ABSTRACT
Most mesenchymal tumors found in the uterine corpus are benign tumors; however, uterine leiomyosarcoma is a malignant tumor with unknown risk factors that repeatedly recurs and metastasizes. In some cases, the histopathologic findings of uterine leiomyoma and uterine leiomyosarcoma are similar and surgical pathological diagnosis using excised tissue samples is difficult. It is necessary to analyze the risk factors for human uterine leiomyosarcoma and establish diagnostic biomarkers and treatments. Female mice deficient in the proteasome subunit low molecular mass peptide 2 (LMP2)/ß1i develop uterine leiomyosarcoma spontaneously. MATERIAL AND METHODS: Out of 334 patients with suspected uterine mesenchymal tumors, patients diagnosed with smooth muscle tumors of the uterus were selected from the pathological file. To investigate the expression status of biomarker candidate factors, immunohistochemical staining was performed with antibodies of biomarker candidate factors on thin-cut slides of human uterine leiomyosarcoma, uterine leiomyoma, and other uterine mesenchymal tumors. RESULTS AND DISCUSSION: In human uterine leiomyosarcoma, there was a loss of LMP2/ß1i expression and enhanced cyclin E1 and Ki-67/MIB1 expression. In human uterine leiomyomas and normal uterine smooth muscle layers, enhanced LMP2/ß1i expression and the disappearance of the expression of E1 and Ki-67/MIB1 were noted. The pattern of expression of each factor in other uterine mesenchymal tumors was different from that of uterine leiomyosarcoma. CONCLUSIONS: LMP2/ß1i, cyclin E1, and Ki-67/MIB1 may be candidate factors for biomarkers of human uterine leiomyosarcoma. Further large-cohort clinical trials should be conducted to establish treatments and diagnostics for uterine mesenchymal tumors.
Subject(s)
Biomarkers, Tumor , Cyclin E , Leiomyoma , Leiomyosarcoma , Oncogene Proteins , Uterine Neoplasms , Humans , Female , Uterine Neoplasms/pathology , Uterine Neoplasms/metabolism , Uterine Neoplasms/diagnosis , Uterine Neoplasms/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Leiomyosarcoma/genetics , Leiomyosarcoma/metabolism , Leiomyosarcoma/pathology , Leiomyosarcoma/diagnosis , Leiomyoma/metabolism , Leiomyoma/pathology , Leiomyoma/diagnosis , Leiomyoma/genetics , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Cyclin E/metabolism , Cyclin E/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Middle Aged , Adult , Cysteine EndopeptidasesABSTRACT
INTRODUCTION: According to WHO, Breast cancer is widely considered to be the first or second cause of cancer-related death almost universally. Cell cycle disruption, either in the form of uncontrolled expression of cyclins or because of the suspension in negative regulatory proteins (CDK inhibitors), was found to cause breast cancer. Palbociclib as specific CDK4/6 inhibitor is used for the treatment of ER+ metastatic cancers. In this study, we are looking to investigate the effect of palbociclib on breast cancer cells and evaluate the changes in the expression of some genes involved in the cell cycle as target genes of miR-141 after treatment with this drug. We used MCF7 as functional estrogen and non-invasive and MDA-MB-231 cell lines as triple-negative type of breast cancer and a model for more aggressive. METHOD & MATERIALS: MCF7 and MDA-MB-231 cell lines were cultured in DMEM medium. After counting cells and measuring viability, Palbociclib was administered at varying doses using the IC50 obtained from MTT, with the treatment given at two time points of 24 and 72 hours. RNA was extracted from untreated and treated cells and RNAs were converted to cDNA in the end. Gene expression changes were investigated by real-time PCR. Data management and analysis were conducted using GraphPad Prism 5.01 software. RESULT AND CONCLUSION: Among investigated genes, E2F3 gene was not significantly affected by Palbociclib in any of cell lines and time points. Besides, the expression of CCNE1 gene was significantly suppressed. It seems this drug was unable to reduce the expression of MDM2 gene significantly in triple negative (MDA-MB-231) cancer cells; however, a decrease was observed in luminal A (MCF-7) cells. CDKN2A and miR-141 genes expression increased significantly after treatment which can be aligned with palbociclib in proliferation inhibition.
