Fibroblast Growth Factor Receptor Inhibitors Decrease Proliferation of Melanoma Cell Lines and Their Activity Is Modulated by Vitamin D.

Regardless of the unprecedented progress in malignant melanoma treatment strategies and clinical outcomes of patients during the last twelve years, this skin cancer remains the most lethal one. We have previously documented that vitamin D and its low-calcaemic analogues enhance the anticancer activity of drugs including a classic chemotherapeutic-dacarbazine-and an antiangiogenic VEGFRs inhibitor-cediranib. In this study, we explored the response of A375 and RPMI7951 melanoma lines to CPL304110 (CPL110), a novel selective inhibitor of fibroblast growth factor receptors (FGFRs), and compared its efficacy with that of AZD4547, the first-generation FGFRs selective inhibitor. We also tested whether 1,25(OH)2D3, the active form of vitamin D, modulates the response of the cells to these drugs. CPL304110 efficiently decreased the viability of melanoma cells in both A375 and RPMI7951 cell lines, with the IC50 value below 1 µM. However, the metastatic RPMI7951 melanoma cells were less sensitive to the tested drug than A375 cells, isolated from primary tumour site. Both tested FGFR inhibitors triggered G0/G1 cell cycle arrest in A375 melanoma cells and increased apoptotic/necrotic SubG1 fraction in RPMI7951 melanoma cells. 1,25(OH)2D3 modulated the efficacy of CPL304110, by decreasing the IC50 value by more than 4-fold in A375 cell line, but not in RPMI7951 cells. Further analysis revealed that both inhibitors impact vitamin D signalling to some extent, and this effect is cell line-specific. On the other hand, 1,25(OH)2D3, have an impact on the expression of FGFR receptors and phosphorylation (FGFR-Tyr653/654). Interestingly, 1,25(OH)2D3 and CPL304110 co-treatment resulted in activation of the ERK1/2 pathway in A375 cells. Our results strongly suggested possible crosstalk between vitamin D-activated pathways and activity of FGFR inhibitors, which should be considered in further clinical studies.

Identification and characterization of aptameric inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.

Preclinical characterization of CPL304110 as a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3 for gastric, bladder, and squamous cell lung cancer.

Fibroblast Growth Factor Receptors (FGFRs) are a family of receptor tyrosine kinases expressed on a plethora of cell membranes. They play crucial roles in both embryonic development and adult tissue functions. There is an increasing amount of evidence that FGFR-mediated oncogenesis is mainly related to gene amplification, activating mutations, or translocation in tumors of various histological types. Dysregulation of FGFRs has been implicated in a wide variety of neoplasms, such as bladder, gastric, and lung cancers. Given their functional significance, FGFRs emerge as promising targets for cancer therapy. Here, we introduce CPL304100, an innovative and highly potent FGFR1-3 kinase inhibitor demonstrating excellent in vitro biological activity. Comprehensive analyses encompassed kinase assays, cell line evaluations, PK/PD studies surface plasmon resonance studies, molecular docking, and in vivo testing in mouse xenografts. CPL304110 exhibited a distinctive binding profile to FGFR1/2/3 kinase domains, accompanied by a good safety profile and favorable ADMET parameters. Selective inhibition of tumor cell lines featuring active FGFR signaling was observed, distinguishing it from cell lines lacking FGFR aberrations (FGFR1, 2, and 3). CPL304110 demonstrated efficacy in both FGFR-dependent cell lines and patient-derived tumor xenograft (PDTX) in vivo models. Comparative analyses with FDA-approved FGFR inhibitors, erdafitinib and pemigatinib, revealed certain advantages of CPL304110 in both in vitro and in vivo assessments. Encouraging preclinical results led the way for the initiation of a Phase I clinical trial (01FGFR2018; NCT04149691) to further evaluate CPL304110 as a novel anticancer therapy.

