Dvl proteins regulate SMAD1, AHR, mTOR, BRD7 protein expression while differentially regulating canonical and non-canonical Wnt signaling pathways in CML cell lines.

Dishevelled (Dvl) is a scaffold protein that transmits Wnt signals to downstream effector molecules via both canonical and non-canonical Wnt signaling pathways. Deregulated activation of Dvl proteins has been reported in various solid tumors. However, it is not clear which pathway and proteins are responsible for observed aberrant activities and their relevance in disease prognosis. In addition, there is relatively limited knowledge on the role Dvl proteins may have in hematologic malignancy etiopathogenesis. In this study, we demonstrated that Dvl genes are not expressed in normal bone marrow but are expressed at different levels in the bone marrow of patients with chronic myeloid leukemia. We showed SMAD1, AHR, mTOR, BRD7 protein expressions are significantly affected by Dvl silencing and overexpression in CML cell lines. Wnt/ß-catenin and Wnt/PCP signaling pathway components are effectively repressed after Dvl silencing in K562 cells, while regulator of Wnt/Ca2+ signaling showed increase in both CML cell lines. Targeting Dvl proteins increases imatinib susceptibility of the K562 and MEG-01 cell lines. In light of our data, Dvl could be a potential therapeutic target in the treatment of CML.

sFRP1 Expression Regulates Wnt Signaling in Chronic Myeloid Leukemia K562 Cells.

BACKGROUND: Wnt signaling cascades play important roles in cell fate decisions and their deregulation has been documented in many diseases, including malignant tumors and leukemia. One mechanism of aberrant Wnt signaling is the silencing of Wnt inhibitors through epigenetic mechanisms. The sFRPs are one of the most studied Wnt inhibitors; and the sFRP1 loss is known in many hematological malignancies. Therefore, we aimed to compare the expression of Wnt related genes in the presence and absence of sFRP1 in a chronic myeloid leukemia (CML) cell line. OBJECTIVE: It is important to understand how sFRP1 and sFRP1 perform their effects on CML to design new agents and strategies for resistant and advanced forms of CML. MATERIALS AND METHODS: We used K562 cells, which normally do not express sFRP1 and its sFRP1 expressing subclone K562s. Total RNA was isolated from K562 and K562s cell lines and converted to cDNA. PCR Array experiments were performed using Human Wnt Signaling Pathway Plus RT2 Profiler™ kit. Wnt signaling pathway activation was studied by western blot for downstream signaling targets. RESULTS: The WNT3, LRP6, PRICKLE1 and BTRC expressions were significantly decreased in the presence of sFRP1; while WNT5B increased. The sFRP1 expression inhibited stabilization of total ß-catenin protein and downstream effector phosphorylation of noncanonical Wnt/PCP signaling; whereas Ca2+/PKC signaling remained active. CONCLUSION: The results suggest that sFRP1 could be a promising therapeutic anticancer agent. Defining these pathway interactions is crucial for designing new agents resistant and advanced forms of CML.

Secreted Wnt antagonists in leukemia: A road yet to be paved.

Wnt signaling has been a topic of research for many years for its diverse and fundamental functions in physiological (such as embryogenesis, organogenesis, proliferation, tissue repair and cellular differentiation) and pathological (carcinogenesis, congenital/genetic diseases, and tissue degeneration) processes. Wnt signaling pathway aberrations are associated with both solid tumors and hematological malignancies. Unregulated Wnt signaling observed in malignancies may be due to a wide spectrum of abnormalities, from mutations in the genes of key players to epigenetic modifications of Wnt antagonists. Of these, Wnt antagonists are gaining significant attention for their potential of being targets for treatment and inhibition of Wnt signaling. In this review, we discuss and summarize the significance of Wnt signaling antagonists in the pathogenesis and treatment of hematological malignancies.

Dishevelled proteins and CYLD reciprocally regulate each other in CML cell lines.