Subject(s)
Cyclin E , Gene Expression Regulation, Neoplastic , MicroRNAs , Oncogene Proteins , Piperazines , Pyridines , Humans , Pyridines/pharmacology , MicroRNAs/genetics , Piperazines/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Cell Line, Tumor , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Cyclin E/genetics , Cyclin E/metabolism , MCF-7 Cells , Female , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Cell Proliferation/drug effectsABSTRACT
Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Breast Neoplasms , Cyclin E , DNA Replication , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Cyclin E/genetics , Cyclin E/metabolism , DNA Replication/genetics , Polymorphism, Single Nucleotide , Cell Proliferation , DNA Copy Number Variations , Middle Aged , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Aged , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , AdultABSTRACT
RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins.
Subject(s)
Adaptor Proteins, Signal Transducing , Cyclic AMP-Dependent Protein Kinases , GRB2 Adaptor Protein , Signal Transduction , src Homology Domains , Humans , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , GRB2 Adaptor Protein/metabolism , GRB2 Adaptor Protein/genetics , HEK293 Cells , Oncogene Proteins/metabolism , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Phosphorylation , Protein BindingABSTRACT
High-grade serous ovarian cancer (HGSC) is an aggressive disease with poor prognosis. The oncoprotein ZNF703 is implicated in driving HGSC pathogenesis, but factors regulating its abundance remain unclear. In this study, we aim to investigate the potential connection between ZNF703 dysregulation and ubiquitin-mediated protein degradation in HGSC. Bioinformatics prediction was performed using BioGRID database. HGSC representative cell lines were utilized for in vitro and in vivo studies. Results showed that ZNF703 protein was stabilized upon proteasome inhibition, suggesting a regulation via ubiquitination. The ubiquitin E3 ligase PARK2 was found to interact with ZNF703 in a dose-dependent manner, promoting its polyubiquitination and subsequent proteasomal degradation. Re-expression of PARK2 in HGSC cells led to reduced ZNF703 levels together with decreased Cyclin D1/E1 abundance and G1 cell cycle arrest. ZNF703 overexpression alone increased S phase cells, Cyclin D1/E1 levels, and xenograft tumor growth, while co-expression with PARK2 mitigated these oncogenic effects. Collectively, our findings identify ZNF703 as a bona fide substrate of PARK2, reveal a tumor suppressive function for PARK2 in attenuating ZNF703-mediated G1/S transition and HGSC growth through instigating its degradation. This study elucidates a pivotal PARK2-ZNF703 axis with therapeutic implications for targeted intervention in HGSC.
Subject(s)
Cell Proliferation , Cystadenocarcinoma, Serous , Ovarian Neoplasms , Proteasome Endopeptidase Complex , Ubiquitin-Protein Ligases , Humans , Female , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Proteasome Endopeptidase Complex/metabolism , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/genetics , Cell Line, Tumor , Animals , Mice , Ubiquitination , Cyclin D1/metabolism , Cyclin D1/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Mice, Nude , Proteolysis , Cyclin E/metabolism , Cyclin E/genetics , Mice, Inbred BALB C , Xenograft Model Antitumor Assays , Gene Expression Regulation, Neoplastic , Carrier ProteinsABSTRACT
The anterior gradient protein 2 (AGR2) plays a crucial role in facilitating the formation of protein disulfide bonds within the endoplasmic reticulum (ER). Research suggests that AGR2 can function as an oncogene, with its heightened expression linked to the advancement of hepatobiliary and pancreatic cancers through invasion and metastasis. Notably, AGR2 not only serves as a pro-oncogenic agent but also as a downstream targeting protein, indirectly fostering cancer progression. This comprehensive review delves into the established functions and expression patterns of AGR2, emphasizing its pivotal role in cancer progression, particularly in hepatobiliary and pancreatic malignancies. Furthermore, AGR2 emerges as a potential cancer prognostic marker and a promising target for immunotherapy, offering novel avenues for the treatment of hepatobiliary and pancreatic cancers and enhancing patient outcomes.
