Maciej Ugorski

Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska‐Okolow M 
(2011) Histopathology 59, 1249–1260 
Podoplanin expression by cancer‐associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor☆

Abstract Characterization of the human placental membrane receptor for human 125 I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10 7 mole −1 , and 2.0 ± 0.16 × 10 15 IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 m M ), and periodate (4 m M ) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 m M or 20 m M ) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of 125 I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.

Characterization of FimH Adhesins Expressed by Salmonella enterica Serovar Gallinarum Biovars Gallinarum and Pullorum: Reconstitution of Mannose-Binding Properties by Single Amino Acid Substitution

ABSTRACT Recombinant FimH adhesins of type 1 fimbriae from Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, in contrast to those of Salmonella enterica serovar Typhimurium, did not bind to high-mannose oligosaccharides or to human colon carcinoma HT-29 cells. However, mutated FimH proteins from biovar Gallinarum and biovar Pullorum, in which the isoleucine at position 78 was replaced by the threonine found in S. enterica serovar Typhimurium, bound well to glycoproteins carrying high-mannose oligosaccharides and colon carcinoma cells. The loss of sugar-binding properties by biovar Gallinarum and biovar Pullorum FimH adhesins, which are a part of the type 1 fimbriae, is most probably the result of a single T78I mutation, as was proven by site-directed mutagenesis of FimH proteins.

Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases.

Background:It was shown recently on the level of gene expression that UGT8, coding UDP-galactose:ceramide galactosyltransferase, is one of six genes whose elevated expression correlated with a significantly increased the risk of lung metastases in breast cancer patients. In this study primary tumours and their lung metastases as well as breast cancer cell lines were analysed for UGT8 expression at the protein level.Methods:Expression of UGT8 in breast cancer tissue specimens and breast cancer cell lines was analysed using IHC, real-time PCR and Western blotting.Results:Comparison of the average values of the reaction intensities (IRS scale) showed a significant difference in UGT8 expression between (1) primary and metastatic tumours (Mann–Whitney U, P<0.05), (2) tumours of malignancy grades G3 and G2 (Mann–Whitney U, P<0.01) as well as G3 and G1 (Mann–Whitney U, P<0.001) and (3) node-positive and node-negative tumours (Mann–Whitney U, P<0.001). The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients (721). Similarly, breast cancer cell lines with the ‘luminal epithelial-like’ phenotype did not express or weakly expressed UGT8, in contrast to malignant, ‘mesenchymal-like,’ cells forming metastases in nude mice.Conclusion:Our data suggest that UGT8 is a significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer.

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le(a) antigen.

Several lines of evidence indicate that sialosyl Le a , tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for a1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le a tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le a and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le a -negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metas-tases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le a antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.© Kluwer Academic Publishers 1998

Liposomal formulation of 5-fluorocytosine in suicide gene therapy with cytosine deaminase – for colorectal cancer

It is generally accepted that successful gene therapy depends on two major factors: tumor-specific expression of a therapeutic gene and the efficient transfer of a therapeutic gene to tumor cells. For gene-directed enzyme prodrug therapy (GDEPT) involving Escherichia coli cytosine deaminase (CD) and 5-fluorocytosine (5-FC), several tumor-specific promoters and virus-based vectors were used. No attention whatsoever was paid to the way of 5-FC delivery to solid tumors, despite the fact that the delivery of drugs to such tumors is generally low because of their insufficient transfer from the blood. To compare the effectiveness of GDEPT with free and liposomal 5-FC, the prodrug was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (1:1). When the liposomal form of 5-FC was administered i.v., mice treated with a dose of 5mg of liposomal 5-FC/kg body weight for 10 days, showed complete regression of transplanted tumors and complete cure was observed, whereas in animals treated with the same amounts of the free prodrug, 50% tumor regression and only insignificantly prolonged median survival were found. In summary, these results showed a remarkable enhancement of the antitumor effects of the liposomal form of 5-FC in comparison with the free prodrug. Therapy with liposomal 5-FC thus represents a new approach to achieving a high local concentration of the prodrug for suicide gene therapy using E. coli CD.

Galactosylceramide Affects Tumorigenic and Metastatic Properties of Breast Cancer Cells as an Anti-Apoptotic Molecule

It was recently proposed that UDP-galactose:ceramide galactosyltransferase (UGT8), enzyme responsible for synthesis of galactosylceramide (GalCer), is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. To further reveal the role of UGT8 and GalCer in breast cancer progression, tumorigenicity and metastatic potential of control MDA-MB-231 cells (MDA/LUC) and MDA-MB-231 cells (MDA/LUC-shUGT8) with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA was studied in vivo in athymic nu/nu mice. Control MDA/LUC cells formed tumors and metastatic colonies much more efficiently in comparison to MDA/LUC-shUGT8 cells with suppressed synthesis of GalCer after their, respectively, orthotopic and intracardiac transplantation. These findings indicate that UGT8 and GalCer have a profound effect on tumorigenic and metastatic properties of breast cancer cells. In accordance with this finding, immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in MDA-MB-231 cells is associated with a much higher proliferative index and a lower number of apoptotic cells in comparison to the MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ.

Impact of SOX18 expression in cancer cells and vessels on the outcome of invasive ductal breast carcinoma.

PurposeSOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development, as well as wound healing processes (together with SOX7 and SOX17). In addition, it has been reported that SOX18 may affect the growth of cancer cells in vitro. Until now, the exact role of SOX18 expression in invasive ductal breast carcinoma (IDC) has remained unknown.MethodsIn this study, we have investigated SOX18 expression in cancer cells and endothelial cells in 122 IDC samples using immunohistochemistry (IHC). SOX18 expression was also determined using real-time PCR and Western blotting in a series of breast cancer-derived cell lines (i.e., MCF-7, BT-474, SK-BR-3, MDA-MB-231, BO2).ResultsUsing IHC, we observed SOX18 nuclear expression in cancer cells, as well as in blood and lymphatic vessels of the IDC samples tested. SOX18 expression in the IDC samples correlated with a higher malignancy grade (Grade 2 and Grade 3 versus Grade 1; p = 0.02 and p = 0.009, respectively) and VEGF-D expression (r = 0.27, p = 0.007). SOX18 expression was also associated with HER2 positivity (p = 0.02). A significantly higher SOX18 expression was found in the HER2-positive cell line BT-474, and a significantly lower expression in the triple negative cell lines MDA-MB-231 and BO2. Laser capture microdissection of IDC samples revealed significantly higher mRNA SOX7, SOX17 and SOX18 expression levels in the vessels as compared to the cancer cells (p = 0.02 and p = 0.0002, p < 0.0001, respectively). SOX18 positive intratumoral and peritumoral microvessel counts (MVC) were associated with higher malignancy grades (p = 0.04 and p = 0.02, respectively). Moreover, peritumoral SOX18 positive MVC were found to act as an independent marker for a poor prognosis (p = 0.04).ConclusionSOX18 expression may serve as a marker for a poor prognosis in IDC.

The high-adhesive properties of the FimH adhesin of Salmonella enterica serovar Enteritidis are determined by a single F118S substitution

The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.