ shRNA lentiviral particles, and orthotopic mouse xenografts, we show that PKCζ-knockout or knockdown from aggressive prostate cancer (PC3 and PC3U) cells, decreasesd tumor growth and lymphatic metastasis in vivo. Intriguingly, PKCζ-knockout or knockdown impaired the activation of AKT, ERK, and NF-κB signaling in prostate cancer cells, thereby impairing the expression of lymphangiogenic factors and macrophage recruitment, resulting in aberrant lymphangiogenesis. Moreover, PKCζ regulated the expression of hyaluronan synthase enzymes, which is important for hyaluronan-mediated lymphatic drainage and tumor dissemination. Thus, PKCζ plays a crucial oncogenic role in the lymphatic metastasis of prostate cancer and is predicted to be a novel therapeutic target for prostate cancer.
Subject(s)
Lymphatic Metastasis/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Kinase C/metabolism , A549 Cells , Animals , Cell Line, Tumor , Humans , Hyaluronan Synthases/metabolism , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphangiogenesis/physiology , Lymphatic Vessels/metabolism , Lymphatic Vessels/pathology , MAP Kinase Signaling System/physiology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Nude , NF-kappa B/metabolism , PC-3 Cells , Prostate/metabolism , Prostate/pathology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiologyABSTRACT
To investigate the significance of expression of HIF-1α, HIF-2α, and SNAIL1 proteins; and TGF-ß signaling pathway proteins in ccRCC, their relation with clinicopathological parameters and patient's survival were examined. We also investigated potential crosstalk between HIF-α and TGF-ß signaling pathway, including the TGF-ß type 1 receptor (ALK5-FL) and the intracellular domain of ALK5 (ALK5-ICD). Tissue samples from 154 ccRCC patients and comparable adjacent kidney cortex samples from 38 patients were analyzed for HIF-1α/2α, TGF-ß signaling components, and SNAIL1 proteins by immunoblot. Protein expression of HIF-1α and HIF-2α were significantly higher, while SNAIL1 had similar expression levels in ccRCC compared with the kidney cortex. HIF-2α associated with poor cancer-specific survival, while HIF-1α and SNAIL1 did not associate with survival. Moreover, HIF-2α positively correlated with ALK5-ICD, pSMAD2/3, and PAI-1; HIF-1α positively correlated with pSMAD2/3; SNAIL1 positively correlated with ALK5-FL, ALK5-ICD, pSMAD2/3, PAI-1, and HIF-2α. Intriguingly, in vitro experiments performed under normoxic conditions revealed that ALK5 interacts with HIF-1α and HIF-2α, and promotes their expression and the expression of their target genes GLUT1 and CA9, in a VHL dependent manner. We found that ALK5 induces expression of HIF-1α and HIF-2α, through its kinase activity. Under hypoxic conditions, HIF-α proteins correlated with the activated TGF-ß signaling pathway. In conclusion, we reveal that ALK5 plays a pivotal role in synergistic crosstalk between TGF-ß signaling and hypoxia pathway, and that the interaction between ALK5 and HIF-α contributes to tumor progression.
Subject(s)
Carcinoma, Renal Cell/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Plasminogen Activator Inhibitor 1/genetics , Receptor, Transforming Growth Factor-beta Type I/genetics , Adult , Carcinoma, Renal Cell/pathology , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Middle Aged , Prognosis , Signal Transduction/genetics , Snail Family Transcription Factors/geneticsABSTRACT
BACKGROUND: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of ß-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling. METHODS: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling. FINDINGS: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of ß-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes. INTERPRETATION: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression. FUND: KAW 2012.0090, CAN 2017/544, Swedish Medical Research Council (2016-02513), Prostatacancerförbundet, Konung Gustaf V:s Frimurarestiftelse and Cancerforskningsfonden Norrland. The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.