Subject(s)
Mucoproteins , Oncogene Proteins , Pancreatic Neoplasms , Humans , Mucoproteins/metabolism , Mucoproteins/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/therapy , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/therapy , Liver Neoplasms/pathology , Animals , Biliary Tract Neoplasms/genetics , Biliary Tract Neoplasms/metabolism , Biliary Tract Neoplasms/drug therapy , Biliary Tract Neoplasms/therapy , Biliary Tract Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/geneticsSubject(s)
Leukemia, Myeloid, Acute , Nuclear Pore Complex Proteins , Humans , Leukemia, Myeloid, Acute/genetics , Nuclear Pore Complex Proteins/genetics , Male , Poly-ADP-Ribose Binding Proteins/genetics , Eyelids/abnormalities , Gene Rearrangement , Oncogene Proteins/genetics , Chromosomal Proteins, Non-HistoneABSTRACT
Twelve DEK-NUP214 fusion gene-positive patients with acute myeloid leukemia and on allo-HSCT treatment at the Hematology Hospital of the Chinese Academy of Medical Sciences from November 2016 to August 2022 were included in the study, and their clinical data were retrospectively analyzed. The patients comprised five men and seven women with a median age of 34 (16-52) years. At the time of diagnosis, all the patients were positive for the DEK-NUP214 fusion gene. Chromosome karyotyping analysis showed t (6;9) (p23;q34) translocation in 10 patients (two patients did not undergo chromosome karyotyping analysis), FLT3-ITD mutation was detected in 11 patients, and high expression of WT1 was observed in 11 patients. Nine patients had their primary disease in the first complete remission state before transplantation, one patient had no disease remission, and two patients were in a recurrent state. All patients received myeloablative pretreatment, five patients received sibling allogeneic hematopoietic stem cell transplantation, and seven patients received haploid hematopoietic stem cell transplantation. The median number of mononuclear cells in the transplant was 10.87 (7.09-17.89) ×10(8)/kg, and the number of CD34(+) cells was 3.29 (2.53-6.10) ×10(6)/kg. All patients achieved blood reconstruction, with a median time of 14 (10-20) days for neutrophil implantation and 15 (9-27) days for platelet implantation. The 1 year transplant-related mortality rate after transplantation was 21.2%. The cumulative recurrence rates 1 and 3 years after transplantation were 25.0% and 50.0%, respectively. The leukemia free survival rates were (65.6±14.0) % and (65.6±14.0) %, respectively. The overall survival rates were (72.2±13.8) % and (72.2±13.8) %, respectively.
Subject(s)
Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Acute , Nuclear Pore Complex Proteins , Transplantation, Homologous , Humans , Male , Female , Adult , Hematopoietic Stem Cell Transplantation/methods , Middle Aged , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/therapy , Adolescent , Retrospective Studies , Young Adult , Nuclear Pore Complex Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins/genetics , Translocation, GeneticABSTRACT
Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics.
Subject(s)
Amino Acid Transport System y+ , Carcinoma, Non-Small-Cell Lung , Ferroptosis , Lung Neoplasms , Proteasome Endopeptidase Complex , Ubiquitin , Ferroptosis/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Animals , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Mice , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Cell Line, Tumor , Ubiquitination , Mice, Nude , Proteolysis/drug effectsABSTRACT
Oncogenic viruses contribute to 15% of global human cancers. To achieve that, virus-encoded oncoproteins deregulate cellular transcription, antagonize common cellular pathways, and thus drive cell transformation. Notably, adenoviruses were the first human viruses proven to induce cancers in diverse animal models. Over the past decades, human adenovirus (HAdV)-mediated oncogenic transformation has been pivotal in deciphering underlying molecular mechanisms. Key adenovirus oncoproteins, encoded in early regions 1 (E1) and 4 (E4), co-ordinate these processes. Among the different adenovirus species, the most extensively studied HAdV-C5 displays lower oncogenicity than HAdV-A12. A complete understanding of the different HAdV-A12 and HAdV-C5 oncoproteins in virus-mediated cell transformation, as summarized here, is relevant for adenovirus research and offers broader insights into viral transformation and oncogenesis.
