Tamara Nikuševa Martić

E‐cadherin and β‐catenin expression patterns in malignant melanoma assessed by image analysis

Background:  We investigated the expression of E‐cadherin and β‐catenin in melanoma. Both proteins are components of adherens junctions but also play signalling roles in the wnt signal transduction pathway.

Changes of AXIN-1 and Beta-Catenin in Neuroepithelial Brain Tumors

In the present study changes of components of Wnt signaling pathway—axin (AXIN1) and beta-catenin (CTNNB1) in a sample of 72 neuroepithelial brain tumors were investigated. AXIN-1 gene was tested by PCR/loss of heterozygosity (LOH). Immunostaining and image analysis revealed the quantity and localization of relevant proteins. Polymorphic marker for AXIN-1, showed LOH in 11.1% of tumors. LOH was distributed to 6.3% of glioblastomas, one was found in neuroepithelial dysembrioplastic tumor and one in medulloblastoma. Down regulation of axin expression and up regulation of beta-catenin were detected in the analyzed tumors. Axin was observed in the cytoplasm in 68.8% of samples, in 28.1% in both the cytoplasm and nucleus and 3.1% had no expression. Beta-catenin was observed mainly in the nucleus and cytoplasm (59.4%). Expression in 34.4% of samples was in the cytoplasm and 6.3% showed no expression. Comparison of mean values of relative increase of axin and beta-catenin showed that they are significantly reversely proportional (P = 0.014). Relative quantity of beta-catenin in patients with gross deletion of AXIN1 was significantly higher in comparison to patients without LOH (P = 0.040). Our results demonstrate that changes of key components of the Wnt signaling play a role in neuroepithelial brain tumors.

Epigenetic alterations of the Wnt signaling pathway in cancer: a mini review

Epigenetic mechanisms play a crucial role in cellular proliferation, migration and differentiation in both normal and neoplastic development. One of the key signaling pathways whose components are altered through the epigenetic mechanisms is the Wnt signaling pathway. In this review, we briefly discuss the key concepts of epigenetics and focus on the recent advances in the Wnt signaling pathway research and its potential diagnostic and therapeutic implications.

Immunohistochemical expression of SFRP1 and SFRP3 proteins in normal and malignant reproductive tissues of rats and humans

Secreted frizzled-related proteins 1 and 3 (SFRP1 and SFRP3) act as Wnt signaling pathway antagonists and play an important role in embryonic development and carcinogenesis. The aim of the present study was to analyze immunohistochemically the distribution of 2 SFRP family proteins, SFRP1 and SFRP3, in an experimental rat model, in normal and intrauterine growth-restricted (IUGR) human placentas, and in a subset of the corresponding human trophoblastic tumors (pure choriocarcinomas and mixed germ cell tumors with choriocarcinoma component). In rats, expression of both SFRP1 and SFRP3 was pronounced in the perimetrium and myometrium, whereas decidual cells showed only occasional positive cytoplasmic staining. The most prominent expression of both proteins was found in blood vessel endothelial cells. Stereological variable of volume density (Vv, mm0) showed statistically higher expression of SFRP1 and SFRP3 in human IUGR placentas than in normal pregnancy placentas (P<0.0001). Compared with adjacent normal/benign tissues, reduced expression of SFRP1 and SFRP3 was observed in human trophoblastic tumors (58.5% and 31.25%, respectively), although none of the examined tumors exhibited complete loss of either protein. Our study indicates that increased expression of both SFRP1 and SFRP3 may contribute to the pathogenesis of IUGR placental dysfunction, whereas the loss of these proteins may be involved in the development of human trophoblastic tumors.

Brain metastases exhibit gross deletions of the APC gene

Candidate genes involved in metastasis to the brain require investigation. In the present study, the adenomatous polyposis coli (APC) gene was analyzed in a set of human brain metastases. Gross deletions of the APC gene were tested by polymerase chain reaction/loss of heterozygosity (LOH) using the restriction fragment length polymorphism method performed by the use of MspI and RsaI genetic markers inside exon 15 and exon 11. Among 21 brain metastases analyzed, 58.8% of samples showed LOH of the APC gene. When assigning the genetic changes to a specific primary tumor type, 6 LOHs were found in metastases originated from lung and 4 LOHs in metastases from colon. The main effector of the wnt signaling, beta-catenin, was upregulated in 42.9% of cases and transferred to the nucleus in 28.6% of metastasis cases. Our findings suggest that genetic changes of the tumor suppressor gene APC, a component of the wnt pathway, represent a part of the brain metastasis genetic profile.

