ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both in vitro and in vivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Deoxycytidine , Gemcitabine , Glucosephosphate Dehydrogenase , MicroRNAs , Pancreatic Neoplasms , Receptors, Prolactin , Animals , Female , Humans , Mice , Antimetabolites, Antineoplastic/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Glucosephosphate Dehydrogenase/metabolism , Glucosephosphate Dehydrogenase/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Receptors, Prolactin/metabolism , Receptors, Prolactin/genetics , Mice, NudeABSTRACT
Endocrine receptors play an essential role in tumor metabolic reprogramming and represent a promising therapeutic avenue in pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by a nutrient-deprived microenvironment. To meet their ascendant energy demands, cancer cells can internalize extracellular proteins via macropinocytosis. However, the roles of endocrine receptors in macropinocytosis are not clear. In this study, we found that progesterone receptor (PGR), a steroid-responsive nuclear receptor, is highly expressed in PDAC tissues obtained from both patients and transgenic LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1-cre (KPC) mice. Moreover, PGR knockdown restrained PDAC cell survival and tumor growth both in vitro and in vivo. Genetic and pharmacological PGR inhibition resulted in a marked attenuation of macropinocytosis in PDAC cells and subcutaneous tumor models, indicating the involvement of this receptor in macropinocytosis regulation. Mechanistically, PGR upregulated CDC42, a critical regulator in macropinocytosis, through PGR-mediated transcriptional activation. These data deepen the understanding of how the endocrine system influences tumor progression via a non-classical pathway and provide a novel therapeutic option for patients with PDAC.
ABSTRACT
Ferroptosis, a nonapoptotic form of cell death, is an emerging potential therapeutic target for various diseases, including cancer. However, the role of ferroptosis in pancreatic cancer remains poorly understood. Pancreatic ductal adenocarcinoma (PDAC) is characterized by a poor prognosis and chemotherapy resistance, attributed to its high Kirsten rats arcomaviral oncogene homolog mutation rate and severe nutritional deficits resulting from a dense stroma. Several studies have linked rat sarcoma (RAS) mutations to ferroptosis, suggesting that inducing ferroptosis may be an effective strategy against oncogenic RAS-bearing tumors. We investigated the role of Family With Sequence Similarity 60 Member A (FAM60A) in this study, a protein closely associated with a poor prognosis and highly expressed in PDAC and tumor tissue from KrasG12D/+;Trp53R172H/+; Pdx1-Cre mice, in regulating ferroptosis, tumor growth, and gemcitabine sensitivity in vitro and in vivo. Our results demonstrate that FAM60A regulates 3 essential metabolic enzymes, ACSL1/4 and GPX4, to protect PDAC cells from ferroptosis. Furthermore, we found that YY1 transcriptionally regulates FAM60A expression by promoting its transcription, and the Hippo-YY1 pathway is restricted in the low-amino-acid milieu in the context of nutrient deprivation, leading to downstream suppression of peroxisome proliferator-activated receptor and ACSL1/4 and activation of GPX4 pathways. Importantly, FAM60A knockdown sensitized PDAC cells to gemcitabine treatment. A new understanding of FAM60A transcriptional regulation pattern in PDAC and its dual function in ferroptosis reliever and chemotherapy resistance is provided by our study. Targeting FAM60A may therefore offer a promising therapeutic approach for PDAC by simultaneously addressing 2 major features of the disease (high RAS mutation rate and tumor microenvironment nutrient deficiency) and preventing tumor cell metabolic adaptation.
ABSTRACT
Ovarian cancer (OV) is a highly aggressive malignancy with poor prognosis due to recurrence and drug resistance. Therefore, it is imperative to investigate the key molecular mechanisms underlying OV progression in order to develop promising diagnostic and therapeutic interventions. Although the importance of hematological and neurological expressed 1 (HN1) protein in hemopoietic cell and neurological development has been well-established, its function in cancer, particularly in OV, remains uncertain. In this study, we compared the expression of HN1 in ovarian cancers and para-tumor tissues and predicted potential related signaling pathways through enrichment analysis. In order to confirm the role of HN1 in vitro and vivo, we carried out a variety of experiments including bioinformation analysis, colony formation, flow cytometry analysis, and subcutaneous tumor models. The results demonstrated that HN1 was upregulated in OV and was negatively associated with clinical prognosis. Moreover, we observed that HN1 enhances cell proliferation, migration, and drug resistance, while suppressing apoptosis in OV cells. Notably, we discovered that HN1 functions as a novel regulator of mTOR pathways. Our findings suggest that HN1-mediated mTOR regulation facilitates OV advancement and targeting HN1 could provide a promising therapeutic approach for clinical OV treatment.