Subject(s)
Adenoviruses, Human , Humans , Adenoviruses, Human/genetics , Adenoviruses, Human/physiology , Animals , Oncogenes , Cell Transformation, Viral , Neoplasms/virology , Neoplasms/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Carcinogenesis/geneticsABSTRACT
NUT (nuclear-protein-in-testis) carcinoma (NC) is a highly aggressive tumor disease. Given that current treatment regimens offer a median survival of six months only, it is likely that this type of tumor requires an extended multimodal treatment approach to improve prognosis. In an earlier case report, we could show that an oncolytic herpes simplex virus (T-VEC) is functional in NC patients. To identify further combination partners for T-VEC, we have investigated the anti-tumoral effects of T-VEC and five different small molecule inhibitors (SMIs) alone and in combination in human NC cell lines. Dual combinations were found to result in higher rates of tumor cell reductions when compared to the respective monotherapy as demonstrated by viability assays and real-time tumor cell growth monitoring. Interestingly, we found that the combination of T-VEC with SMIs resulted in both stronger and earlier reductions in the expression of c-Myc, a main driver of NC cell proliferation, when compared to T-VEC monotherapy. These results indicate the great potential of combinatorial therapies using oncolytic viruses and SMIs to control the highly aggressive behavior of NC cancers and probably will pave the way for innovative multimodal clinical studies in the near future.
Subject(s)
Biological Products , Oncolytic Virotherapy , Oncolytic Viruses , Humans , Oncolytic Viruses/physiology , Oncolytic Viruses/genetics , Oncolytic Virotherapy/methods , Cell Line, Tumor , Combined Modality Therapy , Biological Products/pharmacology , Biological Products/therapeutic use , Cell Proliferation/drug effects , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Carcinoma/therapy , Cell Survival/drug effects , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Neoplasm Proteins , Herpesvirus 1, HumanABSTRACT
Cyclin E overexpression as a result of CCNE1 amplification is a critical driver of genomic instability in gastric cancer, but its clinical implication is largely unknown. Thus, we integrated genomic, transcriptomic, and immune profiling analysis of 7,083 esophagogastric tumors and investigated the impact of CCNE1 amplification on molecular features and treatment outcomes. We identified CCNE1 amplification in 6.2% of esophageal adenocarcinoma samples, 7.0% of esophagogastric junction carcinoma, 4.2% of gastric adenocarcinoma samples, and 0.8% of esophageal squamous cell carcinoma. Metastatic sites such as lymph node and liver showed an increased frequency of CCNE1 amplification relative to primary tumors. Consistent with a chromosomal instability phenotype, CCNE1 amplification was associated with decreased CDH1 mutation and increased TP53 mutation and ERBB2 amplification. We observed no differences in immune biomarkers such as PD-L1 expression and tumor mutational burden comparing CCNE1-amplified and nonamplified tumors, although CCNE1 amplification was associated with changes in immune populations such as decreased B cells and increased M1 macrophages from transcriptional analysis. Real-world survival analysis demonstrated that patients with CCNE1-amplified gastric cancer had worse survival after trastuzumab for HER2-positive tumors, but better survival after immunotherapy. These data suggest that CCNE1-amplified gastric cancer has a distinct molecular and immune profile with important therapeutic implications, and therefore further investigation of CCNE1 amplification as a predictive biomarker is warranted. SIGNIFICANCE: Advanced gastric cancer has a relatively dismal outcome with a 5-year overall survival of less than 10%. Furthermore, while comprehensive molecular analyses have established molecular subtypes within gastric cancers, biomarkers of clinical relevance in this cancer type are lacking. Overall, this study demonstrates that CCNE1 amplification is associated with a distinct molecular profile in gastric cancer and may impact response to therapy, including targeted therapy and/or immunotherapy.
Subject(s)
Cyclin E , Esophageal Neoplasms , Gene Amplification , Oncogene Proteins , Stomach Neoplasms , Humans , Cyclin E/genetics , Oncogene Proteins/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/immunology , Stomach Neoplasms/pathology , Esophageal Neoplasms/genetics , Esophageal Neoplasms/mortality , Esophageal Neoplasms/immunology , Esophageal Neoplasms/pathology , Receptor, ErbB-2/genetics , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Biomarkers, Tumor/genetics , Mutation , Male , Esophagogastric Junction/pathology , Female , Trastuzumab/therapeutic use , Tumor Suppressor Protein p53/genetics , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/immunology , Esophageal Squamous Cell Carcinoma/pathology , Esophageal Squamous Cell Carcinoma/mortality , Antigens, CD/genetics , CadherinsABSTRACT
BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.
