Faculty Opinions recommendation of Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling.

Lu Xiao, Haiqing Bai, James Boyer, Bo Ye, Ning Hou, Haodong Xu, and Faqian Li Department of Pathology and Laboratory Medicine and Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Backgrounds: Canonical Wnt signaling appears to have multiphasic and often antagonistic roles in cardiac development. The molecular mechanism for these opposing actions is not clear. We hypothesized that alternative splicing of TCF7L2, a nuclear interaction partner of beta-catenin is involved in the specificity of canonical Wnt signaling. Methods: RT-PCR were performed on embryonic (E16.5) and neonatal (day 8) hearts with primers spanning the end of first exon and the beginning of last exon and the products were cloned and sequenced. Result: There are totally 18 exons identified so far in TCF7L2. We sequenced 56 clones and 53 clones (29 from day 8) and (24 from E16.5) contained TCF7L2 sequences. No exon 6 or exon 17 was found in TCF7L2 transcripts of mouse hearts. Most clones (more than 80%) from E16.5 and day 8 hearts excluded exon 4. Both E16.5 and day 8 hearts had one clone with exon 9 deletion which does not change reading frame and another with alterations in exon 3 that lead to reading frame shift and premature stop codon. As reported in other organs, there were extensive alternative splicing in the C-terminal exons 14, 15 and 16. The inclusion of exon 14 was more frequently in day 8 (18 of 29, 62%) than in E16.5 (8 of 24, 33%) hearts. The peptide encoded by exon 14 has conserved functional motif. Additionally, this alternative exon usage can change the C-terminus of TCF7L2 to include or exclude the so-called E tail with two binding motifs for C-terminal binding protein. Conclusion: The isoform switch of TCF7L2 occurs in neonatal mouse hearts and may have a role in the terminal differentiation of cardiac myocytes during this period.

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Beta-Catenin Depended and Independent Effects Induced by Myeloma Cells in Human and Murine Osteoblasts and Osteoblast Progenitors.

Blood ◽

10.1182/blood.v108.11.3433.3433 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3433-3433

Author(s):

Francesca Morandi ◽

Sara Tagliaferri ◽

Sabrina Bonomini ◽

Mirca Lazzaretti ◽

Luca Ferrari ◽

...

Keyword(s):

Wnt Signaling ◽

Cell Lines ◽

Osteoblastic Cells ◽

Beta Catenin ◽

Canonical Wnt Signaling ◽

Osteoprogenitor Cell ◽

Significant Difference ◽

Bone Biopsies ◽

Canonical Wnt ◽

Osteoblast Progenitors

Abstract Osteoblast impairment occurs within myeloma (MM) cell infiltration into the bone marrow (BM). Wnt signaling is involved in the regulation of osteoblast formation. Canonical Wnt signaling pathway is activated by Wnt 1/3a that induce the activation of GSK3/Axin complex leading to the stabilization and nuclear translocation of beta-catenin that in turn activates the transcription system Lef1/TCF. Recently it has been reported that MM cells produce the Wnt inhibitors DKK-1 demonstrating a correlation between its expression and the presence of bone lesions in MM patients. However the effect of MM cells on Wnt signaling cascade in osteoblasts and osteoblast progenitors has not been investigated. To clarify this issue, first we checked DKK-1 production by human myeloma cell lines (HMCLs), purified CD138+ MM cells and BM plasma of MM patients by PCR and ELISA. Following we performed a co-culture system with HMCLs or CD138+ MM cells and either human osteoblast line (HOBIT) and with BM osteoprogenitor cells (PreOB) obtained after differentiation from mesenchymal cells or murine osteoprogenitor cell lines C2C12 and MC3T3. Both DKK-1 positive HMCLs (XG-1 and JJN3) and negative ones (RPMI-8226, OPM-2) have been used in co-culture as well as DKK-1 positive and negative purified CD138+ MM cells. Similarly we tested the effect of BM plasma of MM patients positive and negative for DKK-1 production on both human and murine cells. Wnt signaling in osteoblasts and osteoblast progenitors was evaluated either at mRNA level by specific human and murine Wnt Array kits and by quantitative PCR or at protein one by Western blot analysis for GSK3b/Axin and LEF-1/TCF expression. We evaluated active de-phosphorylated beta-catenin and inactive phosphorilated one by westernblot and by ELISA in cytosolic and nuclear extracts. DKK-1 median levels detected in the conditioned media of XG-1 and JJN3, MM cells and in BM plasma of DKK-1 positve MM patients were 0.60 ng/mL and 0.38 and 8.84 (range: 1.55–91) ng/mL respectively. Any significant inhibitory effect on WNT signaling and active beta-catenin expression and levels was not observed in HOBIT and human PreOB after co-culture with both HMCLs and MM cells or BM plasma independently to DKK-1 expression. On the contrary DKK-1 positive MM cells or BM plasma suppressed active beta-catenin expression in murine osteoprogenitor cell lines in presence of BMP-2. Consistently Wnt3a stimulation as well as anti-DKK-1 abs. did not restore the inhibitory effects on osteoblast formation and differentiation induced by MM cells in human PreOB. Consistently any significant difference was not detected on beta-catenin expression by stromal/osteoblastic cells on bone biopsies by immunohistochemistry between osteolytic (n°=10) and non-osteolytic (N°=10) MM patients. The different behavior between human and murine osteoblastic cells was further investigated. We found that both cells expressed significant levels of active beta-catenin however DKK-1 suppressed active nuclear and cytosol beta-catenin at concentration of 20–30 ng/mL in C2C12 and MC3T3 whereas only DKK-1 concentrations higher to 500 ng/mL are able to inhibited beta-catenin in HOBIT and human PreOB as well as osteoblast formation and differentiation in human BM cultures. In conclusion our data indicate that MM cells block canonical Wnt signaling in murine osteoblastic cells but not in human osteoblasts and osteoblast progenitors. Beta-catenin independent mechanisms could be involved in DKK-1 mediated bone destruction in MM patients.