FGFR1-4 RNA-Based Gene Alteration and Expression Analysis in Squamous Non-Small Cell Lung Cancer.

While fibroblast growth factor receptors (FGFRs) are involved in several biological pathways and FGFR inhibitors may be useful in the treatment of squamous non-small cell lung cancer (Sq-NSCLC), FGFR aberrations are not well characterized in Sq-NSCLC. We comprehensively evaluated FGFR expression, fusions, and variants in 40 fresh-frozen primary Sq-NSCLC (stage IA3−IV) samples and tumor-adjacent normal tissues using real-time PCR and next-generation sequencing (NGS). Protein expression of FGFR1−3 and amplification of FGFR1 were also analyzed. FGFR1 and FGFR4 median gene expression was significantly (p < 0.001) decreased in tumors compared with normal tissue. Increased FGFR3 expression enhanced the recurrence risk (hazard ratio 4.72, p = 0.029), while high FGFR4 expression was associated with lymph node metastasis (p = 0.036). Enhanced FGFR1 gene expression was correlated with FGFR1 protein overexpression (r = 0.75, p = 0.0003), but not with FGFR1 amplification. NGS revealed known pathogenic FGFR2,3 variants, an FGFR3::TACC3 fusion, and a novel TACC1::FGFR1 fusion together with FGFR1,2 variants of uncertain significance not previously reported in Sq-NSCLC. These findings expand our knowledge of the Sq-NSCLC molecular background and show that combining different methods increases the rate of FGFR aberrations detection, which may improve patient selection for FGFRi treatment.

Two-Front War on Cancer-Targeting TAM Receptors in Solid Tumour Therapy.

Receptor tyrosine kinases (RTKs) are transmembrane receptors that bind growth factors and cytokines and contain a regulated kinase activity within their cytoplasmic domain. RTKs play an important role in signal transduction in both normal and malignant cells, and their encoding genes belong to the most frequently affected genes in cancer cells. The TAM family proteins (TYRO3, AXL, and MERTK) are involved in diverse biological processes: immune regulation, clearance of apoptotic cells, platelet aggregation, cell proliferation, survival, and migration. Recent studies show that TAMs share overlapping functions in tumorigenesis and suppression of antitumour immunity. MERTK and AXL operate in innate immune cells to suppress inflammatory responses and promote an immunosuppressive tumour microenvironment, while AXL expression correlates with epithelial-to-mesenchymal transition, metastasis, and motility in tumours. Therefore, TAM RTKs represent a dual target in cancer due to their intrinsic roles in tumour cell survival, migration, chemoresistance, and their immunosuppressive roles in the tumour microenvironment (TME). In this review, we discuss the potential of TAMs as emerging therapeutic targets in cancer treatment. We critically assess and compare current approaches to target TAM RTKs in solid tumours and the development of new inhibitors for both extra- and intracellular domains of TAM receptor kinases.

p38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer.

FGFR signalling is one of the most prominent pathways involved in cell growth and development as well as cancer progression. FGFR1 amplification occurs in approximately 20% of all squamous cell lung carcinomas (SCC), a predominant subtype of non-small cell lung carcinoma (NSCLC), indicating FGFR as a potential target for the new anti-cancer treatment. However, acquired resistance to this type of therapies remains a serious clinical challenge. Here, we investigated the NSCLC cell lines response and potential mechanism of acquired resistance to novel selective FGFR inhibitor CPL304110. We found that despite significant genomic differences between CPL304110-sensitive cell lines, their resistant variants were characterised by upregulated p38 expression/phosphorylation, as well as enhanced expression of genes involved in MAPK signalling. We revealed that p38 inhibition restored sensitivity to CPL304110 in these cells. Moreover, the overexpression of this kinase in parental cells led to impaired response to FGFR inhibition, thus confirming that p38 MAPK is a driver of resistance to a novel FGFR inhibitor. Taken together, our results provide an insight into the potential direction for NSCLC targeted therapy.

