ABSTRACT
Systemic modalities are crucial in the management of disseminated malignancies and liquid tumours. However, patient responses and tolerability to treatment are generally poor and those that enter remission often return with refractory disease. Combination therapies provide a methodology to overcome chemoresistance mechanisms and address dose-limiting toxicities. A deeper understanding of tumorigenic processes at the molecular level has brought a targeted therapy approach to the forefront of cancer research, and novel cancer biomarkers are being identified at a rapid rate, with some showing potential therapeutic benefits. The Karyopherin superfamily of proteins is soluble receptors that mediate nucleocytoplasmic shuttling of proteins and RNAs, and recently, nuclear transport receptors have been recognized as novel anticancer targets. Inhibitors against nuclear export have been approved for clinical use against certain cancer types, whereas inhibitors against nuclear import are in preclinical stages of investigation. Mechanistically, targeting nucleocytoplasmic shuttling has shown to abrogate oncogenic signalling and restore tumour suppressor functions through nuclear sequestration of relevant proteins and mRNAs. Hence, nuclear transport inhibitors display broad spectrum anticancer activity and harbour potential to engage in synergistic interactions with a wide array of cytotoxic agents and other targeted agents. This review is focussed on the most researched nuclear transport receptors in the context of cancer, XPO1 and KPNB1, and highlights how inhibitors targeting these receptors can enhance the therapeutic efficacy of standard of care therapies and novel targeted agents in a combination therapy approach. Furthermore, an updated review on the therapeutic targeting of lesser characterized karyopherin proteins is provided and resistance to clinically approved nuclear export inhibitors is discussed.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Active Transport, Cell Nucleus/physiology , Exportin 1 Protein , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Karyopherins/genetics , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Drug Therapy, CombinationABSTRACT
Previous studies have identified increased expression of members of the nuclear transport protein family in cancer cells. Recently, certain nuclear transport proteins have been reported to be secreted by cells and found in the serum. The aims of our study were to investigate the levels of multiple nuclear transport proteins secreted from cancer cells, and to determine their potential as diagnostic markers for cervical and oesophageal cancer. Mass spectrometry identified 10 nuclear transport proteins in the secretome and exosomes of cultured cancer cells, and Western blot analysis confirmed increased secreted levels in cancer cells compared to normal. To investigate their presence in patient serum, enzyme-linked immunosorbent assays were performed and revealed significantly increased levels of KPNß1, CRM1, CAS, IPO5 and TNPO1 in cervical and oesophageal cancer patient serum compared to non-cancer controls. Significantly elevated KPNα2 and RAN levels were also identified in oesophageal cancer serum samples. Logistics regression analyses revealed IPO5 and TNPO1 to be the best performing individual candidate biomarkers in discriminating between cancer cases and controls. The combination of KPNß1, CRM1, KPNα2, CAS, RAN, IPO5 and TNPO1 as a panel of biomarkers had the highest diagnostic capacity with an area under the curve of 0.944 and 0.963, for cervical cancer and oesophageal cancer, and sensitivity of 92.5% at 86.8% specificity and 95.3% sensitivity at 87.5% specificity, respectively. These results suggest that nuclear transport proteins have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of protein family members being the best predictor.
