Vascular endothelial growth factor (VEGF) has a central role in breast cancer development and progression, but the mechanisms that control its expression are poorly understood. Breast cancer tissue ...microarrays revealed an inverse correlation between the Forkhead transcription factor Forkhead box class O (FOXO)3a and VEGF expression. Using the lapatinib-sensitive breast cancer cell lines BT474 and SKBR3 as model systems, we tested the possibility that VEGF expression is negatively regulated by FOXO3a. Lapatinib treatment of BT474 or SKBR3 cells resulted in nuclear translocation and activation of FOXO3a, followed by a reduction in VEGF expression. Transient transfection and inducible expression experiments showed that FOXO3a represses the proximal VEGF promoter, whereas another Forkhead member, FOXM1, induces VEGF expression. Chromatin immunoprecipitation and oligonucleotide pull-down assays showed that both FOXO3a and FOXM1 bind a consensus Forkhead response element (FHRE) in the VEGF promoter. Upon lapatinib stimulation, activated FOXO3a displaces FOXM1 bound to the FHRE before recruiting histone deacetylase 2 (HDAC2) to the promoter, leading to decreased histones H3 and H4 acetylation, and concomitant transcriptional inhibition of VEGF. These results show that FOXO3a-dependent repression of target genes in breast cancer cells, such as VEGF, involves competitive displacement of DNA-bound FOXM1 and active recruitment of transcriptional repressor complexes.
The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is ...known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.
FOXM1 has been implicated in taxane resistance, but the molecular mechanism involved remains elusive. In here, we show that FOXM1 depletion can sensitize breast cancer cells and mouse embryonic ...fibroblasts into entering paclitaxel-induced senescence, with the loss of clonogenic ability, and the induction of senescence-associated β-galactosidase activity and flat cell morphology. We also demonstrate that FOXM1 regulates the expression of the microtubulin-associated kinesin KIF20A at the transcriptional level directly through a Forkhead response element (FHRE) in its promoter. Similar to FOXM1, KIF20A expression is downregulated by paclitaxel in the sensitive MCF-7 breast cancer cells and deregulated in the paclitaxel-resistant MCF-7Tax(R) cells. KIF20A depletion also renders MCF-7 and MCF-7Tax(R) cells more sensitive to paclitaxel-induced cellular senescence. Crucially, resembling paclitaxel treatment, silencing of FOXM1 and KIF20A similarly promotes abnormal mitotic spindle morphology and chromosome alignment, which have been shown to induce mitotic catastrophe-dependent senescence. The physiological relevance of the regulation of KIF20A by FOXM1 is further highlighted by the strong and significant correlations between FOXM1 and KIF20A expression in breast cancer patient samples. Statistical analysis reveals that both FOXM1 and KIF20A protein and mRNA expression significantly associates with poor survival, consistent with a role of FOXM1 and KIF20A in paclitaxel action and resistance. Collectively, our findings suggest that paclitaxel targets the FOXM1-KIF20A axis to drive abnormal mitotic spindle formation and mitotic catastrophe and that deregulated FOXM1 and KIF20A expression may confer paclitaxel resistance. These findings provide insights into the underlying mechanisms of paclitaxel resistance and have implications for the development of predictive biomarkers and novel chemotherapeutic strategies for paclitaxel resistance.
FOXM1 is implicated in genotoxic drug resistance but its mechanism of action remains elusive. We show here that FOXM1-depletion can sensitize breast cancer cells and mouse embryonic fibroblasts ...(MEFs) into entering epirubicin-induced senescence, with the loss of long-term cell proliferation ability, the accumulation of γH2AX foci, and the induction of senescence-associated β-galactosidase activity and cell morphology. Conversely, reconstitution of FOXM1 in FOXM1-deficient MEFs alleviates the accumulation of senescence-associated γH2AX foci. We also demonstrate that FOXM1 regulates NBS1 at the transcriptional level through an forkhead response element on its promoter. Like FOXM1, NBS1 is overexpressed in the epirubicin-resistant MCF-7Epi(R) cells and its expression level is low but inducible by epirubicin in MCF-7 cells. Consistently, overexpression of FOXM1 augmented and FOXM1 depletion reduced NBS1 expression and epirubicin-induced ataxia-telangiectasia mutated (ATM)phosphorylation in breast cancer cells. Together these findings suggest that FOXM1 increases NBS1 expression and ATM phosphorylation, possibly through increasing the levels of the MRN(MRE11/RAD50/NBS1) complex. Consistent with this idea, the loss of P-ATM induction by epirubicin in the NBS1-deficient NBS1-LBI fibroblasts can be rescued by NBS1 reconstitution. Resembling FOXM1, NBS1 depletion also rendered MCF-7 and MCF-7Epi(R) cells more sensitive to epirubicin-induced cellular senescence. In agreement, the DNA repair-defective and senescence phenotypes in FOXM1-deficent cells can be effectively rescued by overexpression of NBS1. Moreover, overexpression of NBS1 and FOXM1 similarly enhanced and their depletion downregulated homologous recombination (HR) DNA repair activity. Crucially, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, demonstrating that NBS1 is indispensable for the HR function of FOXM1. The physiological relevance of the regulation of NBS1 expression by FOXM1 is further underscored by the strong and significant correlation between nuclear FOXM1 and total NBS1 expression in breast cancer patient samples, further suggesting that NBS1 as a key FOXM1 target gene involved in DNA damage response, genotoxic drug resistance and DNA damage-induced senescence.
