ABSTRACT
Estrogen receptor-positive breast cancer patients have a good prognosis, but 30% of these patients will experience recurrence due to the development of resistance through various signaling pathways. This study aimed to evaluate the mode of anticancer effects of 1'-acetoxychavicol acetate, which is isolated from the rhizomes of Alpinia galanga in estrogen receptor positive (MCF7) human epidermal growth factor receptor 2-overexpressed (MCF7/HER2), and endocrine-resistant breast cancer cells (MCF7/LCC2 and MCF7/LCC9). 1'-Acetoxychavicol acetate showed antiproliferation in a concentration- and time-dependent fashion and had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines. This was associated with down-regulation of human epidermal growth factor receptor 2, pERK1/2, pAKT, estrogen receptor coactivator, cyclin D1, and MYC proto-oncogene while in vivo and significant reduction in the tumor mass of 1'-acetoxychavicol acetate-treated zebrafish-engrafted breast cancer groups. The anti-invasive effects of 1'-acetoxychavicol acetate were confirmed in vitro by the matrigel invasion assay and with down-regulation of Câ-âX-C chemokine receptor type 4, urokinase plasminogen activator, vascular endothelial growth factor, and basic fibroblast growth factor 2 genes. The down-regulation of urokinase plasminogen activator and fibroblast growth factor 2 proteins was also validated by molecular docking analysis. Moreover, 1'-acetoxychavicol acetate-treated cells exhibited lower expression levels of the anti-apoptotic Bcl-2 and Mcl-1 proteins in addition to enhanced stress-activated kinases/c-Jun N-terminal kinase 1/2 and poly-ADP ribose polymerase cleavage, indicating apoptotic cell induction by 1'-acetoxychavicol acetate. Moreover, 1'-acetoxychavicol acetate had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines regarding its inhibition on human epidermal growth factor receptor 2, pAKT, pERK1/2, PSer118, and PSer167-ERα proteins. Our findings suggest 1'-acetoxychavicol acetate mediates its anti-cancer effects via human epidermal growth factor receptor 2 signaling pathway.
Subject(s)
Alpinia , Apoptosis , Benzyl Alcohols/pharmacology , Breast Neoplasms , Alpinia/chemistry , Animals , Breast Neoplasms/drug therapy , Cell Proliferation , Female , Humans , MCF-7 Cells , Molecular Docking Simulation , Signal Transduction , ZebrafishABSTRACT
Breast cancer brain metastases are an increasing clinical problem. Studies have shown that brain metastases from breast cancer have a distinct genomic landscape to that of the primary tumour, including the presence of mutations that are absent in the primary breast tumour. In this Review, we aim to review and evaluate genomic sequencing data for breast cancer brain metastases by searching PubMed, Embase, and Scopus for relevant articles published in English between database inception and May 30, 2020. Extracted information includes data for mutations, receptor status (eg, immunohistochemistry and Prediction Analysis of Microarray 50 [PAM50]), and copy number alterations from published manuscripts and supplementary materials. Of the 431 articles returned by the database search, 13 (3%) breast cancer brain metastases sequencing studies, comprising 164 patients with sequenced brain metastases, met all our inclusion criteria. We identified 268 mutated genes that were present in two or more breast cancer brain metastases samples. Of these 268 genes, 22 (8%) were mutated in five or more patients and pathway enrichment analysis showed their involvement in breast cancer-related signalling pathways, regulation of gene transcription, cell cycle, and DNA repair. Actionability analysis using the Drug Gene Interaction Database revealed that 15 (68%) of these 22 genes are actionable drug targets. In addition, immunohistochemistry and PAM50 data showed receptor discordancy between primary breast cancers and their paired brain metastases. This systematic review provides a detailed overview of the most commonly mutated genes identified in samples of breast cancer brain metastases and their clinical relevance. These data highlight the differences between primary breast cancers and brain metastases and the importance of acquiring and analysing brain metastasis samples for further study.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Breast Neoplasms/genetics , DNA Copy Number Variations , Gene Dosage , Mutation , Signal Transduction/genetics , Brain Neoplasms/drug therapy , Brain Neoplasms/secondary , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Female , Gene Regulatory Networks , Genetic Predisposition to Disease , Humans , Molecular Targeted Therapy , Phenotype , Precision Medicine , Signal Transduction/drug effectsABSTRACT