Subject(s)
Biomarkers, Tumor/metabolism , Carrier Proteins/metabolism , Cell Nucleus/metabolism , Esophageal Neoplasms/diagnosis , Nuclear Proteins/metabolism , Secretome/metabolism , Uterine Cervical Neoplasms/diagnosis , Active Transport, Cell Nucleus , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Esophageal Neoplasms/metabolism , Esophageal Squamous Cell Carcinoma/diagnosis , Esophageal Squamous Cell Carcinoma/metabolism , Female , Follow-Up Studies , Humans , Male , Middle Aged , Prognosis , Tumor Cells, Cultured , Uterine Cervical Neoplasms/metabolism , Young AdultABSTRACT
Karyopherin beta 1 (Kpnß1) is a major nuclear import receptor that mediates the import of cellular cargoes into the nucleus. Recently it has been shown that Kpnß1 is highly expressed in several cancers, and its inhibition by siRNA induces apoptotic cancer cell death, while having little effect on non-cancer cells. This study investigated the effect of a novel small molecule, Inhibitor of Nuclear Import-60 (INI-60), on cancer cell biology, as well as nuclear import activities associated with Kpnß1, and cancer progression in vivo using cervical and oesophageal cancer cell lines. INI-60 treatment resulted in the inhibition of cancer cell proliferation, colony formation, migration and invasion, and induced a G1/S cell cycle arrest, followed by cancer cell death via apoptosis. Non-cancer cells were minimally affected by INI-60 at concentrations that inhibited cancer cells. INI-60 treatment altered the localisation of Kpnß1 and its cargoes, NFκB/p65, NFAT and AP-1, and the overexpression of Kpnß1 reduced INI-60 cytotoxicity. INI-60 also inhibited KYSE 30 oesophageal cancer cell line growth in vivo. Taken together, these results show that INI-60 inhibits the nuclear import of Kpnß1 cargoes and interferes with cancer cell biology. INI-60 presents as a potential therapeutic approach for cancers of different tissue origins and warrants further investigation as a novel anti-cancer agent.
Subject(s)
Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Gene Expression Regulation, Neoplastic/drug effects , beta Karyopherins/antagonists & inhibitors , Active Transport, Cell Nucleus/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/genetics , beta Karyopherins/geneticsABSTRACT
BACKGROUND: Inhibition of nuclear import via Karyopherin beta 1 (Kpnß1) shows potential as an anti-cancer approach. This study investigated the use of nuclear import inhibitor, INI-43, in combination with cisplatin. METHODS: Cervical cancer cells were pre-treated with INI-43 before treatment with cisplatin, and MTT cell viability and apoptosis assays performed. Activity and localisation of p53 and NFκB was determined after co-treatment of cells. RESULTS: Pre-treatment of cervical cancer cells with INI-43 at sublethal concentrations enhanced cisplatin sensitivity, evident through decreased cell viability and enhanced apoptosis. Kpnß1 knock-down cells similarly displayed increased sensitivity to cisplatin. Combination index determination using the Chou-Talalay method revealed that INI-43 and cisplatin engaged in synergistic interactions. p53 was found to be involved in the cell death response to combination treatment as its inhibition abolished the enhanced cell death observed. INI-43 pre-treatment resulted in moderately stabilized p53 and induced p53 reporter activity, which translated to increased p21 and decreased Mcl-1 upon cisplatin combination treatment. Furthermore, cisplatin treatment led to nuclear import of NFκB, which was diminished upon pre-treatment with INI-43. NFκB reporter activity and expression of NFκB transcriptional targets, cyclin D1, c-Myc and XIAP, showed decreased levels after combination treatment compared to single cisplatin treatment and this associated with enhanced DNA damage. CONCLUSIONS: Taken together, this study shows that INI-43 pre-treatment significantly enhances cisplatin sensitivity in cervical cancer cells, mediated through stabilization of p53 and decreased nuclear import of NFκB. Hence this study suggests the possible synergistic use of nuclear import inhibition and cisplatin to treat cervical cancer.
Subject(s)
Active Transport, Cell Nucleus/drug effects , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Cisplatin/pharmacology , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Pyrroles/pharmacology , Quinoxalines/pharmacology , Uterine Cervical Neoplasms/drug therapy , beta Karyopherins/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Apoptosis , Biomarkers, Tumor/genetics , Cell Movement , Cell Proliferation , Cisplatin/therapeutic use , Drug Therapy, Combination , Female , Humans , Pyrroles/therapeutic use , Quinoxalines/therapeutic use , Tumor Cells, Cultured , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathologyABSTRACT
Transcription factors control numerous cellular processes, including proliferation, apoptosis, differentiation, and inflammation. Abnormal transcription factor activity has been implicated in a variety of diseases, especially cancer. The correct subcellular localization of transcription factors determines their activation status, implicating the nuclear transport receptors as key players in regulating transcription factor function. Dysregulation of the nuclear transport machinery has been described in numerous cancer types. This review summarizes how altered nuclear transport activity affects transcription factor localization and activity, and contributes to cancer development. Furthermore, the potential of targeting nuclear transporters for cancer therapy is discussed.