Antiparkinsonian-like effects of CPL500036, a novel selective inhibitor of phosphodiesterase 10A, in the unilateral rat model of Parkinson's disease.

Phosphodiesterase 10A (PDE10A), the enzyme which catalyzes hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is located almost exclusively in striatal γ-amino-butyric acid (GABA)ergic medium spiny neurons (MSNs). Since dopaminergic deficiency in Parkinson's disease (PD) leads to functional imbalance of striatal direct and indirect output pathways formed by MSNs, PDE10A seems to be of special interest as a potential therapeutic target in PD. The aim of the present study was to examine the influence of 7-{5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-phenylimidazo[1,2-a]pyrimidine (CPL500036), a novel selective inhibitor of PDE10A, on sensorimotor deficits and therapeutic effects of L-3,4-dihydroxyphenylalanine (L-DOPA) in hemiparkinsonian rats. Animals were unilaterally lesioned with 6-hydroxydopamine, and their sensorimotor deficits were examined in the stepping, cylinder, vibrissae and catalepsy tests. CPL500036 (0.1 and 0.3 mg/kg) was administered either acutely or chronically (2 weeks), alone or in combination with L-DOPA/benserazide (6 mg/kg/6 mg/kg). Acute treatment with CPL500036 reversed the lesion-induced impairments of contralateral forelimb use in the stepping and cylinder tests but did not influence deficits in the vibrissae test and the lesion-induced catalepsy. Moreover, CPL500036 did not diminish the therapeutic effects produced by acute and chronic treatment with L-DOPA in these tests. The present study suggests a potential use of CPL500036 as a co-treatment to L-DOPA in PD therapy.

MET-Pyk2 Axis Mediates Acquired Resistance to FGFR Inhibition in Cancer Cells.

Deregulation of fibroblast growth factor receptors (FGFRs) signaling, as a result of FGFR amplification, chromosomal translocation, or mutations, is involved in both initiation and progression of a wide range of human cancers. Clinical data demonstrating the dependence of cancer cells on FGFRs signaling clearly indicate these receptors as the molecular targets of anti-cancer therapies. Despite the increasing number of tyrosine kinase inhibitors (TKIs) being investigated in clinical trials, acquired resistance to these drugs poses a serious therapeutic problem. In this study, we focused on a novel pan-FGFR inhibitor-CPL304110, currently being investigated in phase I clinical trials in adults with advanced solid malignancies. We analyzed the sensitivity of 17 cell lines derived from cancers with aberrant FGFR signaling, i.e. non-small cell lung cancer, gastric and bladder cancer to CPL304110. In order to explore the mechanism of acquired resistance to this FGFR inhibitor, we developed from sensitive cell lines their variants resistant to CPL304110. Herein, for the first time we revealed that the process of acquired resistance to the novel FGFR inhibitor was associated with increased expression of MET in lung, gastric, and bladder cancer cells. Overexpression of MET in NCI-H1703, SNU-16, RT-112 cells as well as treatment with HGF resulted in the impaired response to inhibition of FGFR activity. Moreover, we demonstrated that cells with acquired resistance to FGFR inhibitor as well as cells overexpressing MET displayed enhanced migratory abilities what was accompanied with increased levels of Pyk2 expression. Importantly, inhibition of both MET and Pyk2 activity restored sensitivity to FGFR inhibition in these cells. Our results demonstrate that the HGF/MET-Pyk2 signaling axis confers resistance to the novel FGFR inhibitor, and this mechanism is common for lung, gastric, and bladder cancer cells. Our study suggests that targeting of MET/Pyk2 could be an approach to overcome resistance to FGFR inhibition.

Discovery and optimization of novel pyrazole-benzimidazole CPL304110, as a potent and selective inhibitor of fibroblast growth factor receptors FGFR (1-3).