Handbook of Pathology Khoo, U S
Hong Kong medical journal = Xianggang yi xue za zhi,
10/2018, Letnik:
24, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Illustrations on the right-hand page showlow-power photomicrographs of liver and spleen sections in waxydegeneration (courtesy of the Hong Kong Museum of Medical SciencesSociety) Unlike conventional ...textbooks, which are usually arranged according to organ systems, or by the commonly described pathological processes, the Handbook is divided into chapters based on aetiological lines or characteristic tissue changes, with lesions of a similar or related nature grouped together for discussion (Fig 3). Wang was a graduate of the Hong Kong College of Medicine, which was the only tertiary-level educational institute in Hong Kong at that time.3 The Licentiate awarded by the College to graduates at that time was not recognised by the General Medical Council of the United Kingdom; therefore, Wang enrolled at the University of Edinburgh, obtaining his MB ChB degree in 1910, after only 2 years of study.1 Motivated by a desire to learn more about scientific and technological advances in the major centres of medical education in the West, Wang then undertook a period of intensive training, obtaining the Edinburgh Diploma of Tropical Health and Hygiene in 1911, the Diploma of Public Health at Manchester in 1912, and the BSc in Bacteriology and Comparative Pathology in 1913, also in Manchester.1 He then worked in the laboratory of the Royal College of Physicians of Edinburgh as an independent researcher from 1913 to 1919.4 His particular interest was in the field of tuberculosis, which was the basis of his MD thesis “A study in some phases of tuberculosis, with special reference to the incidence of bovine infection and the question of latency and prevalence of the disease,” for which he was awarded a gold medal from the University of Edinburgh in 19161 (Fig 3). Tragically, Wang contracted tuberculosis at the height of his career.6 When he could no longer speak on account of tuberculosis affecting his larynx, he wrote down his lectures and asked an assistant to read it out of him.6 With little time left for research, his interest in tuberculosis and laboratory medicine nevertheless continued, with publication of a series of papers in the Lancet on different aspects of laboratory testing.7 8 9 He passed away at the young age of 42.6 He was remembered as a kind and conscientious teacher interested in the welfare of his students (personal communication, Dr Chiu-kwong Yu; 1998).
In the published version of this article, the images for cytoplasmic and nuclear FGF7 in MDA-MB-231 cells were duplicated and mistaken for total FGF7 in SKBR-3 and MDA-MB-231 cells.
CXC chemokine receptor type 4 (CXCR4) plays a prominent role in cancer progression and metastasis. However, its association with breast cancer subtypes is still unknown. In the current study, we ...analyzed the expression level and the cellular location of CXCR4 in 175 cases of human breast tumors, including 75 cases of triple-negative breast cancers (TNBCs), 41 cases of luminal-subtypes and 60 cases of HER2-positive breast cancers by using immmunohistochemistry (IHC). We found that CXCR4 was expressed more frequently in the TNBCs than in other subtypes (71% for TNBC vs 44% for HER2-positive and 37% for luminal subtype, p < 0.001). In the TNBC group, CXCR4 positive patients have a significantly higher rate of visceral metastasis (liver, lung and brain). The expression level of CXCR4 is also significantly related to tumor size, advanced TNM stage, shorter overall- and disease-free survival. However, in luminal or HER2-positive breast cancer groups, CXCR4 is not correlated with such clinico-pathological characteristics and survival. Taken together, our data indicate that CXCR4 might exert its function exclusively in TNBC patients, suggesting that targeting CXCR4 might be an effective therapy for TNBC patients.
FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated ...with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.
Objective. The aim of this study is to investigate the prevalence of promotor CpG island methylation of the death-associated protein kinase (
DAPK),
p16, and
O
6-methylguanine-DNA methyltransferase (
...MGMT) genes in both tumor and plasma samples of cervical cancers.
Methods. Methylation-specific PCR (MSP) was employed to detect promotor CpG island methylation of the
DAPK,
p16, and
MGMT genes in 85 surgical tumor tissue samples and 40 pretreatment plasma samples from cervical cancers.
Results. Promotor CpG island methylation of
DAPK,
p16, and
MGMT was detectable, respectively, in 60%, 28.2%, and 18.8% of cases of cervical tumor DNA; and in 40%, 10%, and 7.5% of cases of patients' plasma DNA. Moreover, at least one of the three methylated genes was detected in 75.3% (64/85) of cases of tumor and in 55% (22/40) of cases of plasma. Higher prevalence of methylation of
DAPK was found in squamous cell carcinoma than in adenocarcinoma in both univariate and multivariate analysis. Methylation of
p16 was significantly associated with that of
MGMT in both univariate and multivariate analysis. The methylation pattern in primary tumor and plasma was found to be concordant in 23 patients with matched tissue and plasma samples. In cases positive for
DAPK and
p16 methylation in tumor, detection in the paired plasma sample was 64.3% (9/14) and 33.3% (3/9), respectively.
Conclusions. Promotor CpG island methylation is a frequent event in cervical carcinogenesis. Detection of the methylated sequences in the circulation suggests that plasma DNA methylation warrants further study to determine its potential role in cancer management.
PDF
