不同分子分型乳腺癌患者血清IGFBP-3、Angptl-2表达水平及其与骨转移、预后的相关性

摘要 目的：分析不同分子分型乳腺癌患者血清胰岛素样生长因子结合蛋白3（IGFBP-3）、生成素养蛋白2（Angptl-2）表达水平及其与骨转移、预后的相关性。方法：选取2018年3月-2021年3月东南大学附属中大医院收治的128例乳腺癌骨转移患者进行研究,其中包括Luminal A型50例、42例Luminal B型（HER-2阴性）42例、HER-2过表达型16例、三阴性乳腺癌（TNBC）20例,并分析4种分子分型乳腺癌的临床病理特征,同时采用酶联免疫吸附法检测其血清IGFBP-3、Angptl-2表达水平;随访24个月后记录两组患者的预后情况,并采用多因素Logistic模型分析影响4种分子分型乳腺癌骨转移患者预后的独立危险因素,以及血清IGFBP-3、Angptl-2与不同分子分型乳腺癌骨转移患者预后的相关性。结果：Luminal A型、Luminal B型、HER-2过表达型、TNBC型TNM分期、淋巴结转移比较,差异有统计学意义（P＜0.05）。与Luminal A型、Luminal B型、TNBC型乳腺癌骨转移患者相比,HER-2过表达型乳腺癌骨转移患者的血清IGFBP-3表达水平较低,Angptl-2表达水平较高（P＜0.05）。Luminal A型、Luminal B型、HER-2过表达型、TNBC型乳腺癌骨转移患者的死亡率分别为13.46%、38.46%、23.08%、25.00%。多因素Logistic结果显示,TNM分期、淋巴结转移、血清IGFBP-3、Angptl-2均是影响不同分子分型乳腺癌骨转移患者预后的独立危险因素（P＜0.05）。血清IGFBP-3异常高表达提示4种分子分型乳腺癌骨转移患者的不良预后,而Angptl-2表达水平与4种分子分型乳腺癌的预后呈正相关性（P＜0.05）。针对不同分子分型乳腺癌骨转移患者的预后预测中,血清IGFBP-3、Angptl-2、IGFBP-3+Angptl-2均呈现AUC＞0.75。结论：血清IGFBP-3、Angptl-2可作为HER-2过表达乳腺癌骨转移患者的潜在生物标志物;同时还可根据血清IGFBP-3、Angptl-2表达水平预测不同分子分型乳腺癌骨转移患者的预后。     

SHCBP1:乳腺癌治疗候选靶点和潜在预后标志物

探讨SHCBP1在乳腺癌及其不同亚型中的表达特征和预后价值。利用Oncomine, bc-GenExMiner v4.2, Kaplan-Meier plotter, GSE41994及STRING数据库分析乳腺癌病人SHCBP1的mRNA表达水平及其与乳腺癌患者生存率的相关性等。结果:(1)乳腺癌组织中SHCBP1的mRNA水平明显高于正常组织样本,但不同分子亚型的乳腺癌患者中SHCBP1的表达水平不同;(2)SHCBP1表达升高可导致Luminal A亚型更短的无转移生存期(Distant metastasis-free survival,DMFS)和无病生存期(Disease-free survival, DFS);(3)在雌激素受体阳性(ER+)、孕酮受体阳性(PR+)亚型、仅接受辅助化疗、仅接受辅助化疗的ER(+)与接受包括或排除内分泌治疗乳腺癌患者中,SHCBP1表达升高则无复发生存率(Relapse-free survival,RFS)显著缩短;(4)SHCBP1和CDC45 mRNA表达水平之间呈正相关,并可能与KIF23等10种蛋白相互作用。结论:SHCBP1是一种很有前景的乳腺癌预后指标和潜在的治疗靶点。     

Molecular subtype is determinant on inflammatory status and immunological profile from invasive breast cancer patients