Subject(s)
Active Transport, Cell Nucleus/physiology , Gene Expression Regulation , Neoplasms/metabolism , Transcription Factors/metabolism , Apoptosis , Cell Differentiation , Humans , Inflammation , Transcription Factors/geneticsABSTRACT
The circadian clock and the ~24 h rhythms it generates are essential in maintaining regular tissue functioning. At the molecular level, the circadian clock comprises a core set of rhythmically expressed genes and gene products that are able to drive rhythmic expression of other genes to generate overt circadian rhythms. It has recently come to light that perturbations of circadian rhythms contribute to the development of pathological states such as cancer, and altered expression and/or regulation of circadian clock genes has been identified in multiple tumour types. This review summarises the important role the circadian system plays in regulating cellular processes, including the cell cycle, apoptosis, DNA repair, the epithelial-to-mesenchymal transition, metabolism and immunity and how its dysregulation has widespread implications and could be a critical player in the development of cancer. Understanding its role in cancer development is important for the field chronotherapy, where the timing of chemotherapy administration is optimised based on differences in circadian clock functioning in normal and cancer cells. This has been found to influence the patient response, minimising the side effects commonly associated with chemotherapy. © 2019 IUBMB Life, 2019.
Subject(s)
Antineoplastic Agents/therapeutic use , Circadian Rhythm Signaling Peptides and Proteins/metabolism , Circadian Rhythm/physiology , Neoplasms/prevention & control , Animals , Circadian Rhythm Signaling Peptides and Proteins/genetics , Humans , Neoplasms/genetics , Neoplasms/metabolismABSTRACT
BACKGROUND: Karyopherin ß1 (Kpnß1) is the main nuclear import protein involved in the transport of cargoes from the cytoplasm into the cell nucleus. Previous research has found Kpnß1 to be significantly overexpressed in cervical cancer and other cancer tissues, and further studies showed that inhibition of Kpnß1 expression by siRNA resulted in cancer cell death, while non-cancer cells were minimally affected. These results suggest that Kpnß1 has potential as an anticancer therapeutic target, thus warranting further research into the association between Kpnß1 expression and cancer progression. Here, the biological effects associated with Kpnß1 overexpression were investigated in order to further elucidate the relationship between Kpnß1 and the cancer phenotype. METHODS: To evaluate the effect of Kpnß1 overexpression on cell biology, cell proliferation, cell cycle, cell morphology and cell adhesion assays were performed. To determine whether Kpnß1 overexpression influences cell sensitivity to chemotherapeutic agents like Cisplatin, cell viability assays were performed. Expression levels of key proteins were analysed by Western blot analysis. RESULTS: Our data revealed that Kpnß1 overexpression, above that which was already detected in cancer cells, resulted in reduced proliferation of cervical cancer cells. Likewise, normal epithelial cells showed reduced proliferation after Kpnß1 overxpression. Reduced cancer cell proliferation was associated with a delay in cell cycle progression, as well as changes in the morphology and adhesion properties of cells. Additionally, Kpnß1 overexpressing HeLa cells exhibited increased sensitivity to cisplatin, as shown by decreased cell viability and increased apoptosis, where p53 and p21 inhibition reduced and enhanced cell sensitivity to Cisplatin, respectively. CONCLUSIONS: Overall, our results suggest that a tight balance of Kpnß1 expression is required for cellular function, and that perturbation of this balance results in negative effects associated with a variety of biological processes.