Subject(s)
Cell Proliferation , Ovarian Neoplasms , Signal Transduction , TOR Serine-Threonine Kinases , Female , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Humans , TOR Serine-Threonine Kinases/metabolism , Cell Line, Tumor , Animals , Disease Progression , Cell Movement , Fucosyltransferases/metabolism , Fucosyltransferases/genetics , Apoptosis/physiology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Mice , Mice, NudeABSTRACT
BACKGROUND & AIMS: Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. METHODS: HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. RESULTS: HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. CONCLUSIONS: Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Homeostasis , Hot Temperature , Insulin-Like Growth Factor I/genetics , Liver Neoplasms/pathology , Mechanistic Target of Rapamycin Complex 1/metabolism , Proteasome Endopeptidase Complex/genetics , Proteome/metabolism , Ribosomes/metabolism , Ribosomes/pathology , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolismABSTRACT
The extracellular matrix (ECM), as an important component of the tumor microenvironment, exerts various roles in tumor formation. Mitochondrial dynamic disorder is closely implicated in tumorigenesis, including hyperfission in HCC. We aimed to determine the influence of the ECM-related protein CCBE1 on mitochondrial dynamics in HCC. Here, we found that CCBE1 was capable of promoting mitochondrial fusion in HCC. Initially, CCBE1 expression was found to be significantly downregulated in tumors compared with nontumor tissues, which resulted from hypermethylation of the CCBE1 promoter in HCC. Furthermore, CCBE1 overexpression or treatment with recombinant CCBE1 protein dramatically inhibited HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, CCBE1 functioned as an inhibitor of mitochondrial fission by preventing the location of DRP1 on mitochondria through inhibiting its phosphorylation at Ser616 by directly binding with TGFßR2 to inhibit TGFß signaling activity. In addition, a higher percentage of specimens with higher DRP1 phosphorylation was present in patients with lower CCBE1 expression than in patients with higher CCBE1 expression, which further confirmed the inhibitory effect of CCBE1 on DRP1 phosphorylation at Ser616. Collectively, our study highlights the crucial roles of CCBE1 in mitochondrial homeostasis, suggesting strong evidence for this process as a potential therapeutic strategy for HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Mitochondrial Dynamics , Liver Neoplasms/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Cell Proliferation , Tumor Microenvironment , Calcium-Binding Proteins/metabolism , Tumor Suppressor ProteinsABSTRACT
BACKGROUND: Liver fibrosis is a wound-healing response to chronic injury, featuring with excess accumulation of extracellular matrix secreted by the activated hepatic stellate cells (HSC). Disulfiram (DSF), also known as Antabuse, has been used for the treatment of alcohol addiction and substance abuse. Recently, overwhelming studies had revealed anti-cancer effects of DSF in multiple cancers, including liver cancer. But the actual effects of DSF on liver fibrosis and liver function remain unknown. METHODS: In this study, we evaluated the effects of low-dose DSF in CCl4- and Bile Duct Ligation (BDL)-induced hepatic fibrosis rat models. Cell proliferation was detected by using the Cell-Light™ EdU Apollo®567 Cell Tracking Kit. Cell apoptosis was analyzed using a TdT-mediated dUTP nick end labeling (TUNEL) kit, viability was measured with Cell Counting Kit-8(CCK8). Relative mRNA expression of pro-fibrogenic was assessed using quantitative RT-PCR. The degree of liver fibrosis, activated HSCs, were separately evaluated through Sirius Red-staining, immunohistochemistry and immunofluorescence. Serum alanine aminotransferase (ALT) and asparagine aminotransferase (AST) activities were detected with ALT and AST detecting kits using an automated analyzer. RESULTS: Liver fibrosis was distinctly attenuated while liver functions were moderately ameliorated in the DSF-treated group. Activation and proliferation of primary rat HSCs isolated from rat livers were significantly suppressed by low-dose DSF. DSF also inhibited the viability of in vitro cultured rat or human HSC cells dose-dependently but had no repressive role on human immortalized hepatocyte THLE-2 cells. Interestingly, upon DSF treatment, the viability of LX-2 cells co-cultured with THLE-2 was significantly inhibited, while that of THLE-2 co-cultured with LX-2 was increased. Further study indicated that HSCs apoptosis was increased in DSF/CCl4-treated liver samples. These data indicated that DSF has potent anti-fibrosis effects and protective effects toward hepatocytes and could possibly be repurposed as an anti-fibrosis drug in the clinic. CONCLUSIONS: DSF attenuated ECM remodeling through suppressing the transformation of quiet HSCs into proliferative, fibrogenic myofibroblasts in hepatic fibrosis rat models. DSF provides a novel approach for the treatment of liver fibrosis.