Subject(s)
14-3-3 Proteins , Actinin , Autophagy , Chemotaxis , Endoplasmic Reticulum Stress , Mammary Neoplasms, Animal , Mucoproteins , Animals , Dogs , 14-3-3 Proteins/metabolism , 14-3-3 Proteins/genetics , Female , Actinin/metabolism , Actinin/genetics , Mammary Neoplasms, Animal/metabolism , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Cell Line, Tumor , Chemotaxis/genetics , Autophagy/genetics , Endoplasmic Reticulum Stress/genetics , Mucoproteins/genetics , Mucoproteins/metabolism , Oncogene Proteins/metabolism , Oncogene Proteins/geneticsSubject(s)
Gene Amplification , Mutation , N-Myc Proto-Oncogene Protein , Neuroblastoma , Nuclear Proteins , Oncogene Proteins , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/complications , N-Myc Proto-Oncogene Protein/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , MaleABSTRACT
Despite considerable therapeutic advancements, the global survival rate for lung cancer patients remains poor, posing challenges in developing an effective treatment strategy. In many cases, microRNAs (miRNAs) exhibit abnormal expression levels in cancers, including lung cancer. Dysregulated miRNAs often play a crucial role in the development and progression of cancer. Therefore, understanding the mechanisms underlying aberrant miRNA expression during carcinogenesis may provide crucial clues to develop novel therapeutics. In this study, we identified and cloned a novel miRNA, hsa-miR-CHA2, which is abnormally downregulated in non-small cell lung cancer (NSCLC)-derived cell lines and tissues of patients with NSCLC. Furthermore, we found that hsa-miR-CHA2 regulates the post-transcriptional levels of Cyclin E1 (CCNE1) by binding to the 3'-UTR of CCNE1 mRNA. CCNE1, a cell cycle regulator involved in the G1/S transition, is often amplified in various cancers. Notably, hsa-miR-CHA2 overexpression led to the alteration of the Rb-E2F pathway, a significant signaling pathway in the cell cycle, by targeting CCNE1 in A549 and SK-LU-1 cells. Subsequently, we confirmed that hsa-miR-CHA2 induced G1-phase arrest and exhibited an anti-proliferative effect by targeting CCNE1. Moreover, in subcutaneous xenograft mouse models, intra-tumoral injection of polyplexed hsa-miR-CHA2 mimic suppressed tumor growth and development. In conclusion, hsa-miR-CHA2 exhibited an anticancer effect by targeting CCNE1 both in vitro and in vivo. These findings suggest the potential role of hsa-miR-CHA2 as an important regulator of cell proliferation in molecular-targeted therapy for NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Cyclin E , Gene Expression Regulation, Neoplastic , Lung Neoplasms , MicroRNAs , Oncogene Proteins , Humans , Cyclin E/genetics , Cyclin E/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Animals , Mice , Cell Proliferation/genetics , Cell Line, Tumor , A549 Cells , Mice, Nude , Xenograft Model Antitumor Assays , 3' Untranslated Regions/genetics , Mice, Inbred BALB C , Signal TransductionABSTRACT
Aurora kinase B (AURKB) initiates the phosphorylation of serine 10 on histone H3 (pH3S10), a crucial process for chromosome condensation and cytokinesis in mammalian mitosis. Nonetheless, the precise mechanisms through which AURKB regulates the cell cycle and contributes to tumorigenesis as an oncogenic factor in colorectal cancer (CRC) remain unclear. Here, we report that AURKB was highly expressed and positively correlated with Ki-67 expression in CRC. The abundant expression of AURKB promotes the growth of CRC cells and xenograft tumors in animal model. AURKB knockdown substantially suppressed CRC proliferation and triggered cell cycle arrest in G2/M phase. Interestingly, cyclin E1 (CCNE1) was discovered as a direct downstream target of AURKB and functioned synergistically with AURKB to promote CRC cell proliferation. Mechanically, AURKB activated CCNE1 expression by triggering pH3S10 in the promoter region of CCNE1. Furthermore, it was showed that the inhibitor specific for AURKB (AZD1152) can suppress CCNE1 expression in CRC cells and inhibit tumor cell growth. To conclude, this research demonstrates that AURKB accelerated the tumorigenesis of CRC through its potential to epigenetically activate CCNE1 expression, suggesting AURKB as a promising therapeutic target in CRC.