Subject(s)
Uterine Cervical Neoplasms/metabolism , beta Karyopherins/metabolism , Apoptosis/drug effects , Apoptosis/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cisplatin/pharmacology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , HeLa Cells , Humans , RNA, Small Interfering/metabolism , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/drug therapyABSTRACT
The Karyopherin superfamily is a major class of soluble transport receptors consisting of both import and export proteins. The trafficking of proteins involved in transcription, cell signalling and cell cycle regulation among other functions across the nuclear membrane is essential for normal cellular functioning. However, in cancer cells, the altered expression or localization of nuclear transporters as well as the disruption of endogenous nuclear transport inhibitors are some ways in which the Karyopherin proteins are dysregulated. The value of nuclear transporters in the diagnosis, prognosis and treatment of cancer is currently being elucidated with recent studies highlighting their potential as biomarkers and therapeutic targets.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/diagnosis , Neoplasms/drug therapy , Small Molecule Libraries/pharmacology , Acetyltransferases/genetics , Acetyltransferases/metabolism , Active Transport, Cell Nucleus/drug effects , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Biomarkers, Tumor/metabolism , Humans , Karyopherins/genetics , Karyopherins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Prognosis , Protein Transport/drug effects , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , alpha Karyopherins/genetics , alpha Karyopherins/metabolism , Exportin 1 ProteinABSTRACT
The karyopherin ß proteins are involved in nuclear-cytoplasmic trafficking and are crucial for protein and RNA subcellular localization. We previously showed that Kpnß1, a nuclear importin protein, is overexpressed in cervical cancer and is critical for cervical cancer cell survival and proliferation, whereas non-cancer cells are less dependent on its expression. This study aimed to identify the mechanisms by which inhibition of Kpnß1 results in cervical cancer cell death. We show that the inhibition of Kpnß1 results in the induction of apoptosis and a prolonged mitotic arrest, accompanied by distinct mitotic defects in cervical cancer cells but not non-cancer cells. In cervical cancer cells, Kpnß1 downregulation results in sustained degradation of the antiapoptotic protein, Mcl-1, and elevated Noxa expression, as well as mitochondrial membrane permeabilization resulting in the release of cytochrome C and activation of associated caspases. Although p53 becomes stabilized in Kpnß1 knockdown cervical cancer cells, apoptosis occurs in a p53-independent manner. These results demonstrate that blocking Kpnß1 has potential as an anticancer therapeutic approach.
Subject(s)
Apoptosis/genetics , Cell Cycle Checkpoints/genetics , Mitosis/genetics , Signal Transduction , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolism , beta Karyopherins/genetics , Cell Line, Tumor , Female , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Potential, Mitochondrial/genetics , Models, Biological , RNA Interference , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , beta Karyopherins/metabolismABSTRACT
Many proteins require transport across the nuclear envelope, the physical barrier separating the nucleus from the cytoplasm. Karyopherin ß (Kpnß1) proteins are the major nuclear receptor proteins in the cell that cargo proteins across the nuclear envelope, allowing them to enter and exit the cell nucleus. Karyopherin ß1, a major nuclear import receptor, plays an integral role in importing transcription factors, cell signaling proteins, cell cycle proteins, and so forth, into the nucleus, thus playing a crucial role in maintaining normal cell homeostasis. However, cancer cells appear to differentially regulate the expression of the Karyopherin ß proteins, presumably in order to maintain increased nuclear transport rates, thus implicating this protein family as a target for cancer therapy. The role of Kpnß1 in cancer is only now being elucidated, and recent work points to its potential usefulness as an anti-cancer target.
Subject(s)
Molecular Targeted Therapy , Neoplasms/genetics , Nuclear Envelope , beta Karyopherins/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/pathology , Neoplasms/therapy , Nuclear Envelope/genetics , Nuclear Envelope/metabolism , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , beta Karyopherins/metabolismABSTRACT
E-boxes are important regulatory elements in the eukaryotic genome. Transcription factors can bind to E-boxes through their basic helix-loop-helix or zinc finger domain to regulate gene transcription. E-box-binding transcription factors (EBTFs) are important regulators of development and essential for physiological activities of the cell. The fundamental role of EBTFs in cancer has been highlighted by studies on the canonical oncogene MYC, yet many EBTFs exhibit common features, implying the existence of shared molecular principles of how they are involved in tumorigenesis. A comprehensive analysis of TFs that share the basic function of binding to E-boxes has been lacking. Here, we review the structure of EBTFs, their common features in regulating transcription, their physiological functions, and their mutual regulation. We also discuss their converging functions in cancer biology, their potential to be targeted as a regulatory network, and recent progress in drug development targeting these factors in cancer therapy.