Subject(s)
Disulfiram , Hepatic Stellate Cells , Animals , Bile Ducts/metabolism , Cell Proliferation , Disulfiram/metabolism , Disulfiram/pharmacology , Disulfiram/therapeutic use , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Liver , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , RatsABSTRACT
Existing evidence indicates that gut fungal dysbiosis might play a key role in the pathogenesis of colorectal cancer (CRC). We sought to explore whether reversing the fungal dysbiosis by terbinafine, an approved antifungal drug, might inhibit the development of CRC. A population-based study from Sweden identified a total of 185 patients who received terbinafine after their CRC diagnosis and found that they had a decreased risk of death (hazard ratio = 0.50) and metastasis (hazard ratio = 0.44) compared with patients without terbinafine administration. In multiple mouse models of CRC, administration of terbinafine decreased the fungal load, the fungus-induced myeloid-derived suppressor cell (MDSC) expansion, and the tumor burden. Fecal microbiota transplantation from mice without terbinafine treatment reversed MDSC infiltration and partially restored tumor proliferation. Mechanistically, terbinafine directly impaired tumor cell proliferation by reducing the ratio of nicotinamide adenine dinucleotide phosphate (NADP+) to reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), suppressing the activity of glucose-6-phosphate dehydrogenase (G6PD), resulting in nucleotide synthesis disruption, deoxyribonucleotide (dNTP) starvation, and cell-cycle arrest. Collectively, terbinafine can inhibit CRC by reversing fungal dysbiosis, suppressing tumor cell proliferation, inhibiting fungus-induced MDSC infiltration, and restoring antitumor immune response.
Subject(s)
Colorectal Neoplasms , Terbinafine , Animals , Antifungal Agents , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Deoxyribonucleotides , Dysbiosis , Glucosephosphate Dehydrogenase , Mice , NADP , Terbinafine/pharmacologyABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, and the patients are generally diagnosed with distant metastasis. Liver is one of the preferred organs of distant metastasis, and liver metastasis is the leading cause of death in PDAC. Diet-induced obesity (DIO) is a risk factor for PDAC, and it remains unclear whether and how DIO contributes to liver metastasis of PDAC. In our study, we found that DIO significantly promoted PDAC liver metastasis compared with normal diet (ND) in intrasplenic injection mouse model. RNA-seq analysis for liver metastasis nodules showed that the various chemokines and several chemokine receptors were altered between ND and DIO samples. The expression levels of CX3CL1 and CX3CR1 were significantly upregulated in DIO-induced liver metastasis of PDAC compared to ND. Increased CX3CL1 promoted the recruitment of CX3CR1-expressing pancreatic tumor cells. Taken together, our data demonstrated that DIO promoted PDAC liver metastasis via CX3CL1/CX3CR1 axis.