BACKGROUND: Among patients with breast carcinoma who have metastatic disease, 15%-30% will eventually develop brain metastases. We examined the genomic landscape of a large cohort of patients with breast carcinoma brain metastases (BCBMs) and compared it with a cohort of patients with primary breast carcinomas (BCs). MATERIAL AND METHODS: We retrospectively analyzed 733 BCBMs tested with comprehensive genomic profiling (CGP) and compared them with 10,772 primary breast carcinomas (not-paired) specimens. For a subset of 16 triple-negative breast carcinoma (TNBC)-brain metastasis samples, programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) was performed concurrently. RESULTS: A total of 733 consecutive BCBMs were analyzed. Compared with primary BCs, BCBMs were enriched for genomic alterations in TP53 (72.0%, 528/733), ERBB2 (25.6%, 188/733), RAD21 (14.1%, 103/733), NF1 (9.0%, 66/733), BRCA1 (7.8%, 57/733), and ESR1 (6.3%,46/733) (p < .05 for all comparisons). Immune checkpoint inhibitor biomarkers such as high tumor mutational burden (TMB-high; 16.2%, 119/733); high microsatellite instability (1.9%, 14/733); CD274 amplification (3.6%, 27/733); and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like mutational signature (5.9%, 43/733) were significantly higher in the BCBM cohort compared with the primary BC cohort (p < .05 for all comparisons). When using both CGP and PD-L1 IHC, 37.5% (6/16) of patients with TNBC brain metastasis were eligible for atezolizumab based on PD-L1 IHC, and 18.8% (3/16) were eligible for pembrolizumab based on TMB-high status. CONCLUSION: We found a high prevalence of clinically relevant genomic alterations in patients with BCBM, suggesting that tissue acquisition (surgery) and/or cerebrospinal fluid for CGP in addition to CGP of the primary tumor may be clinically warranted. IMPLICATIONS FOR PRACTICE: This study found a high prevalence of clinically relevant genomic alterations in patients with breast carcinoma brain metastasis (BCBM), suggesting that tissue acquisition (surgery) and/or cerebrospinal fluid for comprehensive genomic profiling (CGP) in addition to CGP of the primary tumor may be clinically warranted. In addition, this study identified higher positive rates for FDA-approved immunotherapy biomarkers detected by CGP in patients with BCBM, opening a possibility of new on-label treatments. Last, this study noted limited correlation between tumor mutational burden and PD-L1 immunohistochemistry (IHC), which shows the importance of testing patients with triple-negative BCBM for immune checkpoint inhibitor eligibility with both PD-L1 IHC and CGP.
Subject(s)
Brain Neoplasms , Triple Negative Breast Neoplasms , Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Genomics , Humans , Retrospective StudiesABSTRACT
PURPOSE: Brain metastases (BM) are an increasing clinical problem. This study aimed to assess paired primary breast cancers (BC) and BM for aberrations within TP53, PIK3CA, ESR1, ERBB2 and AKT utilising the MassARRAY® UltraSEEK® technology (Agena Bioscience, San Diego, USA). METHODS: DNA isolated from 32 paired primary BCs and BMs was screened using the custom UltraSEEK® Breast Cancer Panel. Data acquisition and analysis was performed by the Agena Bioscience Typer software v4.0.26.74. RESULTS: Mutations were identified in 91% primary BCs and 88% BM cases. TP53, AKT1, ESR1, PIK3CA and ERBB2 genes were mutated in 68.8%, 37.5%, 31.3%, 28.1% and 3.1% respectively of primary BCs and in 59.4%, 37.5%, 28.1%, 28.1% and 3.1% respectively of BMs. Differences in the mutations within the 5 genes between BC and paired BM were identified in 62.5% of paired cases. In primary BCs, ER-positive/HER2-negative cases harboured the most mutations (70%), followed by ER-positive/HER2-positive (15%) and triple-negatives (13.4%), whereas in BMs, the highest number of mutations was observed in triple-negative (52.5%), followed by ER-positive/HER2-negative (35.6%) and ER-negative/HER2-positive (12%). There was a significant association between the number of mutations in the primary BC and breast-to-brain metastasis-free survival (p = 0.0001) but not with overall survival (p = 0.056). CONCLUSION: These data demonstrate the discordancy between primary BC and BM, as well as the presence of clinically important, actionable mutations in BCBM. The UltraSEEK® Breast Cancer Panel provides a tool for BCBM that can be utilised to direct more tailored treatment decisions and for clinical studies investigating targeted agents.