Breast cancer consists in a chronic inflammatory disease with multiple biological and clinical behaviors. Based on high throughput technologies data, this disease is currently classified according to the molecular expression of estrogen (ER), progesterone (PR) and human epidermal growth factor (HER-2) receptors. In this study, we defined the inflammatory profile of the main molecular subtypes of breast cancer patients: luminal (ER and PR positive, HER-2 negative), HER-2 enriched (HER-2 positive) and triple negative (ER, PR and HER-2 negative). Cytokines panel was assessed by measurement of TNF-α, TGF-β, IL-1, IL-10 and IL-12 plasmatic levels. Oxidative profile was assessed by determination of lipid peroxidation, total antioxidant capacity of plasma, malondialdehyde levels, carbonyl content and nitric oxide (NO). Clinical data were correlated with inflammatory findings. Our findings demonstrated that patients bearing the luminal subtype displayed high TNF-α, TGF-β and enhanced oxidative stress levels associated with reduced IL-12. HER-2-enriched group exhibited higher levels of TNF-α, IL-12 and TGF-β associated with enhanced oxidative stress. Triple-negative subtype exhibited the most aggressive profile of disease behavior, with reduction in both TNF-α and TGF-β, with high levels of lipid peroxidation and NO. The clinical importance of our findings lies in the fact that the inflammatory status varies in distinct ways due to molecular subtype of breast cancer, opening potential therapeutic targets to future therapies.     

Clinical utility of determination of HER-2/neu and EGFR fragments in serum of patients with metastatic breast cancer

INTRODUCTION: The growth factor receptors EGFR and HER-2/neu are targets for new treatment strategies and are of potential use as prognostic and predictive factors. However, the optimal method of determination in order to obtain clinically relevant information remains a source of controversy. METHODS: HER-2/neu and EGFR expression was examined by immunohistochemistry in primary tumors of patients with breast cancer. In addition, serum was tested for the extracellular domains of HER-2/neu (HER-2/neu ECD) and EGFR (sEGFR) before initiation of therapy for metastatic disease (n=76). The course of disease from the time of metastasis with regard to these parameters was evaluated by univariate and multivariate analyses. RESULTS: HER-2/neu ECD levels at the time of metastatic disease were correlated with HER-2/neu expression determined by immunohistochemistry from primary tumors (p=0.001). No correlation was observed between expression of EGFR in primary tumors and sEGFR serum levels. HER-2/neu ECD and sEGFR levels at the onset of metastatic disease did not show a significant impact on overall survival. CONCLUSIONS: Determination of HER-2/neu ECD levels in the serum measured by ELISA at the onset of metastatic disease could offer an alternative to immunohistochemistry of the primary tumor since serum levels are correlated with protein expression in primary tumors. In contrast, no such correlation was observed for EGFR.     

不同临床病理特征的乳腺非特殊型浸润性癌分子亚型分析

目的:探讨不同临床病理特征的乳腺非特殊型浸润性癌的分子亚型分布特点。方法:回顾性分析309例乳腺非特殊型浸润性癌患者的临床资料,根据患者年龄、绝经情况、组织学分级和淋巴结转移情况分组,每组再根据免疫组化雌激素受体(ER)、孕激素受体(PR)、人类表皮生长因子受体2(HER-2)结果分为4种不同的分子亚型,分析各组中不同分子亚型的分布特点。结果:309例乳腺非特殊型浸润性癌中Lumina A型为137例(44.3%),Lumina B型为64例(20.7%),HER-2/neu型为63例(20.4%),basal cell-like型为45例(14.6%)。不同年龄段、绝经与否、不同组织学分级和淋巴结转移情况患者的分子亚型差异均具有统计学意义(P0.05)。结论:非特殊型浸润性癌的分子亚型以Luminal A型为主,其中异型性高,转移广泛的basal cell-like型的年龄分布呈现年轻化趋势。     

Basal breast cancer: a complex and deadly molecular subtype

During the last decade, gene expression profiling of breast cancer has revealed the existence of five molecular subtypes and allowed the establishment of a new classification. The basal subtype, which represents 15-25% of cases, is characterized by an expression profile similar to that of myoepithelial normal mammary cells. Basal tumors are frequently assimilated to triple-negative (TN) breast cancers. They display epidemiological and clinico-pathological features distinct from other subtypes. Their pattern of relapse is characterized by frequent and early relapses and visceral locations. Despite a relative sensitivity to chemotherapy, the prognosis is poor. Recent characterization of their molecular features, such as the dysfunction of the BRCA1 pathway or the frequent expression of EGFR, provides opportunities for optimizing the systemic treatment. Several clinical trials dedicated to basal or TN tumors are testing cytotoxic agents and/or molecularly targeted therapies. This review summarizes the current state of knowledge of this aggressive and hard-to-treat subtype of breast cancer.     

