Expanding the protein catalogue in the proteome reference map of human breast cancer cells

In this report we present a catalogue of 162 proteins (including isoforms and variants) identified in a prototype of proteomic map of breast cancer cells. This work represents the prosecution of previous studies describing the protein complement of breast cancer cells of the line 8701-BC, which has been well characterized for several parameters, providing to be a useful model for the study of breast cancer-associated candidate biomarkers. In particular, 110 spots were identified ex novo by PMF, or validated following previous gel matching identification method; 30 were identified by N-terminal microsequencing and the remaining by gel matching with maps available from our former work. As a consequence of the expanded number of proteins, we have updated our previous classification extending the number of protein groups from 4 to 13. In order to facilitate comparative proteome studies of different kinds of breast cancers, in this report we provide the whole complement of proteins so far identified and grouped into the new classification. A consistent number of them were not described before in other proteomic maps of breast cancer cells or tissues, and therefore they represent a valuable contribution for breast cancer protein databases and for future application in basic and clinical researches.     

血清长链非编码RNA MALAT-1 在卵巢癌诊断中的临床意义

目的:探讨血清长链非编码RNA MALAT-1检测在卵巢癌患者中的诊断作用。方法:收集187例在我院行卵巢肿瘤切除术患者术前血清。应用实时定量PCR检测血清中MALAT-1 m RNA的表达。MALAT-1 m RNA的拷贝数使用绝对定量(标准曲线)计算。MALAT-1的诊断价值采用ROC-AUC曲线和logistic回归分析并与血清CA125进行比较。结果:1本研究有效样本比例为88.77%(166/187)。卵巢良性肿瘤和恶性肿瘤比例分别为62.05%(103/166)和37.95%(63/166)。卵巢恶性肿瘤患者MALAT-1明显高于良性肿瘤患者(p0.001)。经ROC曲线分析,血清CA125和MALAT-1曲线下面积分别为0.726和0.844,且血清MALAT-1的诊断价值明显优于血清CA125(p=0.023)。经logistic回归分析,应用血清MALAT-1和CA125的预测准确性分别为78.92%和68.07%,应用血清MALAT-1可提高预测准确性10.85%。进一步的分析得出,取血清MALAT-1截断点为36.5时,其预测的敏感性和特异性分别高达84.1%和67.0%。结论:血清长链非编码RNA MALAT-1检测对卵巢肿瘤的性质具有良好的预测作用,可作为卵巢癌早期诊断的分子标志物。本研究结果需大样本前瞻性的研究进一步验证。     

肺癌患者血清TGF-β1浓度检测及临床意义分析

目的 探讨检测肺癌患者血清中转化生长因子β1( transforming growth factorβtype1,TGF-β1)的临床应用价值.方法 收集98例肺癌患者血清标本及40例健康对照者血清标本,运用酶联免疫吸附试验(ELISA)检测两组标本血清TGF-β1浓度.分析二者之间的差异及其与肺癌患者临床特征之间的关系.结果 肺癌患者的血清TGF-β1浓度明显高于健康对照者,差异有统计学意义(66848 pg/mL±37178 pg/mL vs 48790 pg/mL±23111 pg/mL,P＜0.01);肺癌患者血清TGF-β1浓度与TNM分期,淋巴结转移,病理分型无相关性(P＞0.05);手术前后,化疗前后血清TGF-β1浓度差异无统计学意义(P＞0.05).结论 血清TGF-β1对肺癌的辅助诊断有一定的临床价值.     

Structure–activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-acetyltransferase 1

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure–activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pK_a, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.     