ABSTRACT
The nuclear exporter protein, Crm1, plays a key role in normal cell functioning, mediating the nucleo-cytoplasmic transport of cargo proteins. Elevated Crm1 expression has recently been identified in various tumours; however, the mechanisms driving its expression have not been investigated to date. In this study we identified the Crm1 promoter and factors associated with its elevated expression and with its repression under conditions of DNA damage. The -1405 to +99 Crm1 promoter region was found to be significantly more active in cancer and transformed cells compared to normal, and the -175 to +99 region identified as responsible for the differential activity. Mutation of two CCAAT boxes and a GC box within this region significantly diminished Crm1 promoter activity and ChIP analysis revealed binding of NFY and Sp1 to these sites, with increased binding in transformed and cancer cells. In addition, p53 was found to repress Crm1 promoter activity, after induction with doxorubicin, with p53 siRNA blocking the effect. Crm1 promoter constructs with mutated CCAAT boxes were significantly less responsive to p53 repression, and in vivo binding of NFY to the CCAAT boxes was diminished upon p53 binding, suggesting that p53 mediates repression of the Crm1 promoter via interfering with NFY. This was confirmed using NFY knock-down cells, in which Crm1 promoter activity was significantly less responsive to p53. In vitro EMSAs revealed that NFY and p53 bind the CCAAT boxes as a single complex under conditions of DNA damage. In summary, this study is a first to analyse Crm1 promoter regulation and reveals NFY and Sp1 as contributors to Crm1 overexpression in cancer. In addition, this study reveals that Crm1 transcription is inhibited by DNA damage and that the mechanism of inhibition involves p53 interfering with NFY function.
Subject(s)
CCAAT-Binding Factor/metabolism , DNA Damage , Karyopherins/metabolism , Neoplasms/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Repressor Proteins/metabolism , Response Elements , Sp1 Transcription Factor/metabolism , Tumor Suppressor Protein p53/metabolism , CCAAT-Binding Factor/genetics , Cell Line, Transformed , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Karyopherins/genetics , Mutation , Neoplasms/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Repressor Proteins/genetics , Sp1 Transcription Factor/genetics , Transcription, Genetic/genetics , Tumor Suppressor Protein p53/genetics , Exportin 1 ProteinABSTRACT
Karyopherin beta 1 (Kpnß1) is the principal nuclear importer of cargo proteins and plays a role in many cellular processes. Its expression is upregulated in cancer and essential for cancer cell viability, thus the identification of its binding partners might help in the discovery of anti-cancer therapeutic targets and cancer biomarkers. Herein, we applied immunoprecipitation coupled to mass spectrometry (IP-MS) to identify Kpnß1 binding partners in normal and cancer cells. IP-MS identified 100 potential Kpnß1 binding partners in non-cancer hTERT-RPE1, 179 in HeLa cervical cancer, 147 in WHCO5 oesophageal cancer and 176 in KYSE30 oesophageal cancer cells, including expected and novel interaction partners. 38 binding proteins were identified in all cell lines, with the majority involved in RNA metabolism. 18 binding proteins were unique to the cancer cells, with many involved in protein translation. Western blot analysis validated the interaction of known and novel binding partners with Kpnß1 and revealed enriched interactions between Kpnß1 and select proteins in cancer cells, including proteins involved in cancer development, such as Kpnα2, Ran, CRM1, CCAR1 and FUBP1. Together, this study shows that Kpnß1 interacts with numerous proteins, and its enhanced interaction with certain proteins in cancer cells likely contributes to the cancer state.