Subject(s)
Carcinoma, Pancreatic Ductal , Liver Neoplasms , Pancreatic Neoplasms , Animals , CX3C Chemokine Receptor 1 , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Chemokine CX3CL1/genetics , Diet , Humans , Liver Neoplasms/secondary , Mice , Obesity , Pancreatic Neoplasms/pathology , Pancreatic NeoplasmsABSTRACT
Pancreatic ductal adenocarcinoma (PDAC), cancer with a high mortality rate and the highest rate of KRAS mutation, reportedly internalizes proteins via macropinocytosis to adapt to low amino acid levels in the tumor microenvironment. Here, we aimed to identify a key regulator of macropinocytosis for the survival of tumor cells in a low amino acid environment in PDAC. FYVE, RhoGEF, and PH domain-containing protein 6 (FGD6) were identified as key regulators of macropinocytosis. FGD6 promoted PDAC cell proliferation, macropinocytosis, and tumor growth both in vitro and in vivo. The macropinocytosis level was decreased with FGD6 knockdown in PDAC cell lines. Moreover, FGD6 promoted macropinocytosis by participating in the trans-Golgi network and enhancing the membrane localization of growth factor receptors, especially the TGF-beta receptor. TGF-beta enhanced macropinocytosis in PDAC cells. Additionally, YAP nuclear translocation induced by a low amino acid tumor environment initiated FGD6 expression by coactivation with YY1. Clinical data analysis based on TCGA and GEO datasets showed that FGD6 expression was upregulated in PDAC tissue, and high FGD6 expression was correlated with poor prognosis in patients with PDAC. In tumor tissue from KrasG12D/+/Trp53R172H/-/Pdx1-Cre (KPC) mice, FGD6 expression escalated during PDAC development. Our results uncover a previously unappreciated mechanism of macropinocytosis in PDAC. Strategies to target FGD6 and growth factors membrane localization might be developed for the treatment of PDAC.
Subject(s)
Carcinoma, Pancreatic DuctalABSTRACT
Metastasis is a major cause of cancer-related deaths. Tumor-intrinsic properties can determine whether tumor metastasis occurs or not. Here, by comparing the gene expression patterns in primary colorectal cancer (CRC) patients with or without metastasis, we found that Collagen Triple Helix Repeat Containing 1 (CTHRC1) in primary CRC served as a metastasis-associated gene. Animal experiments verified that CTHRC1 secreted by CRC cells promoted hepatic metastasis, which was closely correlated with macrophage infiltration. Depletion of macrophages by liposomal clodronate largely abolished the promoting effect of CTHRC1 on CRC liver metastasis. Furthermore, we demonstrated that CTHRC1 modulated macrophage polarization to M2 phenotypes through TGF-ß signaling. A mechanistic study revealed that CTHRC1 bound directly to TGF-ß receptor II and TGF-ß receptor III, stabilized the TGF-ß receptor complex, and activated TGF-ß signaling. The combination treatment of CTHRC1 monoclonal antibody and anti-PD-1 blocking antibody effectively suppressed CRC hepatic metastasis. Taken together, our data demonstrated that CTHRC1 is an intrinsic marker of CRC metastasis and further revealed that CTHRC1 promoted CRC liver metastasis by reshaping infiltrated macrophages through TGF-ß signaling, suggesting that CTHRC1 could be a potential biomarker for the early prediction of and a therapeutic target of CRC hepatic metastasis.
Subject(s)
Antibodies, Monoclonal/pharmacology , Colorectal Neoplasms/metabolism , Extracellular Matrix Proteins/metabolism , Liver Neoplasms/secondary , Macrophages/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Databases, Genetic , Disease Models, Animal , Extracellular Matrix Proteins/antagonists & inhibitors , Extracellular Matrix Proteins/genetics , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Macrophages/immunology , Macrophages/pathology , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Staging , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Survival Rate , Treatment OutcomeABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the most dangerous cancers, and the overall 5-year survival rate is only 8%. The microenvironment of PDAC, which promotes tumorigenesis, disease development and metastasis, consists of fibroblasts, immune cells, pancreatic stellate cells (PaSCs), adipocytes and extracellular matrix (ECM). Because the microenvironment is a part of the tumor, it is also an important target for PDAC treatment. Several therapeutic regimens targeting PDAC microenvironment factors or cells have been investigated, but the treatment effects were poor. More research on the physiological and pathological mechanisms and clinical treatment of PDAC is necessary.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , Immunotherapy , Pancreatic Neoplasms/drug therapy , Tumor Microenvironment/drug effects , Animals , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Humans , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Tumor Microenvironment/immunology , Pancreatic NeoplasmsABSTRACT
Acetyl-CoA synthetase 2 (ACSS2) generates acetyl-CoA from acetate is important for histone acetylation and gene expression. ACSS2 fulfills distinct functions depending on its cellular location in tumor cells. The role and cellular localization of ACSS2 in hepatocellular carcinoma (HCC) remains to be studied. Herein, we identified that the alternative transcription start site selection of ACSS2 was significantly different between HCC and corresponding adjacent tissues. Alternative transcription start site selection produced two different ACSS2 transcripts, ACSS2-S1 and ACSS2-S2. The two isoforms of ACSS2 had different subcellular localization and different functions. Overexpression of ACSS2-S2 promoted cell proliferation and invasion, but ACSS2-S1 did not. The ACSS2-S1 was mainly present in cytoplasm, and ACSS2-S2 was distributed in both nucleus and cytoplasm. Finally, we demonstrated that alternative transcription start site selection of ACSS2 correlates ribosome biogenesis in HCC. Our findings reveal an oncogenic role of ACSS2-S2 in HCC progression via increase of ribosome biogenesis, and suggest ACSS2-S2 might be a potential therapeutic target against the HCC.