中国北方地区血清钙、维生素D水平与乳腺癌的相关性研究

目的:探讨中国北方地区血清钙、维生素D水平与乳腺癌及相关临床因素的关系.方法:选取2007年12月至2012年7月哈尔滨医科大学附属肿瘤医院794例女性乳腺癌患者及976例乳腺良性肿瘤患者,并以128例健康妇女为对照,取空腹血清采用原子吸收分光光度法检测三组血清钙含量,采用放免法检测三组中162例血清25(OH)D含量,结合相关临床资料进行分析.结果:乳腺癌组血清钙含量为2.26± 0.12 mmol/L,乳腺良性肿瘤组血清钙含量为2.26±0.09 mmol/L,正常对照组血清钙含量为2.25±0.24 mmol/L,经方差分析,三组总体均数差别无统计学意义(P＞0.05);乳腺癌患者的血清钙水平与年龄、TNM分期、BMI、绝经情况、乳腺癌家族遗传史无关(P＞0.05).乳腺癌组血清25(OH)D含量为41.91±7.55 ng/mL,乳腺良性肿瘤血清25(OH)D含量为54.62±7.48 ng/mL,正常对照组血清25(OH)D含量为56.15±8.87 ng/mL,经方差分析,乳腺癌患者血清25(OH)D含量低于乳腺良性肿瘤组,差别有统计学意义(P＜0.05),乳腺良性肿瘤组与正常对照组差别无统计学意义(P＞0.05);乳腺癌患者的维生素D水平与年龄、TNM分期、BMI、绝经情况有关(P＜0.05),而与乳腺癌家族遗传史无关(P＞0.05).结论:中国北方地区的乳腺癌患者血清钙水平与乳腺良性肿瘤患者无明显差异.乳腺癌患者的维生素D水平低于乳腺良性肿瘤患者,并且与年龄、TNM分期、BMI、绝经情况有关.维生素D水平降低可能与乳腺癌的发生有关,高水平的维生素D可能会降低女性患乳癌的风险.     

Clinical and biomarker endpoint analysis in neoadjuvant endocrine therapy trials

Neoadjuvant endocrine therapy trials for breast cancer are now a widely accepted investigational approach for oncology cooperative group and pharmaceutical company research programs. However, there remains considerable uncertainty regarding the most suitable endpoints for these studies, in part, because short-term clinical, radiological or biomarker responses have not been fully validated as surrogate endpoints that closely relate to long-term breast cancer outcome. This shortcoming must be addressed before neoadjuvant endocrine treatment can be used as a triage strategy designed to identify patients with endocrine therapy “curable” disease. In this summary, information from published studies is used as a basis to critique clinical trial designs and to suggest experimental endpoints for future validation studies. Three aspects of neoadjuvant endocrine therapy designs are considered: the determination of response; the assessment of surgical outcomes; and biomarker endpoint analysis. Data from the letrozole 024 (LET 024) trial that compared letrozole and tamoxifen is used to illustrate a combined endpoint analysis that integrates both clinical and biomarker information. In addition, the concept of a “cell cycle response” is explored as a simple post-treatment endpoint based on Ki67 analysis that might have properties similar to the pathological complete response endpoint used in neoadjuvant chemotherapy trials.     

Proteomic analysis of estrogen response of premalignant human breast cells using a 2-D liquid separation/mass mapping technique

A 2-D liquid-phase separation method based on chromatofocusing and nonporous silica RP-HPLC followed by ESI-TOF-MS was used to analyze proteins in whole cell lysates from estrogen-treated and untreated premalignant, estrogen-responsive cell line MCF10AT1 cells. 2-D mass maps in the pH range 4.6-6.0 were generated with good correlation to theoretical M(r) values for intact proteins. Proteins were identified based on intact M(r), pI and PMF, or MS/MS sequencing. About 300 unique proteins were identified and 120 proteins in mass range 5-75 kDa were quantified upon treatment of estrogen. Around 40 proteins were found to be more highly expressed (>four-fold) and 17 were down-regulated (>four-fold) in treated cells. In our study, we found that many altered proteins have characteristics consistent with the development of a malignant phenotype. Some of them have a role in the ras pathway or play an important role in signal pathways. These changed proteins might be essential in the estrogen regulation mechanism. Our study highlights the use of the MCF10AT1 cell line to examine estrogen-induced changes in premalignant breast cells and the ability of the 2-D mass mapping technique to quantitatively study protein expression changes on a proteomic scale.     

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).     

Molecular mapping the presence of druggable targets in preinvasive and precursor breast lesions: A comprehensive review of biomarkers related to therapeutic interventions

The analysis of clinical breast samples using biomarkers is integral to current breast cancer management. Currently, a limited number of targeted therapies are standard of care in breast cancer treatment. However, these targeted therapies are only suitable for a subset of patients and resistance may occur. Strategies to prevent the occurrence of invasive lesions are required to reduce the morbidity and mortality associated with the development of cancer. In theory, application of targeted therapies to pre-invasive lesions will prevent their progression to invasive lesions with full malignant potential. The diagnostic challenge for pathologists is to make interpretative decisions on early detected pre-invasive lesions. Overall, only a small proportion of these pre-invasive lesions will progress to invasive carcinoma and morphological assessment is an imprecise and subjective means to differentiate histologically identical lesions with varying malignant potential. Therefore differential biomarker analysis in pre-invasive lesions may prevent overtreatment with surgery and provide a predictive indicator of response to therapy. There follows a review of established and emerging potential druggable targets in pre-invasive lesions and correlation with lesion morphology.     