Subject(s)
Esophageal Neoplasms , Uterine Cervical Neoplasms , Female , Humans , beta Karyopherins , Mass Spectrometry , Immunoprecipitation , Cell Cycle Proteins , Apoptosis Regulatory Proteins , DNA-Binding Proteins , RNA-Binding ProteinsABSTRACT
AP-1, a transcription factor comprised primarily of Jun and Fos family proteins, regulates genes involved in proliferation, differentiation and oncogenesis. Previous studies demonstrated that elevated expression of Jun and Fos family member proteins is associated with numerous human cancers and in cancer-relevant biological processes. In this study we used a dominant-negative mutant of c-Jun, Tam67, which interferes with the functional activity of all AP-1 complexes, to investigate the requirement of AP-1 in the proliferation and cell cycle progression of cervical cancer cells. Transient and stable expression of Tam67 in CaSki cervical cancer cells resulted in decreased AP-1 activity that correlated with a significant inhibition of cell proliferation and anchorage-independent colony formation. Inhibiting AP-1 activity resulted in a two-fold increase in cells located in the G(2)/M phase of the cell cycle and an accompanying increase in the expression of the cell cycle regulatory protein, p21. The increase in p21 was associated with a decrease in HPV E6 expression and an increase in p53. Importantly, blocking the induction of p21 in CaSki-Tam67-expressing cells accelerated their proliferation rate to that of CaSki, implicating p21 as a key player in the growth arrest induced by Tam67. Our results suggest a role for AP-1 in the proliferation, G(2)/M progression and inhibition of p21 expression in cervical cancer.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Transcription Factor AP-1/metabolism , Uterine Cervical Neoplasms/metabolism , Blotting, Western , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Female , HeLa Cells , Humans , Peptide Fragments/genetics , Peptide Fragments/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor AP-1/genetics , Uterine Cervical Neoplasms/geneticsABSTRACT
Lamina-associated polypeptide 2 alpha (LAP2α) plays a role in maintaining nuclear structure, in nuclear assembly/disassembly, and in transcriptional regulation. Elevated LAP2α mRNA expression has been previously reported to associate with certain cancer types. The aim of this study was to investigate LAP2α expression in cervical cancer and transformed cells and to identify factors that associate with its differential expression. LAP2α expression was found to be elevated in cervical cancer tissue by microarray, qRT-PCR, and immunofluorescence analyses. LAP2α also showed elevated expression in cervical cancer cell lines and in transformed fibroblasts compared with normal cells. To determine factors associated with elevated LAP2α in cervical cancer, the effect of inhibiting HPV E7 and E6 oncoproteins was investigated. E7 inhibition resulted in a decrease in phosphorylated Rb and an associated decrease in LAP2α, suggesting a role for E2F in regulating LAP2α expression. This finding was confirmed by inhibiting DP1, a co-activator of E2F, which resulted in decreased LAP2α levels. Inhibition of E6 resulted in elevated p53 and an associated decrease in LAP2α, suggesting that p53 associates with the negative regulation of LAP2α expression. This hypothesis was tested by inhibiting p53 in normal cells, and a resultant increase in LAP2α expression was observed. In conclusion, this study provides evidence for elevated LAP2α expression in cervical cancer and suggests that E2F and p53 activities associate with the positive and negative regulation of LAP2α expression, respectively.
Subject(s)
Carcinoma/genetics , DNA-Binding Proteins/genetics , E2F Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Membrane Proteins/genetics , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/genetics , Carcinoma/metabolism , Carcinoma/pathology , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins/metabolism , Female , Gene Expression , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Membrane Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , RNA Interference , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathologyABSTRACT
There is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study, the circadian clock was investigated in cervical and esophageal cancers, to determine whether it is disrupted in these cancer types. Oncomine datamining revealed downregulation of multiple members of the circadian clock gene family in cancer patient tissue compared with matched normal epithelium. Real-time RT-PCR analysis confirmed significant downregulation of CLOCK, PER1, PER2, PER3, CRY1, CRY2, REV-ERBα, and RORα in esophageal tumor tissue. In cell line models, expression of several circadian clock genes was significantly decreased in transformed and cancer cells compared with noncancer controls, and protein levels were dysregulated. These effects were mediated, at least in part, by methylation, where CLOCK, CRY1, and RORα gene promoter regions were found to be methylated in cancer cells. Overexpression of CLOCK and PER2 in cancer cell lines inhibited cell proliferation and activation of RORα and REV-ERBα using agonists resulted in cancer cell death, while having a lesser effect on normal epithelial cells. Despite dysregulated circadian clock gene expression, cervical and esophageal cancer cells maintain functional circadian oscillations after Dexamethasone synchronization, as revealed using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are intact. IMPLICATIONS: This study is a first to describe dysregulated, yet oscillating, circadian clock gene expression in cervical and esophageal cancer cells, and knowledge of circadian clock functioning in these cancer types has the potential to inform chronotherapy approaches, where the timing of administration of chemotherapy is optimized on the basis of the circadian clock.
