Tomasz Owczarek

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

Modelling bladder cancer in mice: opportunities and challenges

The prognosis and treatment of bladder cancer have improved little in the past 20 years. Bladder cancer remains a debilitating and often fatal disease, and is among the most costly cancers to treat. The generation of informative mouse models has the potential to improve our understanding of bladder cancer progression, as well as to affect its diagnosis and treatment. However, relatively few mouse models of bladder cancer have been described, and in particular, few that develop invasive cancer phenotypes. This Review focuses on opportunities for improving the landscape of mouse models of bladder cancer.

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.

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.

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.

MUC1 in human and murine mammary carcinoma cells decreases the expression of core 2 β1,6-N-acetylglucosaminyltransferase and β-galactoside α2,3-sialyltransferase

A good correlation between the expression of mucin1 (MUC1) and T antigen was found in breast cancer tumors and breast cancer cell lines, especially after treatment with neuraminidase. The association between the appearance of T antigen and the overexpression of MUC1 was further confirmed by transfecting MDA-MB-231 cells and murine 4T1 mammary carcinoma cells with cDNA for MUC1 and using an RNAi approach to inhibit the expression of MUC1 gene in T47D cells. Furthermore, we discovered that in 4T1 cells which express the sialyl Le(X) antigen, overexpression of MUC1 caused not only appearance of T antigen, but also loss of the sialyl Le(X) structure. As the observed changes in O-glycan synthesis can be associated with changes in the expression of specific glycosyltransferases, core 1 β1,3-galactosyltransferase, core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) and β-galactoside α2,3-sialyltransferase (ST3Gal I), we studied their expression in parental, vector-transfected and MUC1-transfected MDA-MB-231 and 4T1 cells as well as T47D cells transduced with small hairpin RNA targeted MUC1 mRNA. It was found that the expression of C2GnT1 and ST3Gal I is highly decreased in MUC1-expressing MDA-MB-231 and 4T1 cells and increased in T47D cells with suppressed expression of MUC1. Therefore, we found that changes in the structure of O-linked oligosaccharides, resulting in the occurrence of T antigen, are at least partially associated with MUC1 overexpression which down-regulates the expression of C2GnT1 and ST3Gal I. We showed also that the overexpression of MUC1 in 4T1 cells changes their adhesive properties, as MUC1-expressing cells do not adhere to E-selectin, but bind galectin-3.

Expression of metallothionein in the liver and kidneys of the red deer (Cervus elaphus L.) from an industrial metal smelting area of Poland

The metallothionein 1 (MT1) coding sequence of red deer was identified and compared to orthologous sequences from other mammals. Over 90% identity was observed between red deer MT1 amino acid sequence and MT1 sequences of other ruminants. Liver and kidney samples of red deer were collected from the industrial zinc smelting site of Miasteczko Slaskie and from the Masuria Lake District serving as a pollution-free control site. The concentrations of cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) were analyzed by the atomic absorption spectrometry technique (AAS). The levels of Cd in the liver of red deer from the metal smelting region was about 8 times higher than for the reference control site. Next, the expression of MT1 mRNA in the liver of red deer was quantified by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the expression of MT1/2 protein in the liver and kidneys was analyzed by immunohistochemistry. Positive correlations were found between expression levels for MT1 mRNA and the concentrations of Cu and Zn in liver of red deer, and with the age of animals. Immunohistochemical staining demonstrated the nuclear and cytoplasmatic expression in both liver and kidney tissues, but with no obvious relationship shown for the expression of MT1/2 protein and tissue metal levels. Our results showed that the analysis of MT expression levels in the red deer could not be used independently as a biomarker for identifying exposure to Cd, but could be co-analyzed with tissue metal levels to give better prognosis for environmental exposure to metals.