钼靶X线摄片联合肿瘤相关标志物检测对乳腺癌的诊断价值

目的:探讨乳腺钼靶X射线摄片与血清糖类抗原15-3(CA15-3)、癌胚抗原(CEA)和骨桥蛋白(OPN)联合检测对乳腺癌的临床诊断价值。方法:选择在我院经手术和病理证实为乳腺癌的患者60例作为研究组,另选取60例健康体检者作为对照组。分别检测两组的血清CA15-3、CEA和OPN水平,并采用乳腺钼靶X射线检查。比较X射与血清学检测单独检测及联合检测的阳性率。结果:研究组患者血清CA15-3、CEA及OPN水平均显著高于对照组,差异具有统计学意义(P0.05);血清CA15-3、CEA、OPN和钼靶X射线摄片联合检测的敏感性显著高于单独检测,差异具有统计学意义(P0.05)。结论:对乳腺癌患者进行钼靶X射线摄片及肿瘤相关标志物检测可提高阳性检出率,有利于乳腺癌的早期诊断及治疗。     

Pattern of serum immunoreactivity against breast cancer cell lysates may predict severity of disease in breast cancer patients

Humoral tumor-specific immunity has been investigated as a potential tool to identify tumor-associated antigens and evaluate cancer diagnosis and prognosis. Using SDS-PAGE and western blotting techniques we investigated the humoral immune response against tumor cell antigens in 36 breast cancer patients, 17 node-positive (NP) and 19 node-negative (NN). As a source of antigens, we prepared protein lysates from four breast cancer cell lines (AU565, BT474, MCF-7 and MDA-MB-231) which in vitro exhibit different features of invasion, estrogen receptor/progesterone receptor status and HER2/neu expression thereby potentially representing mild to aggressive forms of clinical disease. A higher number of immunocomplexes Ag–Ab were formed when serum from NN patients was immunoreacted against lysates from AU565 and MCF-7 in comparison to serum from NP patients (P < 0.01). BT474 cells were not a good antigenic source. MDA-MB-231 cells could not significantly discriminate between NN and NP patients since both groups showed higher amounts of reactivity against the lysate. However, comparative analysis of protein preparations purified from MCF-7 and MDA-MB-231 cells and immunodetected concomitantly with the same serum samples showed that serum from patients with cancers with worse prognosis (stage, nodality, HER2/neu and hormonal status) reacted more intensely to proteins purified from the relatively more invasive cell line MDA-MB-231 compared to MCF-7. These findings suggest that the study of serum antibody reactivity to antigens purified from breast cancer cell lines with different invasive properties should be further investigated for its potential in providing beneficial prognostic information in breast cancer. Supported by the United States Military Cancer Institute and the Department of Clinical Investigation at Walter Reed Army Medical Center. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.     

Urinary estrogen metabolites and breast cancer: differential pattern of risk found with pre- versus post-treatment collection

INTRODUCTION: The products of estrogen metabolism may affect breast carcinogenesis. The 16alpha-hydroxyestrone (16-OHE) metabolite has a higher affinity for the estrogen receptor (ER) than the 2-hydroxyestrone (2-OHE) metabolite, while conjugated 2-OHE metabolite may inhibit angiogenesis. We investigated the association between the relative concentrations of these metabolites in urine (2-OHE/16-OHE) and breast cancer in a case-control study of Chinese women living in Shanghai. METHODS: Incident breast cancer cases between 25 and 65 years of age (n=110) were identified from hospital or population tumor registries in Shanghai, China. Controls (n=110) were randomly selected from a complete registry of the Shanghai population, and individually matched to cases by menopausal status, age, and pre-treatment or post-treatment urine collection time. Urine samples were collected prior to any breast cancer treatment or surgery among 78 case-control pairs, while urine was collected after surgery, and perhaps other treatments, among 32 case-control pairs. A commercial enzyme-immunoassay kit was used to measure urinary estrogen metabolite concentrations. Conditional logistic regression was used to calculate odds ratios summarizing the 2-OHE/16-OHE and breast cancer association within subjects providing either pre-treatment or post-treatment urine samples. RESULTS: Subjects with a higher urinary 2-OHE/16-OHE ratio were less likely to be diagnosed with breast cancer, but only when urine samples were collected prior to breast cancer treatment (OR(Tertile3(T3)versusTertile1(T1))=0.5, 95% CI (0.2, 1.1)). In contrast, a higher 2-OHE/16-OHE ratio was significantly associated with breast cancer among subjects providing urine specimens after treatment initiation (OR(T3versusT1)=8.7, 95% CI (1.6, 47.1)). This observed cross-over modification occurred within both pre-menopausal and post-menopausal women, and independent of body mass index or recent dietary intake. CONCLUSION: Cross-study differences in urine collection protocols may explain observed inconsistencies in the 2-OHE/16-OHE and breast cancer association. Our case-control analysis using pre-treatment urine samples suggested that a lower 2-OHE/16-OHE ratio was associated with an increased risk of pre-menopausal and post-menopausal breast cancer diagnosis among Chinese women.     

