Metallothionein isoform expression by breast cancer cells

Expression of metallothionein (MT) isoforms by a human breast cancer cell line, PMC42, which retains many characteristics of normal breast epithelial cells and expresses functional estrogen receptors, was examined because it has been proposed that human breast cancer cells which are estrogen receptor positive can be differentiated from those which are estrogen receptor negative, by failure to express MT-1E [J.A. Friedline, S.H. Garrett, S. Somji, J.H. Todd, D. A. Sens, Differential expression of the MT-1E gene in estrogen-receptor positive and -negative breast cancer cell lines, Am. J. Pathol. 152 (1998) 23-27]. Using RT-PCR, PMC42 cells were found to transcribe genes for the MT isoforms IE, IX and 2A but not 1A or 1H. In order to examine which of the expressed isoforms might protect against metal toxicity, the cells were challenged with high concentrations of zinc and copper. Using competitive RT-PCR, cells resistant to 500 microM zinc showed 7+/-2 fold (SD, n=3) increases in expression of MT-1X and 6+/-3 fold increases in expression of MT-2A compared to control cells in normal media. For cells resistant to 250 microM copper the corresponding increases were 37+/-13 and 60+/-20 fold, whilst for control cells treated with 250 microM copper for only 6 h, increases were 10+/-3 and 6+/-3 fold. There was only a low level of expression of MT-1E in untreated cells and but a >120 fold increase in copper- resistant cells. Thus estrogen receptor positive cells cannot, in general, be differentiated from estrogen receptor negative cells by failure to express MT-1E, as suggested by Friedline et al. (1998). Increased expression of MT-1E, as well as MT-1X and MT-2A, protects against metal toxicity in PMC42 breast cancer cells.     

Quantitation of Human Metallothionein Isoforms: A Family of Small,Highly Conserved,Cysteine-rich Proteins

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.The metallothioneins (MTs)¹ are a family of small, highly conserved proteins with the specific capacity to bind metal ions (1–3). Mammalian MTs, typically 61 to 68 amino acid residues in length, contain 20 invariant cysteine residues that form two distinct metal-binding domains. Up to seven or eight metal ions may be coordinated per MT. Many functions have been attributed to this redox-active protein, including zinc homeostasis; heavy metal detoxification; metal exchange; metal transfer; and protection against oxidative damage, inflammatory responses, and other cellular stresses (4–6). Changes in MT expression have been associated with human pathologies including cadmium-induced renal toxicity (7), neurodegeneration (8), and many forms of cancer (9, 10). The understanding of these changes is complicated by the 11 functional MT genes, seven pseudogenes, and four MT-like genes encoded in the genome, most of which contain only small differences in amino acid sequence (11). Seventeen of the 18 genes and pseudogenes are clustered together on chromosome 16, which is known to be enriched for intrachromosomal duplications (12). The various MT gene products differ in their patterns of mRNA and protein expression in human tissues and cell lines. Immunohistochemical detection using antibodies that do not discriminate between MT-1 and MT-2 isoforms indicates wide tissue and cell type distribution of MTs, as illustrated with the MT-1A entry of the Human Protein Atlas (13, 14). Measurements of individual MT mRNA levels, however, clearly demonstrate differential expression of specific MT-1 isoforms in human tissues and cell lines (15–17). The MT-3 (18, 19) and MT-4 (20) mRNAs are expressed in even narrower ranges of cell types.An abundance of immunohistochemical and mRNA measurements show that alteration of MT isoform expression is correlated with a variety of cancers (9, 10). For example, several studies show that the expression of specific MT isoforms is altered in invasive ductal breast carcinomas. Elevated MT-2A (21) or MT-1F (22) is correlated with increased proliferation or tumor grade, respectively. Expression of MT-3 is associated with poor prognosis (23, 24). The MT-1E isoform is found in estrogen-receptor-negative (ER⁻), but not estrogen-receptor-positive (ER⁺), tumors (25) and cell lines (26). Parallel assessment of changes in MT protein expression via immunohistochemistry supports the mRNA data up to a point. Except for antibodies specific for the MT-3 isoform (27), all commercially available MT antibodies are pan-specific for the MT-1, MT-2, and MT-4 protein isoforms (28). This is because epitopes recognized by antibodies raised against MT-1 or MT-2 are limited to the first five residues of the acetylated N terminus, which are invariant among all MT-1, MT-2, and MT-4 isoforms (29–31). This includes the commercially available E9 antibody that has been used to demonstrate the overexpression of MT in a wide variety of human cancers (28, 32, 33). In general, the overexpression of MT in various cancers has been associated with resistance to anticancer therapies and linked to a poor prognosis.The mounting evidence that specific MT isoforms may be useful prognostic and diagnostic markers for cancers highlights the need for alternative approaches to the assessment of MT isoform expression at the protein level. A few mass-spectrometry-based studies have succeeded in identifying the complement of MT isoforms in human cells (34, 35). Though top-down approaches hold promise for the quantitation of MTs based on the unique masses of intact isoforms (34, 36), this has yet to be exploited. Inductively coupled plasma MS has been used to quantify total metal-bound MTs in cells and tissues, but it cannot assign relative abundance values of MT isoforms because the proteins are reduced to their elemental composition with this technique. Thus far, MALDI-MS has been used in parallel with inductively coupled plasma MS for the qualitative identification of isoforms (35). Bottom-up quantitative approaches specifically targeting MTs have not yet been reported.The use of mass spectrometry to quantify MT isoforms is not straightforward. The N-terminal tryptic peptide of each human MT isoform encompasses the only sequence that distinguishes all 12 and therefore may be used for their identification and quantitation in complex biological samples from cells and tissues (34). Any attempt at quantitation of this family of small, highly conserved, cysteine-rich proteins therefore requires reproducible detection of these signature peptides.An optimized bottom-up proteomic method is presented here that is capable of identifying and quantifying all isoforms that constitute the human MT gene family in a single experiment. The approach is comparable in sensitivity and dynamic range to quantitative PCR methods used to measure mRNA levels. Quantitative and qualitative differences between mRNA and protein expression indicate that isoform-specific measurements of protein levels complement and extend our understanding of MT isoform expression in complex biological samples. The method was applied to the characterization of MT isoforms in ER⁺ and ER⁻ breast cancer cell lines. Protein and mRNA measurements showed the same complement of isoform expression, confirming differential MT expression between ER⁺ and ER⁻ cell lines. The mass spectrometry assay further showed dramatic differences in the abundance of protein and mRNA in specific isoforms, an observation that has not been previously reported.     