Glycosylation pattern and axin expression in normal and IUGR placentae

Abstract Objective: The aim of this study was to investigate the protein glycosylation pattern and AXIN1 protein expression in human placentae of normal pregnancies and compare them with placentae of pregnancies complicated with intrauterine growth restriction (IUGR). Methods: A total of 38 placentae (17 placentae of IUGR fetuses from singleton pregnancies and gestational age-matched 21 control placentae from normal singleton pregnancies) were collected from the Clinical Hospital Sveti Duh, Department of Gynecology and Obstetrics, Zagreb, Croatia. Gestational age was determined according to the last menstrual period (LMP) and by ultrasound measurements. Expression of glycoproteins was measured by Western blotting with SNA, UEA-I, PHA-E and DBA lectins as probes whereas expression of AXIN1 was determined by immunohistochemistry. Results: Comparison of detected sugars revealed differences in protein glycosylation between normal and IUGR placentae. Higher expression of AXIN1 protein located mostly in the cytoplasm of syncytiotrophoblast and to a lesser extent in its nuclei was found in IUGR placentae. Conclusion: Results of our study suggest that changes in glycoprotein content may contribute to restricted placenta growth and development. Higher expression of AXIN1 protein in IUGR placentae indicates a role of Wnt/β-catenin signaling pathway in pathology of placental development.

ELF5 transcription factor expression during gestation in humans and rats - an immunohistochemical analysis.

Abstract Objective: The purpose of this study was to measure immunohistochemically the expression of ELF5 protein in term human and rat placentas and in human placentas associated with gestational diabetes (GD) and intrauterine growth restriction (IUGR). Methods: The results were quantitated stereologically using the stereological variable of volume density. A semiquantitative analysis was performed independently by a certified pathologist. Results: Total expression of ELF5 protein was higher in pathological pregnancies than in corresponding control term placentas, with both methods of quantifications showing similar results. In addition, ELF5 expression was also higher in connective tissue and blood vessels in chorionic villi in IUGR placentas (but not in GD placentas) compared to healthy controls. ELF5 is higher in placenta as a whole and in most of its components in both pathologies. The two exceptions are chorionic plates in IUGR and decidua in GD, where its expression is lower than in healthy controls. Conclusions: We have shown that IUGR and GD are associated with significantly increased levels of ELF5 protein in placentas, which suggests that ELF5 may play an important role in normal placentation. However, these are term placentas and to study ELF5 in premature births would give better insight into human placentation in health and disease.

The expression of SFRP1, SFRP3, DVL1, and DVL2 proteins in testicular germ cell tumors

Germ cell tumors of the testis are a heterogeneous group of neoplasms that affect male adolescents and young adults. Wnt signaling pathway components have been shown to be actively involved in normal and malignant germ cell differentiation and progression. In this study, we aimed to explore the expression patterns of the secreted frizzled‐related protein (SFRP) and Disheveled protein family (DVL) in a subset of testicular germ cell tumors. Eighty‐five formalin‐fixed, paraffin‐embedded tissue samples of the primary germ cell tumors of the testis were stained against SFRP1, SFRP3, DVL1, and DVL2 proteins using immunohistochemistry. SFRP1 and SFRP3 exhibited lower expression in both seminomas and mixed/non‐seminomatous tumors, compared with atrophic/benign tissue (p < 0.001). SFRP3 expression was lower than SFRP1 expression within the seminoma group (p = 0.004), but not within the mixed/non‐seminomatous group (p = 0.409). The majority of the tested cases (27/28, 96%) exhibited low DVL1 protein expression (median 0%, range 0–90%). In contrast, 20 out of 22 tested cases (91%) exhibited strong expression of DVL2 protein (median 80%, range 0–100%). No significant difference in DVL1 and DVL2 protein expression was observed between seminomas and mixed/non‐seminomatous tumors (p = 0.68 and 0.29). The secreted frizzled‐related protein and disheveled protein family members appear to be actively involved in the pathogenesis of primary testicular germ cell tumors.

Structural changes in the rat placenta during the last third of gestation discovered by stereology

Structural changes in the rat placenta during the last third of gestation were for the first time assessed by stereology. Fischer female rats were euthanized on the day 16 or day 19 of gestation, and 35 placentas were collected. Three randomly selected placentas from each group were stereologically analyzed for the absolute volume. The proportion of the glycogenic cells and the trophoblast giant cells (TGC) in the basal part of the placenta was calculated using volume density. The absolute volume of the rat placenta on the day 16 of gestation was determined as 0.0638 cm3. The labyrinth comprised 0.0274 cm3, the basal plate 0.0271 cm3 and the decidua 0.0093 cm3. On the day 19 of gestation, the absolute volume of the placenta was 0.1627 cm3, the labyrinth occupied 0.0922 cm3, the basal plate 0.0596 cm3 and the decidua 0.0109 cm3. The volume density of trophoblast giant cells was 0.174 cm0 on the day 16 and 0.107 cm0 on the day 19 of gestation. The glycogenic cells comprised 0.379 percentage of the basal plate on the day 16 and 0.236 on the day 19 of gestation. We conclude that the absolute volume of the whole placenta and the labyrinth has increased from day 16 to the day 19 of gestation. In contrast, the volume density of glycogenic cells and trophoblast giant cells was higher on the day 16 than on the day 19 of gestation, probably due to the intensive trophoblast invasion during that time.