The FGFR family is characterized by four receptors (FGFR 1-4), binding to 18 ligands called fibroblast growth factors (FGFs). Aberrant activation of FGFs and their FGFRs has been implicated in a broad spectrum of human tumors. We employed the scaffolds hybridization approach, scaffold-hopping concept to synthesize a series of novel pyrazole-benzimidazole derivatives 56 (a-x). Compound 56q (CPL304110) was identified as a selective and potent pan-FGFR inhibitor for FGFR1, -2, -3 with IC50s of 0.75 nM, 0.50 nM, 3.05 nM respectively, whereas IC50 of 87.90 nM for FGFR4. Due to its favorable pharmacokinetic profile, low toxicity and potent anti-tumor activity in vivo, compound 56q is currently under evaluation in phase I clinical trial for the treatment of bladder, gastric and squamous cell lung cancers (01FGFR2018; NCT04149691).

Virtual screening-driven discovery of dual 5-HT6/5-HT2A receptor ligands with pro-cognitive properties.

A virtual screening campaign aimed at finding structurally new compounds active at 5-HT6R provided a set of candidates. Among those, one structure, 4-(5-{[(2-{5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl}ethyl)amino]methyl}furan-2-yl)phenol (1, 5-HT6R Ki = 91 nM), was selected as a hit for further optimization. As expected, the chemical scaffold of selected compound was significantly different from all the serotonin receptor ligands published to date. Synthetic efforts, supported by molecular modelling, provided 43 compounds representing different substitution patterns. The derivative 42, 4-(5-{[(2-{5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl}ethyl)amino]methyl}furan-2-yl)phenol (5-HT6R Ki = 25, 5-HT2AR Ki = 32 nM), was selected as a lead and showed a good brain/plasma concentration profile, and it reversed phencyclidine-induced memory impairment. Considering the unique activity profile, the obtained series might be a good starting point for the development of a novel antipsychotic or antidepressant with pro-cognitive properties.

2-Aminoimidazole-based antagonists of the 5-HT6 receptor - A new concept in aminergic GPCR ligand design.

A new strategy in the design of aminergic GPCR ligands is proposed - the use of aromatic, heterocyclic basic moieties in place of the evergreen piperazine or alicyclic and aliphatic amines. This hypothesis has been tested using a benchmark series of 5-HT6R antagonists obtained by coupling variously substituted 2-aminoimidazole moieties to the well established 1-benzenesulfonyl-1H-indoles, which served as the ligands cores. The crystallographic studies revealed that upon protonation, the 2-aminoimidazole fragment triggers a resonance driven conformational change leading to a form of higher affinity. This molecular switch may be responsible for the observed differences in 5-HT6R activity of the studied chemotypes with different amine-like fragments. Considering the multiple functionalization sites of the embedded guanidine fragment, diverse libraries were constructed, and the relationships between the structure and activity, metabolic stability, and solubility were established. Compounds from the N-(1H-imidazol-2-yl)acylamide chemotype (10a-z) exhibited high affinity for 5-HT6R and very high selectivity over 5-HT1A, 5-HT2A, 5-HT7 and D2 receptors (negligible binding), which was attributed to their very weak basicity. The lead compound in the series 4-methyl-5-[1-(naphthalene-1-sulfonyl)-1H-indol-3-yl]-1H-imidazol-2-amine (9i) was shown to reverse the cognitive impairment caused by the administration of scopolamine in rats indicating procognitive potential.

Epithelial-mesenchymal transition confers resistance to selective FGFR inhibitors in SNU-16 gastric cancer cells.