MT1F mRNA在结肠癌组织中的表达及其临床病理意义

目的:探讨MT1F mRNA在结肠癌组织中的表达及其临床病理意义。方法:采用Taq Man探针实时荧光定量Real-time PCR检测40例结肠癌及对应正常粘膜组织中MT1F mRNA的表达,并通过免疫组化检测Metallothionein(MT)的蛋白表达。结果:82.5%(33/40例)的结肠癌组织MT1F mRNA表达较对应正常组织明显下调,降低2.4倍至113倍不等。MT1F mRNA水平与结肠癌患者的年龄、性别、肿瘤部位、肉眼形态、直径、分化及Dukes分期无关。在MT1F mRNA低表达的病例中,癌组织MT蛋白表达低于对应正常组织(P0.05)。结论:结肠癌组织MT1F mRNA水平显著下调,但与结肠癌的临床病理特征无关。     

Metallothionein as a prognostic biomarker in breast cancer

Breast cancer is the most common cancer in women, with a general upward trend in incidence. Basic and clinical breast cancer research has continued at a rapid pace, in the endeavor to understand the biology of the disease so as to improve management of patients. Besides traditional pathological indicators, expression of molecular markers in breast cancer has also been comprehensively investigated. This paper will focus on the prognostic utility of metallothioneins (MTs), a family of low molecular weight metal binding proteins encoded by at least 10 functional MT genes that are associated with cell proliferation in breast cancer. Evidence that MT is a potential prognostic biomarker for breast cancer is supported by many reports in the literature. Expression of the MT protein has been detected by immunohistochemistry in a significant portion of invasive ductal breast cancers. MT expression has also been well studied in association with traditional clinico-pathological parameters of breast cancers. Generally, higher MT expression in breast cancers is predictive of worse patient outcomes. The relationship of MT isoforms to histological grade, estrogen receptor (ER) status, and prognosis will also be discussed.     

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.     

Identification of BCOX1, a novel gene overexpressed in breast cancer

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 2,4920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.     

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.     

Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas.

