ABSTRACT
BACKGROUND: The glutamate (Glu) and gamma aminobutyric acid (GABA) hypotheses of schizophrenia were proposed in the 1980s. However, current findings on those metabolite levels in schizophrenia have been inconsistent, and the relationship between their abnormalities and the pathophysiology of schizophrenia remains unclear. To summarize the nature of the alterations of glutamatergic and GABAergic systems in schizophrenia, we conducted meta-analyses of proton magnetic resonance spectroscopy (1H-MRS) studies examining these metabolite levels. METHODS: A systematic literature search was conducted using Embase, Medline, PsycINFO, and PubMed. Original studies that compared four metabolite levels (Glu, glutamine [Gln], Glx [Glu+Gln], and GABA), as measured by 1H-MRS, between individuals at high risk for psychosis, patients with first-episode psychosis, or patients with schizophrenia and healthy controls (HC) were included. A random-effects model was used to calculate the effect sizes for group differences in these metabolite levels of 18 regions of interest between the whole group or schizophrenia group and HC. Subgroup analysis and meta-regression were performed based on the status of antipsychotic treatment, illness stage, treatment resistance, and magnetic field strength. RESULTS: One-hundred-thirty-four studies met the eligibility criteria, totaling 7993 participants with SZ-spectrum disorders and 8744 HC. 14 out of 18 ROIs had enough numbers of studies to examine the group difference in the metabolite levels. In the whole group, Glx levels in the basal ganglia (g = 0.32; 95% CIs: 0.18-0.45) were elevated. Subgroup analyses showed elevated Glx levels in the hippocampus (g = 0.47; 95% CIs: 0.21-0.73) and dorsolateral prefrontal cortex (g = 0.25; 95% CIs: 0.05-0.44) in unmedicated patients than HC. GABA levels in the MCC were decreased in the first-episode psychosis group compared with HC (g = -0.40; 95% CIs: -0.62 to -0.17). Treatment-resistant schizophrenia (TRS) group had elevated Glx and Glu levels in the MCC (Glx: g = 0.7; 95% CIs: 0.38-1.01; Glu: g = 0.63; 95% CIs: 0.31-0.94) while MCC Glu levels were decreased in the patient group except TRS (g = -0.17; 95% CIs: -0.33 to -0.01). CONCLUSIONS: Increased glutamatergic metabolite levels and reduced GABA levels indicate that the disruption of excitatory/inhibitory balance may be related to the pathophysiology of schizophrenia-spectrum disorders.
Subject(s)
Schizophrenia , Glutamic Acid/metabolism , Glutamine/metabolism , Humans , Magnetic Resonance Spectroscopy , Proton Magnetic Resonance Spectroscopy/methods , Schizophrenia/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
Cervical adenocarcinoma (ADC) is the second most common pathological subtype of cervical cancer after squamous cell carcinoma. It accounts for approximately 20% of cervical cancers, and the incidence has increased in the past few decades, particularly among young patients. The persistent infection of high-risk human papillomavirus (HPV) is responsible for most cervical ADC. However, almost all available in vitro models are designed to study the carcinogenesis of cervical squamous cell carcinoma. To gain better insights into molecular background of ADC, we aimed to establish an in vitro carcinogenesis model of ADC. We previously reported the establishment of an in vitro model for cervical squamous cell carcinoma by introducing defined viral and cellular oncogenes, HPV16 E6 and E7, c-MYC, and activated RAS to human cervical keratinocytes. In this study, the expression of potential lineage-specifying factors and/or SMAD4 reduction was introduced in addition to the defined four oncogenes to direct carcinogenesis toward ADC. The cell properties associated with the cell lineage were analyzed in monolayer and organoid cultures and the tumors in mouse xenografts. In the cells expressing Forkhead box A2 (FOXA2), apparent changes in cell properties were observed, such as elevated expression of columnar cell markers and decreased expression of squamous cell markers. Strikingly, the histopathology of tumors expressing FOXA2 resembled cervical ADC, proposing that FOXA2 plays a vital role in dictating the histopathology of cervical cancers.