T-cell factor 1 expression in germ cell tumors with trophoblastic differentiation.

To the Editor: T-cell factor 1 (TCF-1) protein is a member of the LEF1/TCF family of transcription factors, which are constituents of the Wnt signaling pathway, that plays an important role in embryonal development, adult stem cell maintenance and tumor growth. TCF-1 binds to the DNA through a high mobility group and at least eight protein isoforms. The canonical Wnt operates in two modes, on and off. In the off mode, the Wnt ligand is absent and cytoplasmic complex, composed of axin, adenomatous polyposis coli (APC), Casein kinase 1 (Ck1) and Glycogen Synthase Kinase (Gsk3), stimulates the destruction of β-catenin. Consequently, as β-catenin fails to enter the nucleus and activates T-cell transcription factors, the TCFs instead bind to a group of co-repressor proteins and inhibit transcription of β-catenin dependent genes. In the on mode, Wnt ligand binding with membrane receptor inhibits cytoplasmic destruction complex, which allows translocation of β-catenin into nucleus and binding with amino-terminal end of TCF. As a consequence of this interaction, TCFs become transcriptional activators which bind to Wnt response elements of target genes, and increased activation of this signaling pathway may lead to development of cancer. Previous studies have indicated that the Wnt signaling pathway plays an important role in balancing cell proliferation, apoptosis and differentiation during the trophoblast differentiation. The β-catenin-induced TCF4 protein activates the GCM1/syncictin signaling pathway, which leads to fusion of the human choriocarcinoma cells. Expression of Wnt antagonist Wnt5a, found normally in human cytotrophoblast cells, is completely absent from choriocarcinoma cell line JAR, whereas exogenous recombinant Wnt5a applied to choriocarcinoma cells decreases their proliferation and induces apoptosis, suggesting it acts as a tumor suppressor for placental choriocarcinomas. Hypermethylation of the APC suppressor gene has been associated with all human choriocarcinoma cell lines. Dickkopf-related protein 1 (DKK1), another Wnt signaling pathway antagonist, is abundantly present in human cytotrophoblast cells, but it is absent from choriocarcinoma cells lines JAR and JEG3. When given exogenously to cells, it reduces proliferation of both choriocarcinoma cells lines and induces apoptosis of JAR cells. This tumor suppressor effect is believed to be accomplished through c-Jun N-terminal kinase, without directly involving Wnt signaling pathway. TCF-1 stimulates the growth of human malignant hematopoietic cells independently of β-catenin binding, i.e. its activity is accomplished through binding to ATF2 [activating transcription factor 2] family of proteins. It is thought that this alternative mechanism is responsible for development of some hematopoietic tumors. Increased expression of TCF-1 protein has been reported recently in various human malignancies including renal cell carcinoma and hepatocellular tumors (adenoma and carcinoma). The status of TCF-1 protein has not been analyzed in germ cell tumors of gonads including the tumors with trophoblastic differentiation. In the present report using immunohistochemistry (Clone: sc-8589, Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:50) we explored TCF-1 expression in a subset of germ cell tumors with trophoblastic differentiation and compared it with adjacent normal/benign reproductive tissues (negative control) and tumor-infiltrating T-lymphocytes (positive control). TCF-1 protein was scored for the intensity and percentage in the choriocarcinoma cells. Tumor-infiltrating lymphocytes retained a strong, nuclear expression of TCF-1 protein (Fig. 1c), whereas normal and benign tissues (testis, ovary, endometrium and myometrium) were negative (Fig. 1a,c). Pure ovarian choriocarcinomas (n = 3) exhibited predominantly strong TCF-1 expression (score 3+) in the range 20–70% of the tumors cells (Fig. 1b). In mixed germ cell tumors of the testis (n = 5), choriocarcinoma cells also showed intense TCF-1 protein expression (Fig. 1d), whereas other germ cell compartments (seminoma, embryonal carcinoma, yolk sac tumor and teratoma) exhibited only weak (score 1+) and focal TCF-1 positivity (<10% of the cells) (Fig. 1c). Our preliminary data indicate that TCF-1 protein may be actively involved in pathogenesis of a subset of germ cell tumors with trophoblastic differentiation. Further functional and clinical studies should validate the relevance of TCF-1 protein in these tumors.