Katalin Dobra

Serglycin Constitutively Secreted by Myeloma Plasma Cells Is a Potent Inhibitor of Bone Mineralization in Vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n = 2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

Kindlin-2 is expressed in malignant mesothelioma and is required for tumor cell adhesion and migration

Kindlin‐2 is a novel integrin‐interacting focal adhesion protein that belongs to the Kindlin family. Focal adhesion proteins control cytoskeleton dynamics and promote cancer cell growth, survival, migration and metastasis. Little is known, however, about expression of Kindlin‐2 in association with human cancer. We now reveal high Kindlin‐2 expression in malignant mesothelioma (MM) cell lines using an affinity‐purified anti‐Kindlin‐2 antibody. Furthermore, we show by immunohistochemistry that Kindlin‐2 is highly expressed in 92 of 102 (90%) MMs with epitheliod; sarcomatoid, biphasic and poorly differentiated morphologies. In addition, Kindlin‐2 expression correlates to cell proliferation, suggesting a role for Kindlin‐2 in tumor growth. We also detect increased expression of Kindlin‐2 at the invasion front of tumors concurrent with increased expression of integrin‐linked kinase, a Kindlin‐binding protein. Besides the high expression of Kindlin‐2 in pleural MMs, pleural metastases of lung adenocarcinoma also express large amounts of Kindlin‐2, but not Kindlin‐1. Notably, in vitro, when endogenous Kindlin‐2 was knocked down with RNAi in MM cells, this impaired cell spreading, adhesion and migration. Overall, our study suggests that heightened expression of Kindlin‐2 might contribute to tumor progression in MM.

An Optimized Battery of Eight Antibodies That Can Distinguish Most Cases of Epithelial Mesothelioma From Adenocarcinoma

An immunocytochemical battery comprising 9 antibodies specifically distinguishes 80% of the epithelial malignant mesotheliomas from adenocarcinomas. The discriminatory power of antibodies to calretinin was tested together with this battery to determine whether the performance thereby could be improved. The study comprises 119 mesotheliomas of epithelial or mixed phenotype and 57 adenocarcinoma metastases in the pleural cavity. The differences between the 2 groups were highly significant for all recorded parameters, but typical reactivity for all parameters was seen in only 6 (5.0%) of the 119 mesotheliomas. An algorithm based on stepwise logistic regression was used to interpret divergent reaction patterns. Most diagnostic information was obtained with 8 of the parameters studied. The resulting algorithm identified almost 90% of the mesotheliomas with high specificity. The battery can be performed in 2 steps: several adenocarcinomas first are diagnosed with a few antibodies, applying the rest of the battery on the remaining unresolved cases.

Adaptation of a commercial fluorescent in situ hybridization test to the diagnosis of malignant cells in effusions

In effusion cytology, adjuvant techniques are often needed for the differentiation of reactive proliferating mesothelial cells and malignant cells. In the case of malignancy the further challenge is to distinguish metastatic tumors from the primary malignant mesothelioma. Fluorescence in situ hybridization (FISH) of cells in interphase is an accurate method to monitor the genetic status of cells, detecting aneuploid signals and gene deletions. Moreover, it has been proposed that a homozygous deletion of the p16(INK4A) gene could more specifically identify malignant mesothelial cells among the exfoliated cells. The first objective of this study was to adapt the commercial FISH-test, UroVysion originally designed for the cytological diagnosis of bladder cancer, to the analysis of cells in effusions. The second objective was to test the clinical utility of the test. Sixty-eight pleural effusions were evaluated. The cytological diagnosis was malignant in 29 cases, inconclusive in 24 cases and benign in 15 cases. The independently verified final diagnoses were mesothelioma in 21 cases, metastatic cancer in 29 and benign in 18 cases. The algorithm for aneuploidy distinguished almost all tested malignant conditions from benign ones, also those with inconclusive cytology. The 9p21 locus, carrying the p16(INK4A) gene, was homozygously deleted in two of the metastatic cancers, while this was seen in 12 of the 21 malignant mesotheliomas. Thus the commercial UroVysion-test can be used to accurately distinguish malignant and reactive cells in effusions, particularly when cytology is inconclusive. The test may also indicate presence of MM.

Syndecan-1 and FGF-2, but not FGF receptor-1, share a common transport route and co-localize with heparanase in the nuclei of mesenchymal tumor cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.

Specific Syndecan-1 Domains Regulate Mesenchymal Tumor Cell Adhesion, Motility and Migration

Background Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression. Methodology/Principal Findings We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration. Conclusions/Significance Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.

Guidelines for cytopathologic diagnosis of epithelioid and mixed type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma (MM). Cytopathologists involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC), who have an interest in the field contributed to this update. Reference material includes peer-reviewed publications and textbooks. This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists, who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in cape town. During the previous IMIG biennial meetings, thorough discussions have resulted in published guidelines for the pathologic diagnosis of MM. However, previous recommendations have stated that the diagnosis of MM should be based on histological material only.[12] Accumulating evidence now indicates that the cytological diagnosis of MM supported by ancillary techniques is as reliable as that based on histopathology, although the sensitivity with cytology may be somewhat lower.[345] Recognizing that noninvasive diagnostic modalities benefit both the patient and the health system, future recommendations should include cytology as an accepted method for the diagnosis of this malignancy.[67] The article describes the consensus of opinions of the authors on how cytology together with ancillary testing can be used to establish a reliable diagnosis of MM.

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

BackgroundMammographic 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.MethodsThree 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.ResultsHistological 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 in situ carcinomas in MAMCs. Stromal deposition of both proteoglycans was related to higher expression of ERα and PR in tumor cells only in MAMCs.ConclusionsThe 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.

Molecular characterization of tumour heterogeneity and malignant mesothelioma cell differentiation by gene profiling.

Malignant mesothelioma is an aggressive tumour, characterized by a variable differentiation pattern and poor prognosis. At present, the clinical outcome in patients with malignant mesothelioma is mainly predicted by the morphological phenotype of the tumour. However, this conventional clinicopathological parameter is of limited value, partly because of the biological heterogeneity of this tumour and poor understanding of the regulatory mechanisms underlying the various patterns of growth. To elucidate the intrinsic molecular programmes that determine tumour differentiation, oligonucleotide arrays were used in an in vitro model of mesothelioma differentiation. The analysis of 2059 genes detected 102 genes that were significantly deregulated. Clustering of these genes into functional categories showed distinctive patterns for the two phenotypes, namely epithelioid and sarcomatoid. The molecular fingerprint of the sarcomatoid tumour component indicates overrepresentation of growth factor receptors and growth factor binding proteins, whereas epithelioid mesothelioma cells express other tumour‐promoting factors involved in differentiation, metabolism, and regulation of apoptosis. These differences in the molecular phenotype may give a better basis for diagnosis and for designing novel therapies. Copyright

Syndecan-1 Enhances Proliferation, Migration and Metastasis of HT-1080 Cells in Cooperation with Syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.