Subject(s)
Acetate-CoA Ligase/genetics , Carcinoma, Hepatocellular/metabolism , Cell Nucleus/metabolism , Liver Neoplasms/metabolism , Ribosomes/metabolism , Transcription Initiation Site , Acetate-CoA Ligase/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation , Cell Survival , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Neoplasm Invasiveness , Prognosis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Ribosomes/geneticsABSTRACT
BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis. METHODS: The Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism. RESULTS: GABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca2+ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression. CONCLUSIONS: Overexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.
Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Receptors, GABA-A/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Chemokines/metabolism , Disease Models, Animal , Humans , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Macrophages/physiology , Mice , Signal Transduction/physiologyABSTRACT
BACKGROUND & AIMS: Agents designed to block or alter cytokinesis can kill or stop proliferation of cancer cells. We aimed to identify cytokinesis-related proteins that are overexpressed in hepatocellular carcinoma (HCC) cells and might be targeted to slow liver tumor growth. METHODS: Using the Oncomine database, we compared the gene expression patterns in 16 cancer microarray datasets and assessed gene enrichment sets using gene ontology. We performed immunohistochemical analysis of an HCC tissue microarray and identified changes in protein levels that are associated with patient survival times. Candidate genes were overexpressed or knocked down with small hairpin RNAs in SMMC7721, MHCC97H, or HCCLM3 cell lines; we analyzed their proliferation, viability, and clone-formation ability and their growth as subcutaneous or orthotopic xenograft tumors in mice. We performed microarray analyses to identify alterations in signaling pathways and immunoblot and immunofluorescence assays to detect and localize proteins in tissues. Yeast 2-hybrid screens and mass spectrometry combined with co-immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation and proximity ligation assays. Chromatin immunoprecipitation, promoter luciferase activity, and quantitative real-time polymerase chain reaction analyses were used to identify factors that regulate transcription of specific genes. RESULTS: The genes that were most frequently overexpressed in different types of cancer cells were involved in cell division processes. We identified 3 cytokinesis-regulatory proteins among the 10 genes most frequently overexpressed by all cancer cell types. Rac GTPase activating protein 1 (RACGAP1) was the cytokinesis-regulatory protein that was most highly overexpressed in multiple cancers. Increased expression of RACGAP1 in tumor tissues was associated with shorter survival times of patients with cancer. Knockdown of RACGAP1 in HCC cells induced cytokinesis failure and cell apoptosis. In microarray analyses, we found knockdown of RACGAP1 in SMMC7721 cells to reduce expression of genes regulated by yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). RACGAP1 reduced activation of the Hippo pathway in HCC cells by increasing activity of RhoA and polymerization of filamentous actin. Knockdown of YAP reduced phosphorylation of RACGAP1 and redistribution at the anaphase central spindle. We found transcription of the translocated promoter region, nuclear basket protein (TPR) to be regulated by YAP and coordinately expressed with RACGAP1 to promote proliferation of HCC cells. TPR redistributed upon nuclear envelope breakdown and formed complexes with RACGAP1 during mitosis. Knockdown of TPR in HCC cells reduced phosphorylation of RACGAP1 by aurora kinase B and impaired their redistribution at the central spindle during cytokinesis. STAT3 activated transcription of RACGAP in HCC cells. CONCLUSIONS: In an analysis of gene expression patterns of multiple tumor types, we found RACGAP1 to be frequently overexpressed, which is associated with shorter survival times of patients. RACGAP1 promotes proliferation of HCC cells by reducing activation of the Hippo and YAP pathways and promoting cytokinesis in coordination with TPR.