Integrating proteomic and phosphoproteomic data for pathway analysis in breast cancer

Background

As protein is the basic unit of cell function and biological pathway, shotgun proteomics, the large-scale analysis of proteins, is contributing greatly to our understanding of disease mechanisms. Proteomics study could detect the changes of both protein expression and modification. With the releases of large-scale cancer proteome studies, how to integrate acquired proteomic and phosphoproteomic data in more comprehensive pathway analysis becomes implemented, but remains challenging. Integrative pathway analysis at proteome level provides a systematic insight into the signaling network adaptations in the development of cancer.

Results

Here we integrated proteomic and phosphoproteomic data to perform pathway prioritization in breast cancer. We manually collected and curated breast cancer well-known related pathways from the literature as target pathways (TPs) or positive control in method evaluation. Three different strategies including Hypergeometric test based over-representation analysis, Kolmogorov-Smirnov (K-S) test based gene set analysis and topology-based pathway analysis, were applied and evaluated in integrating protein expression and phosphorylation. In comparison, we also assessed the ranking performance of the strategy using information of protein expression or protein phosphorylation individually. Target pathways were ranked more top with the data integration than using the information from proteomic or phosphoproteomic data individually. In the comparisons of pathway analysis strategies, topology-based method outperformed than the others. The subtypes of breast cancer, which consist of Luminal A, Luminal B, Basal and HER2-enriched, vary greatly in prognosis and require distinct treatment. Therefore we applied topology-based pathway analysis with integrating protein expression and phosphorylation profiles on four subtypes of breast cancer. The results showed that TPs were enriched in all subtypes but their ranks were significantly different among the subtypes. For instance, p53 pathway ranked top in the Basal-like breast cancer subtype, but not in HER2-enriched type. The rank of Focal adhesion pathway was more top in HER2- subtypes than in HER2+ subtypes. The results were consistent with some previous researches.

Conclusions

The results demonstrate that the network topology-based method is more powerful by integrating proteomic and phosphoproteomic in pathway analysis of proteomics study. This integrative strategy can also be used to rank the specific pathways for the disease subtypes.

     

Co-expression and prognostic value of gross cystic disease fluid protein 15 and mammaglobin in primary breast cancer

Gross cystic disease fluid protein (GCDFP-15) and mammaglobin are both widely used and accepted markers for epithelia of breast origin. We aimed to evaluate their relation of expression on parallel whole tissue sections in primary breast cancer by immunohistochemistry and also to correlate it with clinico-pathological parameters including patient survival. Primary breast carcinomas from 165 patients with a mean clinical follow-up of 73 months were immunostained using commercially available antibodies against GCDFP-15 and mammaglobin. An immunoreactive score (IRS) was calculated based on the cytoplasmic staining intensity and the number of cells stained. Cytoplasmic expression of GCDFP-15 and mammaglobin was observed in 73.3% and 72.1% of invasive breast carcinomas respectively. 91.8% of breast cancer cases expressed at least one of both markers. Both markers strongly correlated with each other and were significantly associated with lower tumour grading. Additionally, GCDFP-15 negativity was significantly associated with shortened disease-free survival times in univariate and multivariate analyses. We demonstrated the strong correlation of GCDFP-15 and mammaglobin with each other and showed that only very few primary breast cancers are completely negative for both markers. The significantly longer disease free survival times for patients with GCDFP-15 positive tumours clearly warrants further study.     

Resveratrol inhibits heregulin-beta1-mediated matrix metalloproteinase-9 expression and cell invasion in human breast cancer cells

The growth factor heregulin-β1 (HRG-β1), which is expressed in breast cancer, activates the HER-2 signaling pathway through induction of heterodimeric complexes of HER-2 with HER-3 or HER-4. It has been shown in many studies that HRG-β1 induces the tumorigenicity and metastasis of breast cancer cells. Matrix metalloproteinase (MMP) 9 is a key enzyme in the degradation of extracellular matrices, and its expression may be dysregulated in breast cancer invasion and metastasis. Resveratrol, a major component in grape, exhibited potential anticarcinogenic activities in both in vitro and in vivo studies. However, the inhibitory effect of resveratrol on HER-2-mediated expression of MMP-9 has not been demonstrated yet.