Expression of metallothioneins I and II in kidney of doxorubicin-treated rats

Metallothioneins I/II (MT) are commonly expressed in mammalian tissues and are highly inducible in the response to stress conditions. Doxorubicin (DOX) intoxication promotes oxidative stress and subsequent apoptosis leading to kidney damage. The present study investigates a correlation between endogenous MT expression and DOX-induced apoptosis in renal tubular cells. Experiments were conducted on Buffalo rats receiving DOX (8 mg/kg b.w. for 3 weeks) versus control rats injected with saline. The histopathological alterations and apoptosis (TUNEL) were evaluated in tissue sections. MT expression and tissue localization was examined using immunohistochemical method (IHC). Western blot (WB) was used to evaluate pro-caspase-3, active caspase-3 and MT expression level in tissue homogenates. Examination of renal tissue revealed severe nephrotoxicity in DOX-treated animals. Apoptosis was observed in distal convoluted tubular cells, whereas MT was detected in proximal tubular cells. A significant increase in pro-caspase-3, active caspase-3 and MT expression levels (WB) were seen in DOX group. Positive correlations between histopathological lesions, apoptosis and MT expression were observed. The results obtained in this study could suggest the protective and antiapoptotic effect of MT expression in renal proximal tubular cells under DOX intoxication.

Elevated PIM2 gene expression is associated with poor survival of patients with acute myeloid leukemia

Abstract The PIM2 gene encodes the serine/threonine kinase involved in cell survival and apoptosis. The aim of the study was to evaluate the expression of the PIM2 gene in acute myeloid leukemia (AML) and to examine its role in apoptosis of the blastic cells. We analyzed the PIM2 expression in 148 patients: 91 with AML, 57 with acute lymphoblastic leukemia and 24 healthy controls by Real-Time PCR and Western blot. Inhibition of the PIM2 gene in human leukemic HL60 cell line was performed with RNAi and apoptosis rate was analyzed. Our results indicate that overexpression of PIM2 in AML is associated with low complete remission rate, high-risk cytogenetics, shorter leukemia-free survival, and event-free survival. Cytometric analysis of HL60/PAC-GFP and HL60/PAC-GFP-shPIM2 cells revealed an increase in the number of apoptotic cells after inhibition of PIM2 gene. In summary, the elevated expression of PIM2 in blastic cells is associated with poor prognosis of AML patients and their resistance to induction therapy.

MP48-09 INTEGRIN SIGNALING MODULATION DEMONSTRATES POTENTIAL THERAPEUTIC STRATEGY IN BLADDER CANCER USING THREE-DIMENSIONAL ORGANOID CULTURE

INTRODUCTION AND OBJECTIVES: Integrin signaling plays an important role in cellular proliferation and migration via interactions with extracellular matrix proteins. Prior studies indicate that integrin signaling facilitates tumor invasion and metastasis, and there are several ongoing clinical trials using agents that modulate this pathway. We recently identified clonal enrichment in mutations in the integrin cell surface interactions pathways in advanced urothelial carcinoma. An ideal strategy for investigating integrin signaling is via 3D organoid culture, maintaining intercellular interactions that replicate the epithelial microenvironment. We hypothesize that pharmacologic integrin signaling modulation will impair organoid growth in bladder cancer cells and demonstrate a therapeutic utility for this approach. METHODS: RT4 human bladder cancer cell line was used as well as a second cell line established from a patient-derived bladder cancer sample (PM748). Cells were grown in 3D organoid culture as previously described. For in vitro integrin modulation, defactinib, an orally-bioavailable selective inhibitor of focal adhesion kinase (FAK, a convergent and conserved enzyme activated by integrin ligand binding), was used. SDS-PAGE and immunoblotting were performed to show in vitro FAK inhibition. Single cell suspensions and organoids were plated in the presence of various concentrations of defactinib to determine the impact on organoid formation and regression. RESULTS: Defactinib caused a dose-dependent decrease in autophosphorylation of FAK for both cell lines, demonstrating effective FAK inhibition. 3D culture of single cells with defactinib produced a dose-dependent decrease in organoid size after 96 hours (mean size for DMSO only, 100nM, 1uM, and 10uM were 128um, 75um, 48um, and 26um, respectively; p<0.0001 versus DMSO for all dilutions). Established organoids showed a dose-dependent regression in size after 72 hours of defactinib exposure (mean size for DMSO, 100nM, 1uM, and 10uM were 225um, 96um, 70um, and 34um, respectively; p<0.0001 versus DMSO). Experiments utilizing Crispr-Cas9-mediated FAK knock-out as well as in vivo studies with FAK inhibitors in xenograft models are currently underway. CONCLUSIONS: Integrin modulation via FAK inhibition with defactinib causes both inhibition of organoid formation as well as regression of formed organoids, and the effects are seen at concentrations well below the cytotoxic range for the drug. This study suggests a utility for these agents in bladder cancer treatment.