Subject(s)
Biomarkers, Tumor/metabolism , Cell Proliferation , Cytokinesis , GTPase-Activating Proteins/metabolism , Liver Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , A549 Cells , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Female , GTPase-Activating Proteins/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HCT116 Cells , Hep G2 Cells , Hippo Signaling Pathway , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , Signal Transduction , Time Factors , Transcription Factors , Tumor Burden , Up-Regulation , YAP-Signaling Proteins , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
Integrin subunit alpha 9 (ITGA9) mediates cell-cell and cell-matrix adhesion, cell migration, and invasion through binding different kinds of extracellular matrix (ECM) components. However, its potential role and underlying molecular mechanisms remain unclear in hepatocellular carcinoma (HCC). Here, we found that ITGA9 expression was obviously decreased in patients with HCC, which was negatively correlated with HCC growth and metastasis. ITGA9 overexpression significantly inhibited cell proliferation and migration in vitro as well as tumor growth and metastasis in vivo. Our data demonstrated that the inhibitory effect of ITGA9 on HCC cell motility was associated with reduced phosphorylation of focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src), disrupted focal adhesion reorganization, and decreased Rac1 and RhoA activity. Our data suggest ITGA9, as a suppressor of HCC, prevents tumor cell migration and invasiveness through FAK/Src-Rac1/RhoA signaling.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Integrin alpha Chains/metabolism , Liver Neoplasms/metabolism , Signal Transduction , rho GTP-Binding Proteins/metabolism , Adult , Aged , Animals , Biomarkers , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Female , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Expression , Gene Expression Profiling , Heterografts , Humans , Immunohistochemistry , Integrin alpha Chains/genetics , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Mice , Middle Aged , Neoplasm Metastasis , PhosphorylationABSTRACT
Tumor-associated macrophages (TAM) represent key regulators of the complex interplay between cancer and the immune microenvironment. Matricellular protein SPON2 is essential for recruiting lymphocytes and initiating immune responses. Recent studies have shown that SPON2 has complicated roles in cell migration and tumor progression. Here we report that, in the tumor microenvironment of hepatocellular carcinoma (HCC), SPON2 not only promotes infiltration of M1-like macrophages but also inhibits tumor metastasis. SPON2-α4ß1 integrin signaling activated RhoA and Rac1, increased F-actin reorganization, and promoted M1-like macrophage recruitment. F-Actin accumulation also activated the Hippo pathway by suppressing LATS1 phosphorylation, promoting YAP nuclear translocation, and initiating downstream gene expression. However, SPON2-α5ß1 integrin signaling inactivated RhoA and prevented F-actin assembly, thereby inhibiting HCC cell migration; the Hippo pathway was not noticeably involved in SPON2-mediated HCC cell migration. In HCC patients, SPON2 levels correlated positively with prognosis. Overall, our findings provide evidence that SPON2 is a critical factor in mediating the immune response against tumor cell growth and migration in HCC.Significance: Matricellular protein SPON2 acts as an HCC suppressor and utilizes distinct signaling events to perform dual functions in HCC microenvironment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/9/2305/F1.large.jpg Cancer Res; 78(9); 2305-17. ©2018 AACR.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Extracellular Matrix Proteins/metabolism , Integrins/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Macrophages/metabolism , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Carcinoma, Hepatocellular/mortality , Cell Line, Tumor , Cell Movement/genetics , Hippo Signaling Pathway , Humans , Liver Neoplasms/mortality , Mice , Models, Biological , Neoplasm Invasiveness , Neoplasm Metastasis , Prognosis , Signal TransductionABSTRACT
Collagen triple helix repeats containing 1 (CTHRC1) participates in vascular remodeling, bone formation, and developmental morphogenesis. Recently, CTHRC1 has been found up-regulated in many solid tumors and contributes to tumorigenesis, but its role in the progression of human colorectal cancer (CRC), remains unclear. In this study, CTHRC1 expression in human CRC cell lines was evaluated by quantitative real-time PCR and immunoblot analyses. The role of CTHRC1 in CRC cell proliferation and extracellular matrix invasion in vitro was analyzed by gene over-expression and recombinant protein. Reporter luciferase assay was used to reveal key relevant signaling pathways involved in CRC cells. The results show that CTHRC1 is secreted both by colorectal epithelia cells and stromal fibroblasts. Recombinant CTHRC1 promotes CRC cell migration and invasion dose-dependently. CTHRC1 overexpression promotes CRC cell migration, invasion and proliferation in vitro. Wnt/PCP signaling but not Wnt/catenin signaling was activates by CTHRC1 in CRC cells. Together, CTHRC1 promotes CRC cell proliferation, migration and invasion in vitro, which is possibly mediated by activating Wnt/PCP pathway.
