Numb在三阴乳腺癌中抑制HDM2泛素化降解p53

目的:探究Numb蛋白在三阴乳腺癌患者中的表达降低情况,及Numb蛋白在三阴乳腺癌中对抑癌因子p53蛋白水平的影响及调控机制,进一步研究Numb蛋白的降低与三阴乳腺癌发生发展的相关性,从而为缺乏有效治疗方法的三阴乳腺癌提供一个潜在的治疗新靶点。方法:40例三阴乳腺癌患者病理组织切片取自重庆医科大学临床病理诊断中心,采用免疫组化法检测Numb蛋白在三阴乳腺癌患者中的表达情况。MCF-10A细胞株和MDA-MB-231细胞株均为ATCC来源,采用qPCR和Western blot法检测对比Numb、HDM2、p53三者的转录水平和蛋白质水平在以上两个细胞株中差异。采用增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)质粒转染的方法在MDA-MB-231细胞中重表达Numb,采用q PCR和Western blot法验证Numb、HDM2、p53三者表达的变化。结果:转染NUMB-EGFP后MDA-MB-231细胞中Numb的mRNA和蛋白质水平均明显上调,HDM2无显著改变,p53在转录水平无明显变化,但在蛋白质水平显著升高。在231细胞中上调Numb蛋白可以在转录后水平调节p53水平,使p53蛋白随之显著升高。结论:Numb蛋白在三阴乳腺癌患者中表达降低的比列很高,为55%,且Numb蛋白在三阴乳腺癌细胞MDA-MB-231中可以调控抑癌因子p53蛋白水平,Numb蛋白水平与p53蛋白水平呈正相关。     

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple‐negative breast cancer

The up‐regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple‐negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down‐regulated in human TNBC, and Claudin15 down‐regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down‐regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non‐TNBC MCF‐7 cells. Conversely, an up‐regulation of Claudin15 remarkably reduced TNBC MDA‐MB‐231 cell migration, invasion and VM formation. We also showed that down‐regulation of Claudin15 was Twist1‐dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15‐deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1‐Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.     

Molecular characterization of circulating tumour cells identifies predictive markers for outcome in primary,triple‐negative breast cancer patients

mRNA profiles of circulating tumour cells (CTCs) were analysed in patients with triple‐negative breast cancer (TNBC) (pts) before (BT) and after therapy (AT) to identify additional treatment options. 2 × 5 mL blood of 51 TNBC pts and 24 non‐TNBC pts (HR+/HER2?; HR?/HER2+) was analysed for CTCs using the AdnaTest EMT‐2/Stem Cell Select?, followed by mRNA isolation and cDNA analysis for 17 genes by qPCR PIK3CA, AKT2, MTOR and the resistance marker AURKA and ERCC1 were predominantly expressed in all breast cancer subtypes, the latter ones especially AT. In TNBC pts, ERBB3, EGFR, SRC, NOTCH, ALK and AR were uniquely present and ERBB2+/ERBB3 + CTCs were found BT and AT in about 20% of cases. EGFR+/ERBB2+/ERBB3 + CTCs BT and ERBB2+/ERBB3 + CTCs AT significantly correlated with a shorter progression‐free survival (PFS; P = 0.01 and P = 0.02). Platinum‐based therapy resulted in a reduced PFS (P = 0.02) and an induction of PIK3CA expression in CTCs AT. In non‐TNBC pts, BT, the expression pattern in CTCs was similar. AURKA+/ERCC1 + CTCs were found in 40% of HR?/HER2 + pts BT and AT. In the latter group, NOTCH, PARP1 and SRC1 were only present AT and ERBB2 + CTCs completely disappeared AT. These findings might help to predict personalized therapy for TNBC pts in the future.     

Aberrant methylation of WD‐repeat protein 41 contributes to tumour progression in triple‐negative breast cancer