Subject(s)
Aurora Kinase B , Cell Proliferation , Colorectal Neoplasms , Cyclin E , Histones , Oncogene Proteins , Humans , Colorectal Neoplasms/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Cyclin E/metabolism , Cyclin E/genetics , Histones/metabolism , Aurora Kinase B/metabolism , Aurora Kinase B/genetics , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Phosphorylation , Animals , Cell Proliferation/genetics , Mice , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Serine/metabolism , Disease Progression , Male , Mice, Nude , FemaleABSTRACT
Lipocalin-2 (LCN2), an effector molecule of the innate immune system that is small enough to be tagged as a reporter molecule, can be coupled with the ferric ion through a siderophore such as enterobactin (Ent). Mintbody (modification-specific intracellular antibody) can track a posttranslational protein modification in epigenetics. We constructed plasmids expressing the LCN2 hybrid of mintbody to examine the potential of LCN2 as a novel reporter for magnetic resonance imaging (MRI). Cells expressing the LCN2 hybrid of mintbody showed proper expression and localization of the hybrid and responded reasonably to Ent, suggesting their potential for in vivo study by MRI.
Subject(s)
Lipocalin-2 , Lipocalins , Lipocalin-2/metabolism , Lipocalin-2/genetics , Humans , Lipocalins/metabolism , Lipocalins/genetics , Magnetic Resonance Imaging , Genes, Reporter , Acute-Phase Proteins/metabolism , Acute-Phase Proteins/genetics , Enterobactin/metabolism , Animals , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/geneticsABSTRACT
CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS.
Subject(s)
Biomarkers, Tumor , Cyclin E , Gene Rearrangement , Oncogene Proteins , Repressor Proteins , Humans , Male , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Female , Adult , Cyclin E/metabolism , Cyclin E/genetics , Middle Aged , Adolescent , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Young Adult , Child , Repressor Proteins/metabolism , Repressor Proteins/genetics , Immunohistochemistry/methods , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Sarcoma, Ewing/genetics , Sarcoma/pathology , Sarcoma/metabolism , Sarcoma/genetics , Sarcoma/diagnosis , In Situ Hybridization, Fluorescence/methods , Aged , Child, Preschool , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , Sarcoma, Small Cell/metabolism , Sarcoma, Small Cell/genetics , Sarcoma, Small Cell/pathology , Sarcoma, Small Cell/diagnosisABSTRACT
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
Subject(s)
N-Myc Proto-Oncogene Protein , Nuclear Proteins , Oncogene Proteins , RNA-Binding Proteins , N-Myc Proto-Oncogene Protein/metabolism , N-Myc Proto-Oncogene Protein/genetics , Humans , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/genetics , Promoter Regions, Genetic , Cell Line, Tumor , Neuroblastoma/metabolism , Neuroblastoma/genetics , Neuroblastoma/pathology , Exosomes/metabolism , Exosomes/genetics , Introns , Protein Binding , Cell Nucleus/metabolism , Exosome Multienzyme Ribonuclease Complex/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Gene Expression Regulation, Neoplastic , RNA/metabolism , RNA/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Cell ProliferationABSTRACT
Circular RNAs (circRNAs) play important roles in cancer occurrence and progression. To explore and elucidate the clinical significance of specific circular RNA in melanoma and its potential molecular mechanism. CircROR1 expression in melanoma cells and tissues was confirmed by qRT-PCR and ISH. qRT-PCR and Western blotting were performed to measure the levels of CCNE1, KAT2A, MMP9 and TIMP2. MTT, Transwell and wound healing assays were performed to evaluate cell proliferation, invasion and metastasis. A xenograft mouse model was established to further verify the CircROR1/CCNE1 axis in vivo. RNA pull-down and RIP assays were performed to detect the direct interaction KAT2A and CircROR1. A ChIP assay was used to investigate the enrichment of H3K9ac acetylation in the CCNE1 promoter. CircROR1 was significantly upregulated in metastatic melanoma cells and tissues, promoting proliferation, invasion and metastasis in vitro and tumour growth in vivo. CircROR1 overexpression increased CCNE1 and MMP9 protein expression and decreased TIMP2 protein expression. Functional rescue assays demonstrated that CircROR1 played a role in promoting malignant progression through CCNE1. CircROR1 specifically bound to the KAT2A protein without affecting its expression. CircROR1 overexpression increased the level of H3K9ac modification in the CCNE1 promoter region by recruiting KAT2A, thus upregulating CCNE1 expression. CircROR1 upregulates CCNE1 expression through KAT2A-mediated histone acetylation. Our research confirms the critical role of CircROR1 in melanoma invasion and metastasis, and CircROR1 could serve as a potential therapeutic target for melanoma treatment.