Subject(s)
Cell Proliferation/genetics , Colorectal Neoplasms/genetics , Extracellular Matrix Proteins/genetics , Neoplasm Invasiveness/genetics , Signal Transduction/genetics , Wnt Proteins/metabolism , beta Catenin/metabolism , Cell Movement/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Invasiveness/pathologyABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder that affects the elderly population. Deposition of beta-amyloid (Aß) in the brain is a hallmark of AD pathology. In our previous study, we have constructed a cell line expressing human APP695 (hAPP695) in SH-EP1 cells stably transfected with human nicotinic receptor (nAChR) α4 subunit and ß2 subunit gene. In present study, we found that activation of α4ß2 nAChR by nicotine and epibatidine decreased secreted Aß level in the cell line and hippocampal neurons, but had no effects on full-length APP695 and sAPP-α. Nicotine also decreases BACE1 and PSEN1 expression, as well as ERK1 and NFκB P65 subunit expression in the cell line. Furthermore, BACE1 promoter activity is, but PSEN1 not, decreased by nicotine in the cell line. All the results suggest that activation of α4ß2 nAChR decreases Aß through regulating BACE1 transcription by ERK1-NFκB pathway. Additionally, analysis of BACE1 promoter activity by dual-luciferase reporter assay may be useful for drug screening as a high throughput method.
Subject(s)
Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases/genetics , Nicotine/pharmacology , Transcription, Genetic/drug effects , Amyloid beta-Protein Precursor/genetics , Cell Line , Enzyme-Linked Immunosorbent Assay , Humans , Polymerase Chain Reaction , Receptors, Nicotinic/geneticsABSTRACT
Multiple lines of evidence have implicated that nicotinic acetylcholine receptor (nAChR) may be an important therapeutic target for the treatment of Alzheimer's disease (AD). Although there are reports suggesting a link between alpha7 nAChR subtype and AD, there has been little report on the mechanism. The present study investigates whether and how α7 nAChR activation affects APP695 processing in SH-EP1 cell model. Cell line co-expressing α7 nAChR gene and human amyloid precursor protein 695 (hAPP695) gene were constructed by stable transfection. Expression of ß-amyloid, α-form of secreted APP (αAPPs) and APP1695 was measured by ELISA, western blotting and real-time PCR respectively. Additionally, α, ß, and γ-secretase activities were also analyzed in constructed SH-EP1-α7 nAChR-hAPP695 cell line. The results showed that SH-EP1-α7 nAChR-hAPP695 cell line, expressing both hAPP695 gene and α7 nAChR subtype gene, was constructed successfully. The secreted Aß was decreased and αAPPs was significantly increased by non-selective nAChR agonist nicotine (10 µM) and specific α7 nAChR agonist GTS-21 (1 µM), and APP expression was not affected. Furthermore, specific α7 nAChR antagonist methyllycaconitine (MLA) reversed the alterations induced by activation of α7 nAChR. CTF-α was increased and CTF-γ was decreased when treated with nicotine (10 µM). In addition, the results of enymatic activity analysis showed that nicotine (1µM) and GTS-21 (0.1, 1 µM) decreased γ-secretase activity, but has no effects on α-secretase activity and ß-secretase activity. Our findings demonstrate that, through regulating γ-secretase activity, α7 nAChR activation reduces APP processing in amyloidogenic pathway, and at the same time enhances APP processing in non-amyloidogenic pathway. The constructed SH-EP1-α7 nAChR-hAPP695 cell line might be useful for screening specific nAChR agonists against AD.