WD‐repeat proteins are implicated in a variety of biological functions, most recently in oncogenesis. However, the underlying function of WD‐repeat protein 41 (WDR41) in tumorigenesis remains elusive. The present study was aimed to explore the role of WDR41 in breast cancer. Combined with Western blotting and immunohistochemistry, the results showed that WDR41 was expressed at low levels in breast cancer, especially in triple‐negative breast cancer (TNBC). Using methylation‐specific PCR (MSP), we observed that WDR41 presented hypermethylation in MDA‐MB‐231 cells. Methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) management increased the expression of WDR41 in MDA‐MB‐231 cells, but not in MCF‐10A (normal mammary epithelial cells) or oestrogen receptor‐positive MCF‐7 breast cancer cells. WDR41‐down‐regulation promoted, while WDR41‐up‐regulation inhibited the tumour characteristics of TNBC cells including cell viability, cell cycle and migration. Further, WDR41‐up‐regulation dramatically suppressed tumour growth in vivo. Mechanistically, WDR41 protein ablation activated, while WDR41‐up‐regulation repressed the AKT/GSK‐3β pathway and the subsequent nuclear activation of β‐catenin in MDA‐MB‐231 cells, and 5‐aza‐dC treatment enhanced this effect. After treatment with the AKT inhibitor MK‐2206, WDR41‐down‐regulation‐mediated activation of the GSK‐3β/β‐catenin signalling was robustly abolished. Collectively, methylated WDR41 in MDA‐MB‐231 cells promotes tumorigenesis through positively regulating the AKT/GSK‐3β/β‐catenin pathway, thus providing an important foundation for treating TNBC.     

Anti‐EGFR antibody‐drug conjugate for triple‐negative breast cancer therapy

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR⁺ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.     

LIPH promotes metastasis by enriching stem‐like cells in triple‐negative breast cancer

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24? stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44⁺/CD24^? stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.     

Development and validation of a novel 15‐CpG‐based signature for predicting prognosis in triple‐negative breast cancer

DNA methylation is an important biological regulatory mechanism that changes gene expression without altering the DNA sequence. Increasing studies have revealed that DNA methylation data play a vital role in the field of oncology. However, the methylation site signature in triple‐negative breast cancer (TNBC) remains unknown. In our research, we analysed 158 TNBC samples and 98 noncancerous samples from The Cancer Genome Atlas (TCGA) in three phases. In the discovery phase, 86 CpGs were identified by univariate Cox proportional hazards regression (CPHR) analyses to be significantly correlated with overall survival (P < 0.01). In the training phase, these candidate CpGs were further narrowed down to a 15‐CpG‐based signature by conducting least absolute shrinkage and selector operator (LASSO) Cox regression in the training set. In the validation phase, the 15‐CpG‐based signature was verified using two different internal sets and one external validation set. Furthermore, a nomogram comprising the CpG‐based signature and TNM stage was generated to predict the 1‐, 3‐ and 5‐year overall survival in the primary set, and it showed excellent performance in the three validation sets (concordance indexes: 0.924, 0.974 and 0.637). This study showed that our nomogram has a precise predictive effect on the prognosis of TNBC and can potentially be implemented for clinical treatment and diagnosis.     

TLR5 is a new reporter for triple‐negative breast cancer indicated by radioimmunoimaging and fluorescent staining

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour that lacks marker for targeted diagnosis. Recently, it was reported that toll‐like receptor 5 (TLR5) was associated with some kind of tumours, especially in TNBC, but whether it could be used as a non‐invasive monitoring target is not fully understood. Here, we established TLR5^? 4T1 cell line with lentivirus‐shRNA‐TLR5 knock‐down transfection (with tag GFP, green fluorescent protein, TLR5^? 4T1) and control TLR5⁺ 4T1 cell line with negative control lentivirus transfection. The effect of TLR5 down‐regulation was detected with qPCR and Western blot. ¹²⁵I‐anti‐TLR5 mAb and control isotype ¹²⁵I‐IgG were prepared and injected to TLR5^+/? 4T1‐bearing mice models, respectively. Whole‐body phosphor‐autoradiography, fluorescence imaging and biodistribution were performed. Furthermore, ex vivo tumour TLR5 expression was proved through immunohistochemistry staining. We found that ¹²⁵I‐anti‐TLR5 mAb could bind to TLR5⁺ 4T1 with high affinity and specificity. Whole‐body phosphor‐autoradiography after ¹²⁵I‐anti‐TLR5 mAb injection showed TLR5⁺ 4T1 tumour images in 24 hours, more clearly in 48 hours. Radioactivities in tumour tissues were positively related with TLR5 expression. Biodistribution assay showed that ¹²⁵I‐anti‐TLR5 mAb was mainly metabolized through the liver and kidney, and ¹²⁵I‐anti‐TLR5 mAb was much more accumulated in TLR5⁺ 4T1 tumour than TLR5^? 4T1. In vivo fluorescence imaging successfully showed tumour tissues clearly both in TLR5⁺ and TLR5^? 4T1 mice compared with lentivirus untreated 4T1 tumour. Immunohistochemistry staining showed that TLR5 expression in tumours was indeed down‐regulated in TLR5^? 4T1 mice. Our results indicated that ¹²⁵I‐antiTLR5 mAb was an ideal agent for non‐invasive imaging of TLR5⁺ tumours; TLR5 may be as a novel molecular target for TNBC non‐invasive diagnosis.     