Subject(s)
Brain Neoplasms , Breast Neoplasms , Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Breast , Breast Neoplasms/genetics , Female , Genomics , Humans , Mutation , Receptor, ErbB-2/geneticsABSTRACT
BACKGROUND: Activating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment. MATERIALS AND METHODS: We knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR. RESULTS: By silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells. CONCLUSION: These data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.
Subject(s)
Activating Transcription Factor 2/metabolism , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/genetics , Activating Transcription Factor 2/genetics , Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , MCF-7 Cells , Oligonucleotide Array Sequence Analysis , Receptors, Estrogen/analysis , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Tamoxifen/pharmacology , Tamoxifen/therapeutic useABSTRACT
The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( Vmax = 3299 pmol/million cells/min, Km = 35.9 µM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.
Subject(s)
Antipsychotic Agents/pharmacology , Clozapine/pharmacology , Schizophrenia/drug therapy , Solute Carrier Proteins/metabolism , Antipsychotic Agents/therapeutic use , Brain/cytology , Brain/metabolism , Cell Line, Tumor , Clozapine/therapeutic use , Endothelial Cells/metabolism , HEK293 Cells , Hepatocytes/metabolism , Humans , Primary Cell Culture , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solute Carrier Proteins/isolation & purificationABSTRACT
Although the prognosis of chronic myeloid leukemia (CML) patients treated with imatinib is good, many fail to develop an optimal response or lose one. This heterogeneity could be attributed to the presence of human organic cation transporter-1 (hOCT1) single nucleotide polymorphisms (SNPs). In the present study, we analyzed the effect of 23 hOCT1 SNPs on imatinib treatment outcome in newly diagnosed CML patients using MassARRAY sequencing and pyrosequencing. The only SNP associated with outcome was M420del (rs35191146), with patients with the M420del demonstrating an increased probability of imatinib treatment failure. In CML cell lines transfected with M420del and/or M408V, M420del significantly decreased imatinib uptake, but this effect was countered if the M408V (rs628031) SNP was also present. A similar effect was seen for the uptake of the hOCT1 substrates TEA(+) and ASP(+). Finally, apparent hOCT1 mRNA levels were studied using both our earlier primers covering the M420del and another set that did not. Different mRNA expression was observed, explaining the disparity in published data on the prognostic importance of hOCT1 mRNA and highlighting the importance of avoiding common SNP sites in primer design. These data demonstrate that the common M420del SNP can modulate the outcome of imatinib treatment.
