Versican but not decorin accumulation is related to malignancy in mammographically detected high density and malignant-appearing microcalcifications in non-palpable breast carcinomas

<p>Abstract</p> <p>Background</p> <p>Mammographic density (MD) and malignant-appearing microcalcifications (MAMCs) represent the earliest mammographic findings of non-palpable breast carcinomas. Matrix proteoglycans versican and decorin are frequently over-expressed in various malignancies and are differently involved in the progression of cancer. In the present study, we have evaluated the expression of versican and decorin in non-palpable breast carcinomas and their association with high risk mammographic findings and tumor characteristics.</p> <p>Methods</p> <p>Three hundred and ten patients with non-palpable suspicious breast lesions, detected during screening mammography, were studied. Histological examination was carried out and the expression of decorin, versican, estrogen receptor α (ERα), progesterone receptor (PR) and c-erbB2 (HER-2/neu) was assessed by immunohistochemistry.</p> <p>Results</p> <p>Histological examination showed 83 out of 310 (26.8%) carcinomas of various subtypes. Immunohistochemistry was carried out in 62/83 carcinomas. Decorin was accumulated in breast tissues with MD and MAMCs independently of the presence of malignancy. In contrast, versican was significantly increased only in carcinomas with MAMCs (median ± SE: 42.0 ± 9.1) and MD (22.5 ± 10.1) as compared to normal breast tissue with MAMCs (14.0 ± 5.8), MD (11.0 ± 4.4) and normal breast tissue without mammographic findings (10.0 ± 2.0). Elevated levels of versican were correlated with higher tumor grade and invasiveness in carcinomas with MD and MAMCs, whereas increased amounts of decorin were associated with <it>in situ </it>carcinomas in MAMCs. Stromal deposition of both proteoglycans was related to higher expression of ERα and PR in tumor cells only in MAMCs.</p> <p>Conclusions</p> <p>The specific accumulation of versican in breast tissue with high MD and MAMCs only in the presence of malignant transformation and its association with the aggressiveness of the tumor suggests its possible use as molecular marker in non-palpable breast carcinomas.</p

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties

Expression of Syndecan-4 and Correlation with Metastatic Potential in Testicular Germ Cell Tumours

Although syndecan-4 is implicated in cancer progression, there is no information for its role in testicular germ cell tumours (TGCTs). Thus, we examined the expression of syndecan-4 in patients with TGCTs and its correlation with the clinicopathological findings. Immunohistochemical staining in 71 tissue specimens and mRNA analysis revealed significant overexpression of syndecan-4 in TGCTs. In seminomas, high percentage of tumour cells exhibited membranous and/or cytoplasmic staining for syndecan-4 in all cases. Stromal staining for syndecan-4 was found in seminomas and it was associated with nodal metastasis (), vascular/lymphatic invasion (), and disease stage (). Reduced tumour cell associated staining for syndecan-4 was observed in nonseminomatous germ cell tumours (NSGCTs) compared to seminomas. This loss of syndecan-4 was associated with nodal metastasis (), vascular/lymphatic invasion (), and disease stage (). Stromal staining for syndecan-4 in NSGCTs did not correlate with any of the clinicopathological variables. The stromal expression of syndecan-4 in TGCTs was correlated with microvessel density (). Our results indicate that syndecan-4 is differentially expressed in seminomas and NSGCTs and might be a useful marker. Stromal staining in seminomas and reduced levels of syndecan-4 in tumour cells in NSGCTs are related to metastatic potential, whereas stromal staining in TGCTs is associated with neovascularization

Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells

Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of similar to 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (similar to 87%), 6-sulfated (similar to 10%) and non-sulfated (similar to 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion

Over-expression of glycanated serglycin increases breast cancer cell migration.

<p>Cells (5×10⁵) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (<i>p</i><0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×10⁵ cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (<i>p</i><0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.</p

Characterization of serglycin expression in stably transfected MCF-7 cells.