Aurora kinase A stabilizes FOXM1 to enhance paclitaxel resistance in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) has a relatively poor outcome. Acquired chemoresistance is a major clinical challenge for TNBC patients. Previously, we reported that kinase‐dead Aurora kinase A (Aurora‐A) could effectively transactivate the FOXM1 promoter. Here, we demonstrate an additional pathway through which Aurora‐A stabilizes FOXM1 by attenuating its ubiquitin in TNBC. Specifically, Aurora‐A stabilizes FOXM1 in late M phase and early G1 phase of the cell cycle, which promotes proliferation of TNBC cells. Knock‐down of Aurora‐A significantly suppresses cell proliferation in TNBC cell lines and can be rescued by FOXM1 overexpression. We observe that paclitaxel‐resistant TNBC cells exhibit high expression of Aurora‐A and FOXM1. Overexpression of Aurora‐A offers TNBC cells an additional growth advantage and protection against paclitaxel. Moreover, Aurora‐A and FOXM1 could be simultaneously targeted by thiostrepton. Combination of thiostrepton and paclitaxel treatment reverses paclitaxel resistance and significantly inhibits cell proliferation. In conclusion, our study reveals additional mechanism through which Aurora‐A regulates FOXM1 and provides a new therapeutic strategy to treat paclitaxel‐resistant triple‐negative breast cancer.     

Long non‐coding RNA ZEB2‐AS1 promotes the proliferation,metastasis and epithelial mesenchymal transition in triple‐negative breast cancer by epigenetically activating ZEB2

The triple‐negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA‐ZEB2‐AS1 was dramatically up‐regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA‐ZEB2‐AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA‐ZEB2‐AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple‐negative breast cancer. It is mainly endo‐nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF‐induced F‐actin polymerization in MDA231 cells can be suppressed by reducing lncRNA‐ZEB2‐AS1 expression. The migration and invasion of triple‐negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA‐ZEB2‐AS1 is an important factor affecting the development of triple‐negative breast cancer and thus a potential oncogene target.     

ABCC9, NKAPL,and TMEM132C are potential diagnostic and prognostic markers in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly heterogeneous disease. The aim of this study is to identify the diagnostic and poor prognostic signatures in TNBC by exploring the aberrant DNA methylation and gene expression. Differential expression and methylation analysis of the TNBC and paracancer samples from The Cancer Genome Atlas were performed. Gene set enrichment and protein–protein interaction (PPI) network analysis was used to explore the mechanisms of TNBC. Methylation‐gene expression correlation analysis was performed, and multivariate Cox analysis and receiver operating characteristics analysis were used to further screen the hub genes for TNBC. We identified 1,525 differentially expressed genes and 150 differentially methylated genes between TNBC and paracancer samples. About 96.64% of the methylation sites were located on the CpG island. A total of 17 Gene Ontology biological process terms and 18 signal pathways were significantly enriched. GNG4, GNG11, PENK, MAOA, and AOX1 were identified as the core genes of the PPI network. Methylation‐expression correlations revealed that ABCC9 (cg06951626), NKAPL (cg18675097, cg01031101, and cg17384889), and TMEM132C (cg03530754) showed promise as diagnostic and prognostic markers in TNBC. ABCC9 (cg06951626), NKAPL (cg18675097, cg01031101, and cg17384889), and TMEM132C (cg03530754) were potential diagnostic and prognostic markers in TNBC.     

抑癌基因p53与肿瘤研究的最新进展

p53基因是迄今为止已发现的与人类肿瘤发生相关性最高的抑癌基因,其主要生物学功能是通过调控DNA修复、细胞周期停滞和诱导细胞凋亡,维持基因组和细胞稳定,抑制肿瘤生长;肿瘤血管再生、微小RNA（microRNA,miRNA）及肿瘤干细胞是近几年来肿瘤发生机理研究领域的热点,本文综述了p53基因在肿瘤血管再生、miRNA、肿瘤干细胞中作用的最新研究进展及其在肿瘤治疗中的应用。