Subject(s)
Adenocarcinoma/pathology , Alphapapillomavirus/pathogenicity , Models, Biological , Uterine Cervical Neoplasms/pathology , Adenocarcinoma/metabolism , Alphapapillomavirus/genetics , Animals , Cell Line, Tumor , Cell Lineage , Cell Transformation, Neoplastic , Female , Hepatocyte Nuclear Factor 3-beta/metabolism , Humans , Mice , Neoplasm Transplantation , Oncogene Proteins, Viral/metabolism , Organoids , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Smad4 Protein/metabolism , Uterine Cervical Neoplasms/metabolismABSTRACT
Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance.
Subject(s)
Genome, Viral , Keratinocytes/virology , Oncogene Proteins, Viral/metabolism , Papillomaviridae/physiology , Papillomavirus Infections/virology , Tumor Suppressor Protein p53/metabolism , Virus Replication , Cells, Cultured , DNA, Viral/genetics , Humans , Keratinocytes/metabolism , Keratinocytes/pathology , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/genetics , Papillomavirus Infections/metabolism , Plasmids , Tumor Suppressor Protein p53/geneticsABSTRACT
Major depressive disorder (MDD) is a mental illness with high socio-economic burden, but its pathophysiology has not been fully elucidated. Recently, the cortical excitatory and inhibitory imbalance hypothesis and neuroplasticity hypothesis have been proposed for MDD. Although several studies have examined the neurophysiological profiles in MDD using transcranial magnetic stimulation (TMS), a meta-analysis of TMS neurophysiology has not been performed. The objective of this study was to compare TMS-electromyogram (TMS-EMG) findings between patients with MDD and healthy controls (HCs). To this end, we examined whether patients with MDD have lower short-interval cortical inhibition (SICI) which reflects gamma-aminobutyric acid (GABA)A receptor-mediated activity, lower cortical silent period (CSP) which represents GABAB receptor-mediated activity, higher intracortical facilitation (ICF) which reflects glutamate N-methyl-D-aspartate receptor-mediated activity, and the lower result of paired associative stimulation (PAS) paradigm which shows the level of neuroplasticity in comparison with HC. Further, we explored the effect of clinical and demographic factors that may influence TMS neurophysiological indices. We first searched and identified research articles that conducted single- or paired-pulse TMS-EMG on patients with MDD and HC. Subsequently, we extracted the data from the included studies and meta-analyzed the data with the comprehensive meta-analysis software. Patients with MDD were associated with lower SICI, lower CSP, potentially higher ICF, and lower PAS compared with HC. Our results confirmed the proposed hypotheses, suggesting the usefulness of TMS neurophysiology as potential diagnostic markers of MDD.
Subject(s)
Depressive Disorder, Major/physiopathology , Depressive Disorder, Major/therapy , Transcranial Magnetic Stimulation/methods , Humans , NeurophysiologyABSTRACT
OBJECTIVE: Cervical cancer is the fourth most common cause of cancer-related deaths in Asian women, due to its poor prognosis. This study aimed to decipher genomic alteration profiles of a cohort of Japanese cervical cancer patients to understand why certain patients benefited from molecular targeted therapies and their prognostic significance. METHODS: During 2008-2018, 154 cervical cancer patients underwent a potentially curative resection procedure at the National Cancer Center Hospital. Genomic DNA samples were analyzed using Ion AmpliSeq™ Cancer Hotspot Panel v2. Alterations in the copy number of PIK3CA, ERBB2, PTEN, and STK11 were detected using the TaqMan assay. HPV-positive results were confirmed by genomic testing and in situ hybridization assay. RESULTS: The frequency of genomic alterations in PIK3CA (36%), STK11 (16%), PTEN (11%), TP53 (11%), and KRAS (8%) was >5%. KRAS mutations were preferentially detected in patients with adenocarcinomas, and the frequency of PIK3CA mutations in patients with squamous cell carcinomas was higher than that in patients with other histological cancer types. HPV-positive results were observed in 139/154 (90.3%) patients, and TP53 mutants were detected in HPV-negative specimens. In this study, the overall survival of patients with genomic alterations in STK11 was worse than in patients with wild-type STK11 (hazard ratio = 10.6, P = 0.0079) and TCGA dataset (hazard ratio = 2.46, P = 0.029). CONCLUSIONS: More than one-third of Japanese cervical cancer patients exhibit mutations targeted by molecular targeted therapies. We have proposed the prognostic value of STK11 genomic alterations.