<Emphasis Type="Italic">Interleukin13</Emphasis> haplotypes and susceptibility of Iranian women to breast cancer

Interleukin-13 (IL-13) is a TH2 cytokine with direct and indirect immunoregulatory functions on cancer cells. The cytokine has been reported to have some polymorphic variations at the gene level associated with some immune related diseases including asthma and allergy. In the present study, association of three IL13 gene polymorphisms at positions −1512 A/C and −1055 C/T in the promoter and +2044 G/A in exon-4 was investigated in Iranian women with breast cancer and healthy controls. Genotyping of IL13 gene polymorphisms were performed by PCR–RFLP methods. Serum level of IL-13 was assessed by ELISA. Haplotypes were constructed from genotypic data using Arlequin 3.1 software package. Haplotype analysis revealed higher frequency of a three-locus haplotype, ACA (−1512A/−1055C/+2044A), in normal women than breast cancer patients (P < 0.025). Haplotype CCA, from the other hand, was observed with more frequency among patients than controls (P < 0.03). No statistically significant differences were found in the frequency of genotypes and alleles between patients and control group. No association was observed between investigated genotypes and other prognostic factors including tumor type, lymph node involvement and tumor size. IL-13 serum level was undetectable in both patients and control subjects. Despite observing no association between breast cancer and the single SNPs, results of this investigation suggest that the presence of CCA haplotype of IL13 gene may be associated with susceptibility of Iranian women to breast cancer.     

Validation of CSN1S1 transcriptional expression,promoter methylation,and prognostic power in breast cancer using independent datasets

Breast cancer ranked second among most frequent cancer in the world playing a significant role in mortality rate. Having prior knowledge on differentially expressed genes in breast cell carcinoma elucidated important indications to understand the molecular mechanism underneath breast carcinogenesis. In this study we have investigated the distinguished CSN1S1 expression in human breast cancer. We have analyzed CSN1S1 mRNA expression between cancer and normal tissues using TCGA datasets. Moreover, analysis including promoter methylation, mutations, prognosis, co-expression, gene ontology, and pathways of CSN1S1 were performed by the TCGA Wanderer, UCSC Xena, cBioPortal, PrognoScan, UALCAN, and Enricher server. We have observed low mRNA expression and high promoter methylation of CSN1S1 in cancer tissues compared to normal tissues. Furthermore, we have also identified low mRNA expression in clinicopathological patients, as well as 9 deleterious mutations with highly co-expressed protein MRC1, and significantly related signaling pathways. We have found a positive correlation between the lower expression of CSN1S1 and patients surviving with breast cancer. Here we have concluded that CSN1S1 acts as a biomarker for the surveillance and prognosis of breast cancer, and also works as a novel therapeutic target at the molecular and pathway levels.     

The role of copper in drug-resistant murine and human tumors

Multidrug resistance (MDR) is still a major threat to successful clinical application of cancer chemotherapy. Copper plays an important role in biological systems, and copper is also involved in carcinogenesis. In the present investigation, we addressed the question whether metal copper might be involved in drug resistance of murine and human tumors. By means of atomic absorption spectroscopy, we determined serum copper concentrations. We found that the blood serum of tumor-bearing mice contained higher amounts of copper than healthy mice with tumors. Secondly, mice bearing doxorubicin-resistant Ehrlich ascites carcinoma- or cyclophosphamide-resistant Lewis lung carcinoma contained more copper in their serum than mice bearing the corresponding drug-sensitive parental tumors. Furthermore, the analysis of patients with breast cancer, colon carcinoma or lung cancer showed that the serum copper contents were higher in patients not responding to chemotherapy when compared to patients whose tumors responded to treatment. The copper levels in serum of healthy volunteers were lower than in cancer patients irrespective of their response to chemotherapy. Our results imply that the level of serum copper may be considered as a biomarker for treatment response.