Subject(s)
Antineoplastic Agents/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Organic Cation Transporter 1/genetics , Piperazines/therapeutic use , Polymorphism, Single Nucleotide , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Adult , Aged , Aged, 80 and over , Alleles , Antineoplastic Agents/pharmacokinetics , Benzamides , Cell Line, Tumor , Female , Gene Expression , Gene Expression Regulation, Leukemic , Genotype , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Male , Middle Aged , Models, Molecular , Organic Cation Transporter 1/chemistry , Organic Cation Transporter 1/metabolism , Piperazines/pharmacokinetics , Protein Conformation , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Treatment Outcome , Young AdultABSTRACT
Activating transcription factor-2 (ATF-2) has been implicated as a tumour suppressor in breast cancer (BC). c-JUN N-terminal kinase (JNK) and p38 MAPK phosphorylate ATF-2 within the activation domain (AD), which is required for its transcriptional activity. To date, the role of ATF-2 in determining response to endocrine therapy has not been explored. Effects of ATF-2 loss in the oestrogen receptor (ER)-positive luminal BC cell line MCF7 were explored, as well as its role in response to tamoxifen treatment. Genome-wide chromatin binding patterns of ATF-2 when phosphorylated within the AD in MCF-7 cells were determined using ChIP-seq. The expression of ATF-2 and phosphorylated ATF-2 (pATF-2-Thr71) was determined in a series of 1,650 BC patients and correlated with clinico-pathological features and clinical outcome. Loss of ATF-2 diminished the growth-inhibitory effects of tamoxifen, while tamoxifen treatment induced ATF-2 phosphorylation within the AD, to regulate the expression of a set of 227 genes for proximal phospho-ATF-2 binding, involved in cell development, assembly and survival. Low expression of both ATF-2 and pATF-2-Thr71 was significantly associated with aggressive pathological features. Furthermore, pATF-2 was associated with both p-p38 and pJNK1/2 (< 0.0001). While expression of ATF-2 is not associated with outcome, pATF-2 is associated with longer disease-free (p = 0.002) and BC-specific survival in patients exposed to tamoxifen (p = 0.01). Furthermore, multivariate analysis confirmed pATF-2-Thr71 as an independent prognostic factor. ATF-2 is important for modulating the effect of tamoxifen and phosphorylation of ATF-2 within the AD at Thr71 predicts for improved outcome for ER-positive BC receiving tamoxifen.
Subject(s)
Activating Transcription Factor 2/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Tamoxifen/pharmacology , Activating Transcription Factor 2/genetics , Breast Neoplasms/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease-Free Survival , Female , Humans , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , MCF-7 Cells , Phosphorylation , Prognosis , Transcription Factors/genetics , Transcription Factors/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
ALOX5 is implicated in chronic myeloid leukemia development in mouse leukemic stem cells, but its importance in human chronic myeloid leukemia is unknown. Functional ALOX5 was assessed using an LTB4 ELISA and ALOX5, and LTB4R1 mRNA expression was determined via a TaqMan gene expression assay. LTB4R1 and 5-LOX protein levels were assessed by cell surface flow cytometry analysis. At diagnosis ALOX5 was below normal in both blood and CD34(+) stem cells in all patients. On treatment initiation, ALOX5 levels increased in all patients except those who were destined to progress subsequently to blast crisis. LTB4 levels were increased despite low ALOX5 expression, suggesting that the arachidonic acid pathway is functioning normally up to the point of LTB4 production. However, the LTB4 receptor (BLT1) protein in newly diagnosed patients was significantly lower than after a period of treatment (P<0.0001). The low level of LTB4R1 at diagnosis explains the downregulation of ALOX5. In the absence of LTB4R1, the arachidonic acid pathway intermediates (5-HEPTE and LTA4) negatively regulate ALOX5. ALOX5 regulation is aberrant in chronic myeloid leukemia patients and may not be important for the development of the disease. Our data suggest caution when extrapolating mouse model data into human chronic myeloid leukemia.
Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Receptors, Leukotriene B4/blood , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Arachidonate 5-Lipoxygenase/metabolism , Blast Crisis , Disease Progression , Down-Regulation , Female , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Leukemia, Myeloid, Chronic-Phase/diagnosis , Leukemia, Myeloid, Chronic-Phase/genetics , Male , Middle Aged , RNA, Messenger/genetics , Receptors, Leukotriene B4/genetics , Treatment Outcome , Young AdultABSTRACT
PURPOSE: Genomic alterations have been identified in patients with breast cancer brain metastases (BCBMs), but large structural rearrangements have not been extensively studied. MATERIALS AND METHODS: We analyzed the genomic profiles of 822 BCBMs and compared them with 11,988 local, breast-biopsied breast cancers (BCs) and 15,516 non-CNS metastases (Non-CNS M) derived from formalin-fixed paraffin-embedded material using targeted capture sequencing. RESULTS: Nine genes with structural rearrangements were more prevalent within BCBMs as compared with local BCs and Non-CNS M (adjusted-P < .05) and displayed a prevalence of >0.5%. The most common rearrangements within BCBMs involves cyclin-dependent kinase 12 (CDK12; 3.53%) as compared with the local BC (0.86%; adjusted-P = 7.1 × 10-8) and Non-CNS M specimens (0.68%; adjusted-P = 3.7 × 10-10). CDK12 rearrangements had a significantly higher frequency within human epidermal growth factor receptor 2 (HER2)-positive BCBMs (14.59%) compared with HER2-positive BCs (7.80%; P = 4.6 × 10-3) and HER2-positive Non-CNS M (7.87%; P = 4.8 × 10-3). CONCLUSION: The most common structural rearrangements involve CDK12 with the higher prevalence in HER2-positive BCBMs. These data support more detailed investigation of the role and importance of CDK12 rearrangements in BCBMs.
Subject(s)
Brain Neoplasms , Breast Neoplasms , Cyclin-Dependent Kinases , Gene Rearrangement , Receptor, ErbB-2 , Humans , Female , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Brain Neoplasms/secondary , Brain Neoplasms/genetics , Receptor, ErbB-2/genetics , Cyclin-Dependent Kinases/genetics , Middle Aged , Adult , AgedABSTRACT
Prospective identification of patients whose chronic myeloid leukemia (CML) will progress to blast crisis is currently not possible. PP2A is a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. Cancerous inhibitor of PP2A (CIP2A) is a recently described inhibitor of PP2A in breast and gastric cancer. The aim of this study was to investigate whether CIP2A played a role in CML and whether PP2A or its inhibitor proteins CIP2A or SET could predict clinical outcome. At the time of diagnosis of CML, patients who will later progress to blast crisis have significantly higher levels of CIP2A protein (P < .0001) than patients who do not progress, suggesting that PP2A is functionally inactive. We show that the potential mechanism for disease progression is via altered phosphorylation of the oncogene c-Myc. Knockdown of CIP2A results in increased PP2A activity, decreased c-Myc levels, and a decrease in BCR-ABL1 tyrosine kinase activity. We demonstrate that CIP2A levels at diagnosis can consistently predict patients who will progress to blast crisis. The data show that CIP2A is biologically and clinically important in CML and may be a novel therapeutic target.
Subject(s)
Autoantigens/physiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Membrane Proteins/physiology , Adult , Autoantigens/genetics , Autoantigens/metabolism , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cells, Cultured , Cohort Studies , Disease Progression , Female , Gene Expression Regulation, Leukemic , Humans , Intracellular Signaling Peptides and Proteins , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Middle Aged , Prognosis , Survival AnalysisABSTRACT
Breast cancer is the commonest malignancy of women and with its incidence on the rise, the need to identify new targets for treatment is imperative. There is a growing interest in the role of lipid metabolism in cancer. Carnitine palmitoyl-transferase-1 (CPT-1); the rate limiting step in fatty acid oxidation, has been shown to be overexpressed in a range of tumours. There are three isoforms of CPT-1; A, B and C. It is CPT-1A that has been shown to be the predominant isoform which is overexpressed in breast cancer. We performed a bioinformatic analysis using readily available online platforms to establish the prognostic and predictive effects related to CPT-1A expression. These include the KM plotter, the Human Protein Atlas, the cBioPortal, the G2O, the MethSurvand the ROC plotter. A Network analysis was performed using the Oncomine platform and signalling pathways constituting the cancer hallmarks, including immune regulation as utilised by NanoString. The epigenetic pathways were obtained from the EpiFactor website. Spearman correlations (r) to determine the relationship between CPT-1A and the immune response were obtained using the TISIDB portal. Overexpression of CPT-1A largely confers a worse prognosis and CPT-1A progressively recruits a range of pathways as breast cancer progresses. CPT-1A's interactions with cancer pathways is far wider than previously realised and includes associations with epigenetic regulation and immune evasion pathways, as well as wild-type moderate to high penetrant genes involved in hereditary breast cancer. Although CPT-1A genomic alterations are detected in 9% of breast carcinomas, both the alteration and the metagene associated with it, confers a poor prognosis. CPT-1A expression can be utilised as a biomarker of disease progression and as a potential therapeutic target.