<p>Detection of serglycin gene transcripts in MCF-7 breast cancer cells carrying empty vector (MCF-7V), vector with serglycin cDNA (MCF-7VSG) and vector coding serglycin cDNA lacking GAG attachment sites (MCF-7VSG/−GAG) by RT-PCR. The PCR products were analyzed on 1% agarose gels stained with GelRed nucleic acid gel stain (A). The ratio of serglycin (SG) to β-actin was determined following quantification of bands’ densities using Scion Image software (B). Statistical significance of variances was calculated using using a one-way ANOVA test (*<i>p</i><0.05). (C) Immunofluorescence staining for serglycin (green) and nuclei (blue) in stably transfected MCF-7V, MCF-7VSG and MCF-7VSG/−GAG cells. Cells were cultured in chamber slides, then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 and incubated with a polyclonal rabbit anti-serglycin antibody. Bars, 25 µm. (D) Equal amounts of protein from concentrated cell culture supernatants from stably transfected MCF-7V, MCF-7VSG and MCF-7VSG/−GAG cells correspond to known number of cells were subjected to Western blot analysis using a polyclonal antibody against serglycin without (D) or after treatment with chondroitinase ABC (E). (D) MCF-7VSG secreted to the culture medium a high molecular mass population, which remained on the top of the separating gel and represents highly glycanated serglycin, and low glycanated sub-populations with molecular masses ranging from 170 kDa to 70 kDa. Furthermore, MCF-7VSG and MCF-7VSG/−GAG cells secreted a core protein of 48 kDa that might represent non-glycanated serglycin core containing GFP-tag. (E) When samples were treated with chondroitinase ABC, MCF-7VSG cells found to secrete a major protein core of 58 kDa (1) that might represent the core protein of highly glycanated serglycin containing oligosaccharides remaining after chondroitinase ABC treatment and the GFP-tag. Minor protein cores of 52–48 kDa (2) secreted from MCF-7VSG and a protein core of 48 kDa (arrowhead) secreted from MCF-7VSG/−GAG cells might represent the low glycanated or/and non-glycanated core proteins of serglycin containing the GFP-tag. Arrow shows the top of the separating gel.</p

Fractionation of PGs secreted from the MDA-MB-231 breast cancer cell line by anion-exchange and gel permeation chromatographies.

<p>One liter of conditioned medium was fractionated on a DEAE-Sephacel column (40 ml bed volume). The column was eluted stepwise with 3 volumes of the formamide buffer described under “Experimental Procedures” containing 0.2 M NaCl and 10 volumes of a NaCl linear gradient ranging from 0.2 to 1.0 M NaCl. Fractions of 6.7 ml were collected, and aliquots were precipitated by the addition of ethanol in the presence of potassium acetate. Precipitates were dissolved in distilled water, analyzed for their GAG content by the DMMB method (A), and subjected to SDS-PAGE analysis using a 4% stacking - 10% separating gel (B). Gels were stained with toluidine blue, followed by staining with coomassie blue. Serglycin was detected throughout the dissolved aliquots by Western blotting analysis after chondroitinase ABC digestion using a polyclonal antibody against serglycin (C) (SG, standard of serglycin used as positive control). A major PG peak was eluted from 0.50 to 0.7 M NaCl and pooled as indicated by the bar. Pooled serglycin was further fractionated by gel permeation chromatography (D). The pooled fractions were chromatographed on a Sepharose CL-4B column. The column was eluted with 4 M guanidine hydrochloride, 50 mM sodium acetate buffer, pH 5.8. Fractions were collected, and PGs were monitored by the DMMB method (D), were subjected to SDS-PAGE following toluidine blue and coomassie staining (E), and were analyzed by Western blotting after chondroitinase ABC digestion (F) (SG, standard of serglycin used as positive control).</p

Immunohistochemical reactivity of serglycin in normal breast and breast carcinoma.

<p>Normal epithelial breast cells in mammary glands were moderately reactive with the polyclonal antibody against serglycin and the reactivity was cytoplasmic (A). Breast cancer cells were stained strongly for serglycin in grade 2 breast carcinomas (B–D). The serglycin reactivity was mainly cytoplasmic, although in some cases cell-surface associated staining was detected in breast cancer cells (D, arrowheads and insert). Bars, 50 µm.</p

Serglycin is secreted to the culture medium by aggressive breast cancer cells.

<p>Equal amounts of protein from concentrated cell culture supernatants of breast cancer cell lines correspond to known number of cells were treated with chondroitinase ABC and subjected to Western blot analysis for serglycin using chondroitinase ABC digested standard serglycin (SG) as positive control (A). (B) Various amounts of standard SG measured as protein from the CAG myeloma cell line and known amounts of protein from concentrated cell culture supernatants from MDA-MB-231 breast cancer cells correspond to known number of cells (two independent experiments) were treated with chondroitinase ABC and were simultaneously analyzed for the presence of SG by Western blotting. (C) A standard curve was created each time by plotting protein band density against the amount of SG subjected to Western blot analysis. A linearity in the standard curve was obtained for amounts up to 24 ng of loaded SG protein (R² = 0.998), and the curve was used for the quantification of SG present in the culture medium of MDA-MB-231 cells.</p