Subject(s)
Protein Serine-Threonine Kinases/genetics , Uterine Cervical Neoplasms/genetics , AMP-Activated Protein Kinase Kinases , Asian People/genetics , DNA Mutational Analysis , DNA, Neoplasm/genetics , DNA, Neoplasm/isolation & purification , Female , Humans , Middle Aged , Papillomaviridae/genetics , Papillomavirus Infections/enzymology , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Predictive Value of Tests , Protein Serine-Threonine Kinases/metabolism , Uterine Cervical Neoplasms/enzymology , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virologyABSTRACT
Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers.
Subject(s)
Cell Death/genetics , Epithelial Cells/pathology , Proto-Oncogene Proteins c-myc/genetics , ras Proteins/genetics , Cell Line, Tumor , Cells, Cultured , Humans , Neoplasms/genetics , Neoplasms/pathology , rac1 GTP-Binding Protein/metabolismABSTRACT
The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG12V ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , Keratinocytes/virology , Membrane Proteins/metabolism , Oncogene Proteins, Viral/metabolism , Repressor Proteins/metabolism , Uterine Cervical Neoplasms/virology , Adaptor Proteins, Signal Transducing , Animals , Blotting, Western , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/physiology , Heterografts , Humans , Keratinocytes/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , PDZ Domains/physiology , Papillomavirus Infections/complications , Papillomavirus Infections/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/pathologyABSTRACT
HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant.
Subject(s)
Cell Transformation, Viral/immunology , Human papillomavirus 16/physiology , Immunity, Innate/immunology , Keratinocytes/immunology , Keratinocytes/virology , Proteome/immunology , Cells, Cultured , Cytokines/immunology , Gene Expression Regulation, Viral/immunology , Human papillomavirus 16/classification , Human papillomavirus 16/isolation & purification , Humans , Species SpecificityABSTRACT
UNLABELLED: NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE: A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.
Subject(s)
Host-Pathogen Interactions , Human papillomavirus 16/immunology , NF-kappa B/immunology , NF-kappa B/metabolism , Oncogene Proteins, Viral/immunology , Oncogene Proteins, Viral/metabolism , Virus Replication , Cells, Cultured , Human papillomavirus 16/physiology , Humans , Keratinocytes/virology , ProteolysisABSTRACT
High-risk human papillomavirus (HR-HPV) infections account for more than 5% of all cancers (11% in women) such as cervical cancer worldwide. HPVs infect to basal cells of the stratified squamous epithelium and establish persistent infection within the basal compartment. HR-HPV infections can persist more than a decade, leading to development of cancers. The life cycle of HPVs is tightly associated with the differentiation processes of the stratified squamous epithelium; the replication of the viral genome and the expression of the viral genes are strictly regulated depending on differentiation of the host keratinocytes. The viral genome is transiently amplified immediately following infection and then maintained at constant copy numbers in the basal cells. In terminally differentiating keratinocytes, the viral genome is drastically amplified. However, molecular mechanisms underlying switching these three stages of viral genome replication in the viral life cycle are poorly understood. Recently, it has become evident that DNA damage response pathways are involved in the regulation of HPV genome replication. In this review, we would like to introduce recent findings describing the associations of DNA damage response with HPV genome replication.