In the present study, we investigated the anti-invasive mechanism of resveratrol in human breast cancer cells. Human breast cancer MCF-7 cells were exposed to resveratrol (2, 5 and 10 μM). The expression activity of MMP-9 was measured by zymogram analysis. Phosphorylated levels of HER-2 and mitogen-activated protein kinase (MAPK)/ERK were measured by Western blot analysis. Total actin was used as internal control for protein expression. HRG-β1 induced the phosphorylation of HER-2/neu receptor and MMP-9 expression in human breast cancer MCF-7 cells. Resveratrol significantly inhibited HRG-β1-mediated MMP-9 expression in human breast cancer cells. MEK inhibitor induced a marked reduction in MMP-9 expression, and it suggested that ERK1/2 cascade could play an important role in HRG-β1-mediated MMP-9 expression. Furthermore, resveratrol significantly suppressed HRG-β1-mediated phosphorylation of ERK1/2 and invasion of breast cancer cells. However, resveratrol had negligible effects on either HRG-β1-mediated phosphorylation of HER-2 receptor or expression of the tissue inhibitor of MMP, tissue inhibitor metalloproteinase protein 1.

Taken together, our results suggest that resveratrol inhibited MMP-9 expression in human breast cancer cells. The inhibitory effects of resveratrol on MMP-9 expression and invasion of breast cancer cells are, in part, associated with the down-regulation of the MAPK/ERK signaling pathway.     

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of cancer.     

Identification and Validation of Oncologic miRNA Biomarkers for Luminal A-like Breast Cancer

Introduction

Breast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.

Methods

Blood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.

Results

Microarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.

Conclusion

This study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.     

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

IntroductionAnnexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s).MethodsBy mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways.ResultsOur in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1.ConclusionsOur data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.     

Purification and characterization of the mammaglobin/lipophilin B complex,a promising diagnostic marker for breast cancer

Mammaglobin, a promising diagnostic marker for breast cancer, forms a covalent complex with lipophilin B. mRNA levels for each component of the complex were determined for a number of breast tumors and normal tissues, and correlation of message expression was highly significant between mammaglobin and lipophilin B (p < 0.0001). The complex was purified by both standard biochemical techniques and immunoaffinity chromatography. N-Terminal sequencing revealed that mammaglobin and lipophilin B are processed as predicted by cleavage of their signal sequence after amino acids 19 and 21, respectively. Three molecular masses-representing the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteins-are detected by ProteinChip array SELDI-TOF mass spectrometry after partial enzymatic deglycosylation. This is consistent with the two predicted N-linked glycosylation sites in the primary sequence of mammaglobin and each site having an attached sugar of approximately 3500 Da. Reducing agents release lipophilin B from mammaglobin, and the free peptides are seen at their predicted molecular masses in the deglycosylated complex. Molecular modeling, secondary structure prediction, and circular dichroism indicate that the complex is a small alpha-helical globule that has three disulfide bridges and a carbohydrate chain at each pole. LC-ESI-MS shows that mammaglobin and lipophilin B are bonded in a head to tail orientation. This work describes the biochemistry of the mammaglobin/lipophilin B complex and lays the framework for use of this complex as a novel protein-based serological marker for breast cancer.     

Coupling proteomics and transcriptomics in the quest of subtype‐specific proteins in breast cancer

Breast‐cancer subtypes present with distinct clinical characteristics. Therefore, characterization of subtype‐specific proteins may augment the development of targeted therapies and prognostic biomarkers. To address this issue, MS‐based secretome analysis of eight breast cancer cell lines, corresponding to the three main breast cancer subtypes was performed. More than 5200 non‐redundant proteins were identified with 23, four, and four proteins identified uniquely in basal, HER2‐neu‐amplified, and luminal breast cancer cells, respectively. An in silico mRNA analysis using publicly available breast cancer tissue microarray data was carried out as a preliminary verification step. In particular, the expression profiles of 15 out of 28 proteins included in the microarray (from a total of 31 in our subtype‐specific signature) showed significant correlation with estrogen receptor (ER) expression. A MS‐based analysis of breast cancer tissues was undertaken to verify the results at the proteome level. Eighteen out of 31 proteins were quantified in the proteomes of ER‐positive and ER‐negative breast cancer tissues. Survival analysis using microarray data was performed to examine the prognostic potential of these selected candidates. Three proteins correlated with ER status at both mRNA and protein levels: ABAT, PDZK1, and PTX3, with the former showing significant prognostic potential.