BACKGROUND: Tissue factor (TF) is a glycoprotein which binds factor VIIa. The TF-VIIa complex serves as a potent initiator of the coagulation pathways. TF, an immediate early gene, may also play a role in cell growth. Expression of TF was correlated with some types of cancers. MATERIALS AND METHODS: Normal, immortalized, and tumor human mammary epithelial cells were used in the experiments. The differential display (DD) technique was used to identify genes differentially expressed in the cells. TF expression patterns were examined by Northern blot analysis, immunofluorescence staining of cultured cells, and immunohistochemical staining in human cryostat sections. RESULTS: In a 5-way display, an amplified polymerase chain reaction (PCR) product was found in normal and immortalized human mammary epithelial cells but not in the breast cancer cells. The PCR fragment was cloned and sequenced. The result showed that the fragment was identical to human tissue factor. Northern blot analysis showed that expression level of tissue factor mRNA remained high in growing, quiescent, and senescent normal mammary epithelial cells. Immunofluorescence staining also confirmed tissue factor expression pattern in the cell lines tested. Immunohistochemical staining showed that tissue factor was expressed in the normal luminal and myoepithelial cells of some ducts but not others. No staining was observed in invasive carcinoma cells. However, myoepithelial cell staining was seen in some residual ductal structures in invasive tumors. CONCLUSIONS: This study shows the use of DD to reveal the loss of TF expression pattern in human breast cancer cell lines. Immunohistochemical staining results showed breast carcinoma cells expressed little TF, if any, suggesting that TF is not required for breast tumor cell invasion. The results also indicated that TF expression was independent of the proliferation status of the expressing cells. The expression pattern of TF may be a meaningful marker in the development of breast cancer.     

Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells.

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.     

Aberrant p63 and WT-1 expression in myoepithelial cells of pregnancy-associated breast cancer: implications for tumor aggressiveness and invasiveness

Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly associated with tumor aggressiveness and invasiveness.     

CD44及CD24在乳腺癌组织中的表达及与临床病理特征的关系研究

目的:探讨肿瘤标志因子CD44及CD24在乳腺癌组织中的表达及与临床病理特征的关系。方法:选择从2015年1月到2017年1月在我院接受手术治疗的乳腺癌患者80例纳入本次研究,另选同期在我院治疗的导管原位癌患者30例,小叶增生患者20例及导管单纯增生患者20例的组织提取标本进行对照,分析CD44及CD24在乳腺癌组织和不同病变类型中的表达,并分析CD44~+/CD24~-细胞在癌症免疫分型中的表达以及CD44~+/CD24~-细胞与乳腺浸润导管癌相关病理特征的关系。结果:乳腺癌组织内的CD44阳性率为52.50%,CD24的阳性率为57.50%,均显著高于癌旁组织的11.25%和15.00%,差异均有统计学意义(均P0.05)。CD44及CD24在导管原位癌及乳腺浸润导管癌中的阳性率高于小叶增生和导管单纯增生,导管原位癌的阳性率高于乳腺浸润导管癌,差异均有统计学意义(均P0.05),且CD44在乳腺浸润导管癌不同分化类型中的阳性率差异有统计学意义(P0.05)。CD24在乳腺浸润导管癌不同分化类型中的阳性率差异不显著(P0.05)。CD44~+/CD24~-细胞在不同癌症免疫分型以及不同分化中的阳性率比较差异均有统计学意义(P0.05)。CD44~+/CD24~-细胞与乳腺浸润导管癌患者的年龄、月经状态、肿瘤直径、淋巴结转移以及远处转移之间均无明显关系(均P0.05)。结论:CD44及CD24在乳腺癌组织内存在较高的阳性率,且CD44~+/CD24~-在乳腺原位癌及低分化的乳腺癌组织内具有更高的阳性率,临床上可尝试通过监测CD44~+/CD24~-的阳性表达情况评价患者的病情及预后。     

The impact of operative approach on outcome of surgery for gastro-oesophageal tumours

Background

Caveolin-1 is thought to have an important impact on both signal transduction and mediation of intracellular processes. Furthermore, it has been suggested that Caveolin-1 may contribute to certain steps of carcinogenesis in various types of cancer. We examined the potential clinical relevance of Caveolin-1 in normal, benign and malignant breast tissue specimens.

Methods

Using tissue microarray (TMA) technology cases of invasive breast cancer, DCIS, benign breast disease (i.e. fibroadenoma, sclerosing adenosis, ductal hyperplasia and radial scar) and normal breast tissue were evaluated for Caveolin-1 expression. Immunohistochemical staining with an anti-Caveolin-1-antibody was performed. Staining intensity was quantified semiquantitatively. In invasive lesions staining results were correlated with clinical and pathological data.

Results

No Caveolin-1 expression was observed in epithelial cells of normal breast tissue (n = 5), benign breast disease (n = 295) and DCIS (n = 108). However, Caveolin-1 expression was found in 32 of 109 cases of invasive breast carcinomas (29.4%). Caveolin-1 expression in invasive breast cancer could neither be correlated with survival parameters such as overall or disease-free survival nor with established clinical and pathological markers.

Conclusion

In this study we demonstrated expression of Caveolin-1 in one third of invasive breast cancers. A significant increase in Caveolin-1 expression was observed comparing invasive breast cancer to both benign breast tissue and non-invasive breast cancer. Since inhibitors of Caveolin-1 signalling are available, targeting Caveolin-1 in breast cancer may represent a potential option for future breast cancer treatment.