Subject(s)
Breast Neoplasms , Carnitine O-Palmitoyltransferase/analysis , Biomarkers , Breast Neoplasms/pathology , Carnitine , Carnitine O-Palmitoyltransferase/metabolism , Computational Biology , Epigenesis, Genetic , Fatty Acids/metabolism , Female , Humans , Protein Isoforms/metabolismABSTRACT
We have previously shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human organic cation transporter 1 (hOCT1; SLC22A1), and that low hOCT1 expression is an important determinant of clinical outcome to imatinib treatment. We hypothesized that dasatinib might be transported differently than imatinib, possibly accounting for its favorable effects in imatinib-resistant patients. (14)C-dasatinib uptake was greater in KCL22-transfected cells with pcDNA3-hOCT1 plasmid (high hOCT1-expressing cells) than in control cells (P = .02). However, hOCT inhibitors did not decrease dasatinib uptake into either control or primary cells, in contrast to their block on imatinib uptake. Dasa-tinib decreased the level of phosphorylated CrkL to 49.9% in control and 40.3% in high hOCT1-expressing cells. Dasa-tinib efflux was investigated in confluent ABCB1-transfected MDCKII cell monolayers. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating that they were transported by ABCB1, which was confirmed using the ABCB1 inhibitor PSC833 (P = .001 and P < .001, respectively). Compared with imatinib, dasatinib achieved superior intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1. Efflux of dasatinib and imatinib appear similar via ABCB1. Dasatinib may therefore offer an advantage over imatinib in patients with low hOCT1 expression.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Organic Cation Transporter 1/physiology , Piperazines/pharmacology , Pyrimidines/pharmacology , Pyrimidines/pharmacokinetics , Thiazoles/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Benzamides , Cell Line, Tumor , Cell Separation , Dasatinib , Dogs , Flow Cytometry , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Models, Biological , Organic Cation Transporter 1/geneticsSubject(s)
Autoantigens/metabolism , Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Membrane Proteins/metabolism , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Cell Line, Tumor , Enzyme Activation/drug effects , Humans , Intracellular Signaling Peptides and Proteins , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , RNA, Small Interfering/genetics , Thiazoles/pharmacology , Thiazoles/therapeutic useABSTRACT
BACKGROUND: Chronic myeloid leukemia is characterized by a reciprocal translocation between chromosomes 9 and 22, creating the fusion gene BCR-ABL. The clinical significance of the type of BCR-ABL transcript in newly diagnosed patients in chronic phase treated with imatinib 400 mg from initial diagnosis remains unknown. DESIGN AND METHODS: We analyzed the clinical outcome of 78 newly diagnosed chronic phase patients, aged 16 or over, treated with imatinib 400 mg. Of these, 71 expressed either e13a2 or e14a2 transcripts. BCR-ABL transcripts were assayed by quantitative real-time polymerase chain reaction. RESULTS: After 12 months of treatment, 54% of the e14a2 patients had achieved a complete cytogenetic response, compared to 25% of the e13a2 patients (p=0.01). Kaplan-Meier analysis of the time to achieve complete cytogenetic response revealed that e14a2 patients had more rapid response rates, compared to e13a2 patients (p=0.006). e14a2 patients had a higher event-free survival rate in the first 12 months of treatment, although overall survival did not differ significantly between the patients with the two types of transcript. Human organic cation transporter protein 1 mRNA levels did not differ between the patients with the two types of transcript. The pre-treatment pCrKL/CrKL ratio (a surrogate marker of BCR-ABL tyrosine kinase activity) was higher in patients with e13a2 transcripts than in those with e14a2 (p=0.017). CONCLUSIONS: Patients expressing the e14a2 transcript type have a higher rate and more rapid complete cytogenetic responses than e13a2-expressing patients, which may be due to higher BCR-ABL tyrosine kinase activity. Knowledge of the transcript type may yield additional prognostic information, although this requires testing on larger datasets.