Dishevelled (Dvl) proteins are activated by Wnt pathway stimulation and have crucial roles in the regulation of ß-catenin destruction complex. CYLD is a tumor suppressor and a deubiquitination enzyme. CYLD negatively regulates the Wnt/ß-catenin signaling pathway by deubiquitinating Dvl proteins. Loss of function and mutations of CYLD were linked to different types of solid tumors. Loss of function in CYLD is associated with Dvl hyper ubiquitination, resulting in the transmission of Wnt signaling to downstream effectors. ß-catenin upregulation is observed during disease progression in chronic myeloid leukemia (CML). Deregulated Dvl signaling may be a reason for ß-catenin activation in CML; and CYLD may contribute to Dvl deregulation. First, we evaluated mRNA expression in three CML cell lines and mRNA expression of the CYLD gene was found to be present in all (K562, MEG01, KU812). Unlike solid tumors sequencing revealed no mutations in the coding sequences of the CYLD gene. DVL genes were silenced by using a pool of siRNA oligonucleotides and gene expression differences in CYLD was determined by RT-PCR and western blot. CYLD protein expression decreased after Dvl silencing. An opposite approach of overexpressing Dvl proteins resulted in upregulated CYLD expression. While previous reports have described CYLD as a regulator of DVL proteins; our data suggests the presence of a more complicated reciprocal regulatory mechanism in CML cell lines.

Forced expression of Wnt antagonists sFRP1 and WIF1 sensitizes chronic myeloid leukemia cells to tyrosine kinase inhibitors.

Chronic myeloid leukemia is a clonal myeloproliferative disorder that arises from the neoplastic transformation of the hematopoietic stem cell, in which the Wnt/ß-catenin signaling pathway has been demonstrated to play an important role in disease progression. However, the role of Wnt signaling antagonists in therapy resistance and disease progression has not been fully investigated. We aimed to study the effects of Wnt/ß-catenin pathway antagonists-secreted frizzled-related protein 1 and Wnt inhibitory factor 1-on resistance toward tyrosine kinase inhibitors in chronic myeloid leukemia. Response to tyrosine kinase inhibitors was analyzed in secreted frizzled-related protein 1 and Wnt inhibitory factor 1 stably transfected K562 cells. Experiments were repeated using a tetracycline-inducible expression system, confirming previous results. In addition, response to tyrosine kinase inhibitor treatment was also analyzed using the secreted frizzled-related protein 1 expressing, BCR-ABL positive MEG01 cell line, in the presence and absence of a secreted frizzled-related protein 1 inhibitor. Our data suggests that total cellular ß-catenin levels decrease in the presence of secreted frizzled-related protein 1 and Wnt inhibitory factor 1, and a significant increase in cell death after tyrosine kinase inhibitor treatment is observed. On the contrary, when secreted frizzled-related protein 1 is suppressed, total ß-catenin levels increase in the cell and the cells become resistant to tyrosine kinase inhibitors. We suggest that Wnt antagonists carry the potential to be exploited in designing new agents and strategies for the advanced and resistant forms of chronic myeloid leukemia.

An In-House Method for Molecular Monitoring of BCR-ABL.

OBJECTIVE: At present, there are a limited number of facilities in Turkey that can provide reliable real-time quantitative(RQ)-PCR BCR-ABL results. The present study aimed to test a cost-effective, in-house method of BCR-ABL quantification,including verification of the method by RQ-PCR validation tests. MATERIAL AND METHODS: BCR-ABL and ABL target sequences were cloned into pJET1.2 vectors, from whichcalibrators were prepared and used as templates in RQ-PCR reactions to generate standard curves. Dilutions of K562cells (representing an in vitro simulation of BCR-ABL transcript reduction) were analyzed. RESULTS: Standard curves were generated from calibrators. These curves were then used to calculate the BCR-ABL andABL copy numbers; in which linear BCR-ABL results were obtained. Repetitive experiments showed that our methodologywas able to detect 1 BCR-ABL positive cell from amnong 1x105 cells. CONCLUSION: The method described herein is suitable for implementation with any RQ-PCR instrument and/or kit forquantify BCR-ABL transcripts. CONFLICT OF INTEREST: None declared.