BACKGROUND: Up to 10 % of primary gastric cancers are characterized by FGFR2 amplification, and fibroblast growth factor receptor (FGFR) inhibitors may represent therapeutic agents for patients with these malignancies. However, long-term benefits of the treatment might be limited owing to the occurrence of drug resistance. METHODS: To investigate the mechanisms of resistance to selective FGFR inhibitors, we established three FGFR2-amplified SNU-16 gastric cancer cell lines resistant to AZD4547, BGJ398, and PD173074, respectively. RESULTS: The resistant cell lines (SNU-16R) demonstrated changes characteristic of epithelial-to-mesenchymal transition (EMT). In addition, they displayed loss of expression of FGFR2 and other tyrosine kinase receptors concurrent with activation of downstream signaling proteins and upregulation of the transforming growth factor ß (TGF-ß) level. However, treatment of parental SNU-16 cells with TGF-ß1 did not evoke EMT, and pharmacological inhibition of TGF-ß receptor I was not sufficient to reverse EMT changes in the resistant cells. Finally, we showed that the SNU-16R cell lines were sensitive to the human epidermal growth factor receptor 2 inhibitor mubritinib and the heat shock protein 90 inhibitor AUY922. CONCLUSION: In conclusion, we provide experimental evidence that EMT-mediated resistance might emerge in gastric cancer patients following treatment with FGFR inhibitors, and mubritinib or AUY922 treatment may be an alternative therapeutic strategy for these patients.

Differences in gene expression and alterations in cell cycle of acute myeloid leukemia cell lines after treatment with JAK inhibitors.

Janus kinase (JAK) inhibitors are a promising treatment strategy in several hematological malignancies and autoimmune diseases. A number of inhibitors are in clinical development, and two have already reached the market. Unfortunately, all of them are burdened with different toxicity profiles. To check if the JAK inhibitors of different selectivity evoke different responses on JAK2-dependent and independent cells, we have used three acute myeloid leukemia cell lines with confirmed JAK2 mutation status. We have found that JAK inhibitors exert distinct effect on the expression of BCLXL, CCND1 and c-MYC genes, regulated by JAK pathway, in JAK2 wild type cells in comparison to JAK2 V617F-positive cell lines. Moreover, cell cycle analysis showed that inhibitors alter the cycle by arresting cells in different phases. Our results suggest that observed effect of JAK2 inhibitors on transcription and cell cycle level in different cell lines are associated not with activity within JAK family, but presumably with other off-target activities.

Cell-based assay for low- and high-scale screening of the Wnt/ß-catenin signaling modulators.

Deregulation of the Wnt/ß-catenin signaling pathway is associated with many serious disorders, including cancer and Alzheimer's disease. The pivotal player is ß-catenin, which avoids degradation after activation of the pathway and is translocated to the nucleus, where it interacts with TCF/LEF transcription factors and induces expression of genes involved in cell cycle and apoptosis regulation. The identification of small molecules that may affect Wnt/ß-catenin signaling remains an important target during the development of novel therapies. We used the TCF/LEF lentiviral vector and the Wnt-independent H1703 cell line to develop a luciferase reporter-based cell assay for screening of the Wnt/ß-catenin pathway modulators. Following the optimization of cell density, concentration of activator, and stimulation time, the reporter system was validated by demonstrating its specific and dose-dependent response to several established modulators of Wnt/ß-catenin signaling such as Wnt3a, small interfering RNA (siRNA) against ß-catenin, glycogen synthase kinase 3 (GSK-3), and ß-catenin/TCF transcription complex inhibitors. Two pilot screens of inhibitors and activators of Wnt/ß-catenin signaling identified potential novel modulators of this pathway. Our findings suggest that the H1703-7TFP assay constitutes a suitable model of low background and high sensitivity for the low- and high-scale screening of the Wnt/ß-catenin pathway modulators.

Wnt/ß-catenin pathway as a potential prognostic and predictive marker in patients with advanced ovarian cancer.