Subject(s)
Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Adult , Aged , Aged, 80 and over , Benzamides , Female , Follow-Up Studies , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Male , Middle Aged , Survival Rate/trends , Transcription, Genetic/drug effects , Transcription, Genetic/geneticsABSTRACT
Breast cancer brain metastasis (BCBM) is an area of unmet clinical need. MicroRNAs (miRNAs) have been linked to the metastatic process in breast cancer (BC). In this study, we aim to determine differentially-expressed miRNAs utilising primary BCs that did not relapse (BCNR, n = 12), primaries that relapsed (BCR) and their paired (n = 40 pairs) brain metastases (BM) using the NanoString™ nCounter™ miRNA Expression Assays. Significance analysis of microarrays identified 58 and 11 differentially-expressed miRNAs between BCNR vs BCR and BCR vs BM respectively and pathway analysis revealed enrichment for genes involved in invasion and metastasis. Four miRNAs, miR-132-3p, miR-199a-5p, miR-150-5p and miR-155-5p, were differentially-expressed within both cohorts (BCNR-BCR, BCR-BM) and receiver-operating characteristic curve analysis (p = 0.00137) and Kaplan-Meier survival method (p = 0.0029, brain metastasis-free survival; p = 0.0007, overall survival) demonstrated their potential use as prognostic markers. Ingenuity pathway enrichment linked them to the MET oncogene, and the cMET protein was overexpressed in the BCR (p < 0.0001) and BM (p = 0.0008) cases, compared to the BCNRs. The 4-miRNAs panel identified in this study could be potentially used to distinguish BC patients with an increased risk of developing BCBM and provide potential novel therapeutic targets, whereas cMET-targeting warrants further investigation in the treatment of BCBM.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/mortality , Breast Neoplasms/mortality , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Biomarkers, Tumor/genetics , Brain Neoplasms/secondary , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Gene Expression Profiling , Humans , Kaplan-Meier Estimate , Neoplasm Invasiveness , Neoplasm Recurrence, Local , Normal Distribution , Oligonucleotide Array Sequence Analysis , Principal Component Analysis , Prognosis , ROC Curve , Risk , Treatment OutcomeSubject(s)
Adaptor Proteins, Signal Transducing/blood , Biomarkers, Tumor/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Nuclear Proteins/blood , Adult , Aged , Antineoplastic Agents/therapeutic use , Benzamides , Female , Humans , Imatinib Mesylate , Male , Middle Aged , Neoplasm Proteins/blood , Piperazines/therapeutic use , Prognosis , Prospective Studies , Pyrimidines/therapeutic use , Treatment Outcome , Young AdultABSTRACT
BACKGROUND: Bosutinib is a recently approved ABL inhibitor. In spite of the well-documented effectiveness of BCR-ABL inhibitors in treating chronic myeloid leukemia, development of resistance is a continuous clinical challenge. Transporters that facilitate drug uptake and efflux have been proposed as one potential source of resistance to tyrosine kinase inhibitor treatment. Our aim was to determine which carriers are responsible for bosutinib transport. METHODS: K562S cells overexpressing the drug transporters ABCB1, ABCG2, and SLC22A1 were generated, characterized and used in proliferation assay and intracellular uptake and retention assay (IUR). In vivo experiments were performed in nude mice injected with K562S, K562DOX cells (overexpressing ABCB1), and K562DOX silenced for ABCB1 (K562DOX/sh P-GP). RESULTS: The IUR assay using C-14 bosutinib showed that only ABCB1 was responsible for active bosutinib transport. K562DOX cells showed the lowest intracellular level of bosutinib, while K562DOX cells treated with the ABCB1 inhibitor verapamil showed intracellular bosutinib levels comparable with parental K562S. Proliferation assays demonstrated that K562DOX are resistant to bosutinib treatment while verapamil is able to restore the sensitivity to the drug. Nude mice injected with K562DOX and treated with bosutinib showed very limited response and quickly relapsed after stopping treatment while K562S as well as K562DOX/sh P-GP remained tumor-free. CONCLUSIONS: Our data suggest that the analysis of ABCB1 expression levels might help determine treatment options for patients exhibiting resistance to bosutinib.