Subject(s)
Circadian Clocks/genetics , Esophageal Neoplasms/genetics , Genes, Tumor Suppressor/physiology , Uterine Cervical Neoplasms/genetics , Cell Proliferation , Down-Regulation , Esophageal Neoplasms/pathology , Female , Humans , Uterine Cervical Neoplasms/pathologyABSTRACT
The Karyopherin proteins are involved in nucleo-cytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest they are important in nuclear envelope component assembly, mitosis and replication. Since these are all critical cellular functions, alterations in the expression of the Karyopherins may have an impact on the biology of cancer cells. In this study, we examined the expression of the Karyopherins, Crm1, Karyopherin beta1 (Kpnbeta1) and Karyopherin alpha2 (Kpnalpha2), in cervical tissue and cell lines. The functional significance of these proteins to cancer cells was investigated using individual siRNAs to inhibit their expression. Microarrays, quantitative RT-PCR and immunofluorescence revealed significantly higher expression of Crm1, Kpnbeta1 and Kpnalpha2 in cervical cancer compared to normal tissue. Expression levels were similarly elevated in cervical cancer cell lines compared to normal cells, and in transformed epithelial and fibroblast cells. Inhibition of Crm1 and Kpnbeta1 in cancer cells significantly reduced cell proliferation, while Kpnalpha2 inhibition had no effect. Noncancer cells were unaffected by the inhibition of Crm1 and Kpnbeta1. The reduction in proliferation of cancer cells was associated with an increase in a subG1 population by cell cycle analysis and Caspase-3/7 assays revealed increased apoptosis. Crm1 and Kpnbeta1 siRNA-induced apoptosis was accompanied by an increase in the levels of growth inhibitory proteins, p53, p27, p21 and p18. Our results demonstrate that Crm1, Kpnbeta1 and Kpnalpha2 are overexpressed in cervical cancer and that inhibiting the expression of Crm1 and Kpnbeta1, not Kpnalpha2, induces cancer cell death, making Crm1 and Kpnbeta1 promising candidates as both biomarkers and potential anticancer therapeutic targets.
Subject(s)
Gene Expression Regulation, Neoplastic , Karyopherins/biosynthesis , Receptors, Cytoplasmic and Nuclear/biosynthesis , Uterine Cervical Neoplasms/metabolism , beta Karyopherins/biosynthesis , Adult , Aged , Cell Nucleus/metabolism , Cell Proliferation , Cell Survival , Female , Humans , Middle Aged , Models, Biological , RNA, Small Interfering/metabolism , Transcription, Genetic , Exportin 1 ProteinABSTRACT
Photolon is a photosensitiser with demonstrated potential as an anti-tumour agent. In this study, an in vitro investigation was performed to determine the mechanism of Photolon-induced cell death. Cell killing was observed in a light-dependent manner and light-activated Photolon resulted in a significant production of reactive oxygen species (ROS), which could be blocked by type I ROS scavengers. Inhibition of ROS production using Trolox prevented Photolon-induced cell death. Light-activated Photolon caused no increase in caspase-3/7 activity, but a rapid increase in lactate dehydrogenase (LDH) release suggesting a loss of membrane integrity and subsequent cell death by necrosis. We conclude that the mechanism of Photolon-induced cell death involves the induction of ROS via a type I mechanism, which is ultimately responsible for cell killing by necrosis.