Subject(s)
Alphapapillomavirus/genetics , Alphapapillomavirus/physiology , DNA Repair/physiology , Gene Expression Regulation, Viral/genetics , Genome, Viral/genetics , Life Cycle Stages/genetics , Uterine Cervical Neoplasms/virology , Virus Replication/genetics , Animals , Cell Differentiation/genetics , DNA Helicases/physiology , DNA Replication , DNA, Viral/genetics , Epithelial Cells/cytology , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Gene Dosage , Genes, Viral/genetics , HumansABSTRACT
Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16.
Subject(s)
Genome, Viral , Human papillomavirus 16/physiology , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/virology , Virus Replication , Cell Line , DNA Replication , Human papillomavirus 16/genetics , Humans , Oncogene Proteins, Viral/geneticsABSTRACT
Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity.
Subject(s)
Oncogene Proteins, Viral , Papillomavirus Infections , Uterine Cervical Neoplasms , Female , Humans , Uterine Cervical Neoplasms/genetics , Human papillomavirus 16/genetics , Oncogene Proteins, Viral/genetics , Cervix Uteri , Carcinogenesis/genetics , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/geneticsABSTRACT
Recently, the specific association between Sinonasal inverted papilloma (SIP) and EGFR exon 20 mutations has been reported. To investigate the link between specific EGFR mutations and SIP development, we established organotypic raft culture system using nasal polyp-derived immortalized NP2 (iNP2) cells expressing EGFR exon 20 mutants or an exon 19 mutant, and SIP-derived iIP4 cells harboring P772_H773insPYNP mutation. In the raft culture, iIP4 cells showed the inverted growth pattern characteristic to SIP. Interestingly, iNP2 cells expressing EGFR exon 20 duplication mutants, S768_D770dup and N771_H773dup, but not of EGFR exon 19 mutant, E746_A750del, showed the inverted growth pattern. Enhanced activation of the PI3K/AKT signaling pathway was observed in iNP2_S768_D770dup and iIP4 cells, while increased MAPK signaling was found in iNP2_N771_H773dup. Increased cell migration and invasion were found in all cells carrying EGFR mutations when compared to iNP2 cells, and this effect was inhibited by either PI3K or MEK inhibitor. Notably, iNP2 cells expressing the N771_H773dup mutant showed the highest migration and invasion abilities. These results suggest that specific mutations in EGFR exon 20 play a crucial role in SIP development, partially though hyper-activation of the PI3K/AKT and MAPK signaling pathways. This study presents the first in vitro model for SIP development, which could facilitate further investigations into SIP pathogenesis and preclinical studies for new therapeutic agents.
Subject(s)
Head and Neck Neoplasms , Papilloma, Inverted , Humans , Papilloma, Inverted/genetics , Papilloma, Inverted/pathology , ErbB Receptors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , MutationABSTRACT
Somatic genetic alteration analysis was performed for post-hysterectomy high-risk early-stage uterine cervical cancer patients who underwent post-operative radiation therapy. Post-operative radiation therapy was performed for patients with pathological features of pelvic lymph node metastasis, parametrium invasion, or positive vaginal margin, which corresponded to the post-operative high-risk category. DNA was extracted from paraffin-embedded surgical specimens, and 50 somatic hotspot genetic alternations were detected using Ion AmpliSeq Cancer Hotspot Panel. The existence of actionable mutation was assessed based on OncoKB evidence level > 3A. Between January 2008 and November 2019, 89 patients who underwent abdominal radical hysterectomy followed by post-operative radiation therapy were identified. The follow-up period for living patients was 82.3 months (range 9.3-153.9), and the 5-year relapse-free survival and overall survival rates were 72.6% and 85.9%, respectively. The most frequently detected somatic mutation was PIK3CA (26 [29.2%] patients); however, no prognostic somatic genetic alterations were identified. Actionable mutations were detected in 30 (33.7%) patients. Actionable mutations were detected in approximately one-third of patients, suggesting that precision medicine can be offered to patients with post-operative high-risk uterine cervical cancer in the near future.