Subject(s)
Aniline Compounds/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Nitriles/therapeutic use , Quinolines/therapeutic use , ATP Binding Cassette Transporter, Subfamily B/drug effects , Aniline Compounds/administration & dosage , Aniline Compounds/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Female , Humans , In Vitro Techniques , Mice , Mice, Nude , Microscopy, Confocal , Nitriles/administration & dosage , Nitriles/metabolism , Quinolines/administration & dosage , Quinolines/metabolism , TransfectionABSTRACT
Gabapentin is used in the treatment of epilepsy and neuropathic pain. Gabapentin has high and saturable permeability across the BBB, but no mechanistic studies underpinning this process have been reported. The aim of the current study was to investigate the transport of gabapentin in a model of the BBB, identify the important drug transporter(s) and to use mathematical modelling to quantify the processes involved. A human brain endothelial cell line (hCMEC/D3) was utilised as an in-vitro model of the BBB. Uptake of radiolabeled gabapentin into cells in the presence of chemical inhibitors, siRNA or overexpressed drug transporters of interest was investigated. Gabapentin was demonstrated to be a LAT1 substrate in brain endothelial cells (LAT1-process; Km=530µM and Vmax=7039pmoles/million cells/min versus other-processes; Km=923µM and Vmax=3656pmoles/million cells/min) and in transfected HEK 293 LAT1 cells (LAT1-process; Km=217µM and Vmax=5192pmoles/million cells/min versus otherprocesses; Km=1546µM and Vmax=3375pmoles/million cells/min). At physiological concentrations of gabapentin, LAT1 mediated transport was 3 or ~10-fold higher than the other transport processes in the two systems, respectively, demonstrating clear selectivity for gabapentin. In-silico structural homology modelling confirmed that LAT1 could have the LeuT conserved fold and functions by the alternative access mechanism. Mathematical modelling of this mechanism revealed revised significance of Vmax and Km so that a low Km may not necessarily imply a high affinity transport process. Gabapentin was negative for OCT like transport and LAT2 activity in the hCMEC/D3 and OCT1 transfected cells. Our data shows that gabapentin is a substrate for the influx transporter LAT1 at therapeutic concentrations.
Subject(s)
Amines/pharmacokinetics , Cyclohexanecarboxylic Acids/pharmacokinetics , Large Neutral Amino Acid-Transporter 1/physiology , gamma-Aminobutyric Acid/pharmacokinetics , Biological Transport , Blood-Brain Barrier , Blotting, Western , Brain/blood supply , Brain/metabolism , Cell Line , Gabapentin , Humans , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Low pretreatment expression of the imatinib uptake transporter human organic cation transporter 1 (hOCT1) is associated with inferior complete cytogenetic response rates, progression-free survival, and overall survival in imatinib-treated chronic myeloid leukemia (CML). Upregulation of hOCT1 can therefore increase the uptake of imatinib. The hOCT1 gene is transactivated by hepatocyte nuclear factor 4α in human liver, and peroxisome proliferator-activated receptors (PPAR) α and γ activation increases OCT1 expression in mouse hepatocytes. Here we report that no isoform of hepatocyte nuclear factor 4α is expressed in CML lines or in CML primary cells. In contrast, both PPARα and γ were expressed in all CML cell lines and primary cells studied. PPARα agonist treatment increased imatinib killing of CML KCL22 cells and primitive CD34(+) cells, and also upregulates hOCT1 gene expression and increases imatinib uptake into KCL22 cells and primary cells. PPARα agonists might potentially be of clinical use in CML patients failing imatinib.