BACKGROUND: ß-catenin is the key protein in the WNT signalling pathway and it forms adherent junctions together with E-cadherin. In ovarian carcinoma, abnormal expression of ß-catenin, E-cadherin and WNT-1 was observed, but their prognostic and predictive role is unclear. The aim of this study was to clarify the prognostic and predictive role of E-cadherin, ß-catenin and WNT-1 in advanced epithelial ovarian carcinoma (AEOC). METHODS: The expression of E-cadherin, ß-catenin and WNT-1 was determined by immunohistochemistry in AEOC. The correlation between expression of these proteins and progression-free survival (PFS) and overall survival (OS) was evaluated. Statistical analyses included Kaplan-Meier estimation, log-rank test, Spearman correlation and Cox proportional-hazards model. RESULTS: In ovarian cancer, intense expression of E-cadherin, ß-catenin and WNT-1 was found. In multivariate analysis, strong membrane ß-catenin expression was an independent unfavourable predictor for PFS (HR 2.19, 95% CI 1.09-4.39; p = 0.028), while in univariate analysis, strong membrane ß-catenin expression was a prognostic factor for OS in patients with AOC (p = 0.039). In multivariate analysis, only resistance to first-line chemotherapy was an adverse independent prognostic factor for OS (HR 16.84; 95% CI 5.07-55.98; p < 0.0001). Additionally, strong membranous ß-catenin expression was associated with resistance to platinum-based chemotherapy (p = 0.027). CONCLUSIONS: These findings support that WNT/ß-catenin pathway and E-cadherin are important factors in advanced epithelial ovarian cancer.

Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma.

PURPOSE: Crizotinib, the first FDA-approved ALK inhibitor, showed significant antitumor activity in young patients with anaplastic large-cell lymphoma (ALCL) frequently displaying ALK rearrangement. However, long-term therapeutic benefits of crizotinib are limited due to development of drug resistance. CH5424802--more potent and selective ALK inhibitor--comprises a good candidate for second-line treatment in crizotinib-relapsed patients. The aim of this study was to determine possible mechanisms of resistance to ALK inhibitors that can appear in ALCL patients. METHODS: ALK+ ALCL cell lines resistant to crizotinib (Karpas299CR) and to CH5424802 (Karpas299CHR) were established by long-term exposure of Karpas299 cells to these inhibitors. Next, alterations in their sensitivity to ALK, HSP90 and mTOR inhibitors were investigated by cell viability and BrdU incorporation assays and immunoblot analysis. RESULTS: cDNA sequencing of ALK kinase domain revealed activating mutations-I1171T in Karpas299CR and F1174C in Karpas299CHR. The resistant cells displayed diminished sensitivity to structurally unrelated ALK inhibitors-crizotinib, CH5424802 and TAE684. Nevertheless, CH5424802 and TAE684 were still more potent against the resistant cells than crizotinib. Moreover, Karpas299CR and Karpas299CHR cells remained sensitive to HSP90 or mTOR inhibitors. CONCLUSIONS: Resistance mediated by activating mutations in ALK kinase domain may emerge in ALCL patients during ALK inhibitors treatment. However, more potent second-generation ALK inhibitors, HSP90 or mTOR inhibitors may represent an effective therapy for relapsed ALK+ ALCL patients.

Transcriptional expression of selected genes associated with excretion of carboxylic acids from aci mutants of Saccharomyces cerevisiae.

INTRODUCTION: Saccharomyces cerevisiae is an excellent model organism for studies of transcriptional regulation of metabolic processes in other eukaryotic cells including human cells. Cellular acid-base balance can be disturbed in pathologic situations such as renal acidosis or cancer. The extracellular pH of malignant solid tumors is acidic in the range of 6.5-6.9. EG07 and EG37 aci mutants of Saccharomyces cerevisiae excessively excrete carboxylic acids to glucose-containing media or distilled water. The excreted acids are Krebs and/or glyoxylate cycle intermediates. The genes restoring the wild-type phenotype have function that does not easily explain theAci+ phenotype. MATERIAL/METHODS: In this study, using real-time PCR we measured relative mRNA expression, in the mutants compared to the wild-type strain, of selected genes associated with both carboxylic acid cycles and two cell transporters, Pma1 and Pdr12, of organic acids. RESULTS: Unexpectedly, we found that the relative expression of the selected Krebs cycle and glyoxylate cycle genes did not change significantly. However, the expression of the two transporter genes was strongly elevated in EG37 and moderately increased in EG07. CONCLUSION: These results indicate that the induction of the two cell transporterg enes plays an important role in acid excretion by the aci mutants.