Subject(s)
Class I Phosphatidylinositol 3-Kinases/genetics , PTEN Phosphohydrolase/genetics , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Protein p53/genetics , Uterine Cervical Neoplasms/genetics , AMP-Activated Protein Kinase Kinases , Adult , Aged , Female , Humans , Hysterectomy , Japan/epidemiology , Middle Aged , Radiotherapy, Intensity-Modulated , Retrospective Studies , Uterine Cervical Neoplasms/mortality , Uterine Cervical Neoplasms/radiotherapy , Uterine Cervical Neoplasms/surgeryABSTRACT
Transcription, replication, and segregation of human papillomaviruses (HPVs) are regulated by various host factors, but our understanding of host proteins that bind to the HPV genome is limited. Here we report the results of a search of cellular proteins that can associate with specific genomic regions of HPV type 16 (HPV16). We found that human nucleolin, an abundant nucleolar protein, was preferentially captured in vitro by an HPV16 genomic fragment from nucleotide positions (nt) 531-780. Electrophoretic mobility shift assays with a bacterially expressed nucleolin revealed that nucleolin bound to an HPV16 genomic region between nt 604 and 614 in a sequence-dependent manner. Chromatin immunoprecipitation analysis showed that both exogenous and endogenous nucleolin bound to a plasmid containing the HPV16 genomic region in HeLa cells. Furthermore, nucleolin associated with the HPV16 genome stably maintained in HPV16-infected W12 cells, suggesting that the nucleolin binding may be involved in the dynamics of the HPV genome in cells.
Subject(s)
DNA, Viral/metabolism , Genome, Viral , Human papillomavirus 16/genetics , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , HeLa Cells , Humans , NucleolinABSTRACT
The success rate of establishing human cancer cell lines is not satisfactory and the established cell lines often do not preserve the molecular and histological features of the original tissues. In this study, we developed a novel culture method which can support proliferation of almost all primary epithelial ovarian cancer cells, as well as primary normal human oviductal epithelial cells. Cancer cells from fresh or frozen specimens were enriched by the anti-EpCAM antibody-conjugated magnetic beads, plated on Matrigel-coated plate and cultivated under the optimized culture conditions. Seventeen newly established ovarian cancer cell lines, which included all four major histotypes of ovarian cancer, were confirmed to express histotype-specific markers in vitro. Some of the cell lines from all the four histotypes, except mucinous type, generated tumors in immune-deficient mice and the xenograft tumor tissues recapitulated the corresponding original tissues faithfully. Furthermore, with poorly tumorigenic cell lines including mucinous type, we developed a novel xenograft model which could reconstruct the original tissue architecture through forced expression of a set of oncogenes followed by its silencing. With combination of the novel culture method and cell-derived xenograft system, virtually every epithelial ovarian cancer can be reconstituted in mice in a timely fashion.
Subject(s)
Carcinoma, Ovarian Epithelial/pathology , Cystadenoma, Mucinous/pathology , Ovarian Neoplasms/pathology , Primary Cell Culture/methods , Adult , Carcinoma, Ovarian Epithelial/genetics , Cell Line, Tumor , Cystadenoma, Mucinous/genetics , Ectopic Gene Expression , Female , Humans , Middle Aged , Oncogenes/genetics , Ovarian Neoplasms/genetics , Ovary/pathology , Xenograft Model Antitumor AssaysABSTRACT
Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs.