Subject(s)
Light , Neoplasms/drug therapy , Neoplasms/pathology , Porphyrins/therapeutic use , Protoporphyrins/therapeutic use , Reactive Oxygen Species/metabolism , Cell Death/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Chlorophyllides , Drug Evaluation, Preclinical , Humans , Necrosis/chemically induced , Photochemotherapy/methods , Porphyrins/chemistry , Porphyrins/pharmacology , Povidone , Protoporphyrins/pharmacology , Radiation-Sensitizing Agents/pharmacology , Radiation-Sensitizing Agents/therapeutic use , Reactive Oxygen Species/pharmacology , Tumor Cells, CulturedABSTRACT
Karyopherin beta 1 (Kpnß1) is a nuclear transport receptor that imports cargoes into the nucleus. Recently, elevated Kpnß1 expression was found in certain cancers and Kpnß1 silencing with siRNA was shown to induce cancer cell death. This study aimed to identify novel small molecule inhibitors of Kpnß1, and determine their anticancer activity. An in silico screen identified molecules that potentially bind Kpnß1 and Inhibitor of Nuclear Import-43, INI-43 (3-(1H-benzimidazol-2-yl)-1-(3-dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine) was investigated further as it interfered with the nuclear localization of Kpnß1 and known Kpnß1 cargoes NFAT, NFκB, AP-1, and NFY and inhibited the proliferation of cancer cells of different tissue origins. Minimum effect on the proliferation of noncancer cells was observed at the concentration of INI-43 that showed a significant cytotoxic effect on various cervical and esophageal cancer cell lines. A rescue experiment confirmed that INI-43 exerted its cell killing effects, in part, by targeting Kpnß1. INI-43 treatment elicited a G2-M cell-cycle arrest in cancer cells and induced the intrinsic apoptotic pathway. Intraperitoneal administration of INI-43 significantly inhibited the growth of subcutaneously xenografted esophageal and cervical tumor cells. We propose that Kpnß1 inhibitors could have therapeutic potential for the treatment of cancer. Mol Cancer Ther; 15(4); 560-73. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , beta Karyopherins/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/drug effects , Computer Simulation , Computers, Molecular , Disease Models, Animal , Drug Discovery , Female , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , Gene Expression , Humans , Mice , Models, Molecular , Molecular Targeted Therapy , Protein Binding , Protein Transport , Small Molecule Libraries , Structure-Activity Relationship , Transcription Factors/metabolism , Xenograft Model Antitumor Assays , beta Karyopherins/chemistry , beta Karyopherins/geneticsABSTRACT
The nuclear export receptor, Crm1 (exportin 1), is involved in the nuclear translocation of proteins and certain RNAs from the nucleus to the cytoplasm and is thus crucial for the correct localisation of cellular components. Crm1 has recently been reported to be highly expressed in certain types of cancers, yet its expression in oesophageal cancer has not been investigated to date. We investigated the expression of Crm1 in normal and tumour tissues derived from 56 patients with human oesophageal squamous cell carcinoma and its functional significance in oesophageal cancer cell line models. Immunohistochemistry revealed that Crm1 expression was significantly elevated in oesophageal tumour tissues compared to normal tissues and its localisation shifted from predominantly nuclear to nuclear and cytoplasmic. Realtime RTPCR revealed that Crm1 expression was elevated at the mRNA level. To determine the functional significance of elevated Crm1 expression in oesophageal cancer, its expression was inhibited using siRNA, and a significant decrease in cell proliferation was observed associated with G1 cell cycle arrest and the induction of apoptosis. Similarly, leptomycin B (LMB) treatment resulted in the effective killing of oesophageal cancer cells at nanomolar concentrations. Normal oesophageal epithelial cells, however, were much less sensitive to Crm1 inhibition with siRNA and LMB. Together, this study reveals that Crm1 expression is increased in oesophageal cancer and is required for the proliferation and survival of oesophageal cancer cells.