[Role of CEACAM in neutrophil activation]. / Rola CEACAM w aktywacji granulocytów obojetnochlonnych.

 Neutrophils express many surface adhesion molecules, including CEACAM1, CEACAM3, CEACAM4, CEACAM6 and CEACAM8 glycoproteins, which play an important role in biological functions of neutrophils such as adhesion, phagocytosis, oxidative burst and degranulation. CEACAM3 activates neutrophils and initiates phagocytosis as a result of binding to bacterial Opa protein. In addition, CEACAM1 and CEACAM6 can delay apoptosis. All neutrophil CEACAMs, except for CEACAM3, can stimulate adhesion of neutrophils to endothelium. One CEACAM family member, CEA, which is not expressed by neutrophils, displays strong chemotactic activity, and probably can prime and/or activate neutrophils to adhesion. Induction of CEACAM signaling can be initiated by dimerization of CEACAMs and/or phosphorylation of their cytoplasmic domains. CEACAM signaling is often associated with an increase in the cytoplasmic calcium level.

Prognostic significance of Wnt-1, ß-catenin and E-cadherin expression in advanced colorectal carcinoma.

Wnt/ß-catenin pathway plays an important role in initiation and progression of colorectal oncogenesis. The aim of this study was to determine expression and localization of E-cadherin, ß-catenin and Wnt-1 proteins in colorectal tumors. Expression of ß-catenin, E-cadherin and Wnt-1 was determined by immunohistochemistry on advanced colorectal cancers. Abnormal expression of E-cadherin, ß-catenin, Wnt-1 was observed. Additionally, we revealed correlations between levels of studied proteins and histoclinical data. In multivariate analysis nuclear ß-catenin, higher carcinoembryonic antigen serum level before treatment, female sex and tumor localized in colon or rectum were independent unfavorable prognostic factors. These findings support the hypothesis that Wnt/ß-catenin pathway plays an important role in advanced colorectal carcinoma.

Silencing of Wnt-1 by siRNA induces apoptosis of MCF-7 human breast cancer cells.

OBJECTIVE: Wnt family of secreted-type glycoproteins plays key role in carcinogenesis and embryogenesis. Signals of Wnts are transduced through seven-transmembrane-type Wnt receptors encoded by Frizzled (Fzd) genes to the beta-catenin-Tcf pathway, the c-Jun-N-terminal kinase (JNK) pathway or the Ca(2+)-releasing pathway. Aberrant activation of the Wnt/beta-catenin signaling pathway is associated with a variety of human cancers. In human breast cancer, evidence of beta-catenin accumulation implies that the canonical Wnt signaling pathway is active in over 50% of carcinomas. RESULTS: We found that in breast cancer cells overexpressing Wnt-1 siRNA anti-Wnt-1 induced apoptosis and caused changes in downstream proteins levels. Among treated cells there were 71% apoptotic cells in comparison to cells treated with scrambled siRNA (6%) and control cells (6%) after 48 h (p < 0.01). METHODS: To examine if Wnt-1 signal is essential for cancer cell survival, we investigated the effect of Wnt-1 gene silencing in triggering of apoptosis in MCF-7 breast cancer cell line. Light microscopy, viability/cytotoxicity tests, flow cytometry, real-time PCR and western blotting were used for evaluation of the morphological features of cell death, percentage of apoptotic cells, Wnt-1 mRNA and protein level. CONCLUSION: Our results significantly indicate that anti-Wnt-1 siRNA inhibits Wnt-1 signaling, inducing apoptosis in human breast cancer MCF-7 cells and thus may serve as a potential anti-cancer drug.