Subject(s)
BRCA2 Protein/metabolism , Chromosomal Instability , Fanconi Anemia Complementation Group D2 Protein/metabolism , Human papillomavirus 16/metabolism , Oncogene Proteins, Viral/metabolism , Apoptosis Regulatory Proteins , Cell Line , Chromatin/metabolism , Humans , Papillomavirus E7 Proteins , Protein BindingABSTRACT
Mitochondrial DNA (mtDNA) mutations are found in many types of cancers and suspected to be involved in carcinogenesis, although the mechanism has not been elucidated. In this study, we report that consecutive C-to-T mutations (hypermutations), a unique feature of mutations induced by APOBECs, are found in mtDNA from cervical dysplasia and oropharyngeal cancers. In vitro, we found that APOBEC3A (A3A) and 3B (A3B) expression, as well as mtDNA hypermutation, were induced in a cervical dysplastic cell line W12 when cultured in a differentiating condition. The ectopic expression of A3A or A3B was sufficient to hypermutate mtDNA. Fractionation of W12 cell lysates and immunocytochemical analysis revealed that A3A and A3B could be contained in mitochondrion. These results suggest that mtDNA hypermutation is induced upon keratinocyte differentiation, and shed light on its molecular mechanism, which involves A3s. The possible involvement of mtDNA hypermutations in carcinogenesis is also discussed.
Subject(s)
Cell Differentiation/genetics , Cytidine Deaminase/genetics , DNA, Mitochondrial/genetics , Keratinocytes/cytology , Keratinocytes/metabolism , Minor Histocompatibility Antigens/genetics , Proteins/genetics , Cell Differentiation/physiology , Cell Line , Female , Humans , In Vitro Techniques , Mutation/genetics , Oropharyngeal Neoplasms/genetics , Uterine Cervical Dysplasia/geneticsABSTRACT
Most human papillomavirus (HPV)-positive cervical cancers contain integrated copies of the viral genome in their chromosomes and express the viral oncoproteins E6 and E7. A virus-encoded transcription factor, E2, is known to repress E6/E7 expression in HPV-positive cancer cells, leading to growth inhibition, which indicates that E6/E7 is required for the survival of the cells. We found that the E2-mediated growth inhibition of HeLa cells, an HPV18-positive cancer cell line, was coupled with a reduction in telomerase activity, an effect which was rescued by the complementation of E7 expression, but not E6 expression, indicating that the cell viability and the telomerase activity in HeLa cells are maintained by an E7-associated function. Analysis of E7 mutants suggested that the binding to the pRB family of pocket proteins was involved in the ability of E7 to rescue the growth potential and telomerase activity inhibited by E2 expression. We also showed that the telomerase activity upregulated by E7 expression was determined by the hTERT promoter activity, and that c-Myc upregulation caused by pRB inactivation could account for the promoter activity. The activation of p53 and consequent accumulation of p21Cip1, which were triggered by the downregulation of E6, appeared not to be essential for the E2-mediated growth arrest.
Subject(s)
DNA-Binding Proteins/physiology , Oncogene Proteins, Viral/physiology , Cell Cycle Proteins , Cell Survival , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genes, myc/genetics , HeLa Cells/physiology , Humans , Nuclear Proteins/metabolism , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Promoter Regions, Genetic/genetics , Repressor Proteins/metabolism , Telomerase/metabolism , Up-RegulationABSTRACT
The papillomavirus life cycle is tied to the differentiation of the stratified squamous epithelium that this virus infects. The ability to study the papillomavirus life cycle is facilitated by organotypic culturing techniques that allow one to closely recapitulate this terminal differentiation process in the laboratory. Current techniques allow for the establishment of recombinant wild-type or mutant human papillomavirus (HPV) genomes in transfected early-passage human foreskin keratinocytes (HFKs). These cells can then be used in organotypic culture to investigate the role of individual viral genes in different aspects of the viral life cycle. When using early-passage HFKs, there is a need for the transfected HPV genome to extend the life span of the cells in order to have sufficient cell generations in which to carry out organotypic culturing. The recent isolation of a spontaneously immortalized HFK cell line that supports the complete HPV life cycle has further allowed investigators to study wild-type or mutant papillomaviral genomes that do not confer immortalization. In this chapter, we describe the methodologies that permit the study of the HPV life cycle in this HFK cell line.