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Expression of Wnt-3a in Myeloma Cells Inhibits the Osteolytic Phenotype In-Vivo.

Blood ◽

10.1182/blood.v108.11.3420.3420 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3420-3420

Author(s):

Ya-Wei Qiang ◽

Shmuel Yaccoby ◽

John D. Shaughnessy

Keyword(s):

Bone Resorption ◽

Wnt Signaling ◽

Myeloma Cell ◽

Beta Catenin ◽

Canonical Wnt Signaling ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Canonical Wnt

Wnt signaling is a highly conserved signal transduction pathway involved in embryonic development. Inappropriate canonical Wnt signaling resulting in beta-catenin stabilization, is associated with several types of human cancers. Multiple myeloma plasma cells express Wnt receptors, Wnt ligands and soluble Wnt inhibitors. Wnt signaling is central to osteoblast and osteoclasts development and secretion of Wnt signaling inhibitors by myeloma cells is thought to contribute to the osteolytic phenotype seen in this disease and prostate cancer. While it is now clear that MM cells can signal through both canonical and non-canonical mechanisms, there are conflicting data as to the direct role of Wnt signaling in myeloma cell biology. Others have shown that Wnts cause proliferation of myeloma cells; while we have shown that canonical Wnts cause morphological changes and migration, but not cell proliferation. To further elucidate the role of canonical Wnt signaling in myeloma and myeloma bone disease we used limiting dilutions in the presence of G418 to create two independent stable clones of the myeloma cell line NCI-H929 expressing Wnt-3A (H929/W3A), which is not expressed in myeloma, and an empty vector (H929/EV). Because Wnt antibodies are not available we cloned Wnt-3A as a fusion protein with hemagglutinin (HA). Western blots against HA revealed a positive band of the expected size only in the H929/W3A clones. GST-E-cadherin binding assay and Western blot analysis revealed elevated levels of total and free beta-catenin in H929/W3A relative to H929/EV, however, there this was not associated with increased growth or proliferation by MTT assay. To determine the in-vivo growth characteristics and effects on bone resorption of Wnt-3A producing cells, we transplanted the lines into a human bone implanted the flank of SCID mice. Tumor growth rate as determined by increased production of human immunoglobulin in mice serum was significantly slower in the Wnt-3A transfected cells relative to controls (P < .05). Loss of bone mineral density (BMD) of the implanted bones engrafted with H929/W3A cells was lower than in bones engrafted with H929/EV cells (P < .05). Reduced tumor burden and BMD loss was also visualized on x-ray radiographs. Taken together these data indicate that all factors promoting bone resorption produced by or elicited by the myeloma cell line H929 are subordinate to canonical Wnt signaling and that prevention of bone destruction may help control myeloma progression.

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cfDNA analysis of mCRPC patients expressing mutations in Wnt signaling.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.6_suppl.256 ◽

2018 ◽

Vol 36 (6_suppl) ◽

pp. 256-256

Author(s):

Ashkan Shahbandi ◽

Elisa Ledet ◽

Bryce Raymon Christensen ◽

Marcus Marie Moses ◽

Peter Steinwald ◽

...

Keyword(s):

Wnt Signaling ◽

Statistical Significance ◽

Positive Association ◽

Cancer Center ◽

Beta Catenin ◽

Canonical Wnt Signaling ◽

Significant Positive Association ◽

Variants Of Unknown Significance ◽

Castrate Resistant ◽

Canonical Wnt

256 Background: Alterations in Wnt signaling have been shown to play a role in the development of castrate resistant prostate cancer. Cell free DNA (cfDNA) isolated from patient plasma can provide a non-invasive way to further assess this role. The goal of this study was to identify patients with cfDNA alterations in major canonical Wnt signaling components (APC and/or beta-catenin), and relate the emergence of these mutations during treatment to alterations in other common mutations. Methods: 134 clinically progressive metastatic CRPC patients from Tulane Cancer Center underwent cfDNA analysis through Guardant360 test (Guardant Health, Redwood City, CA). This analysis consisted of exonic coverage of 70 genes as well as amplifications in 18 genes, with mutations categorized as either pathologic, non-pathologic or as variants of unknown significance (VUS). Clinical annotation of prior treatment history was recorded. Results: 21.6% (29/134) of the mCRPC patients evaluated had a canonical Wnt signaling (APC and/or CTNNB) alteration. Of these patients, 62.1% (18/29) had mutations identified as pathologic. 77.8% (14/18) of patients identified with a pathologic Wnt mutation were treated with abiraterone and/or enzalutamide prior to Guardant360 testing. To determine potential associations between Wnt signaling alterations and other detected changes in cfDNA, the relationship between Wnt mutations (APC and/or CTNNB1) with pathologic TP53 mutations, AR mutations, BRAF amplifications, and MYC amplifications was assessed using a patient’s latest Guardant360 test. A significant positive association was found between Wnt mutations (n = 18) and MYC amplifications (n = 22), (p = 0.0373). 33.3% (6/18) of patients with a pathologic Wnt mutation were found to have amplification in MYC. Other notable associations included Wnt mutations and AR mutations (n = 74), which approached statistical significance (p = 0.112). Conclusions: While the understanding of the role of Wnt signaling in the treatment of mCRPC is still evolving, co-segregation of Wnt-signaling alterations with other oncogenic alterations, particularly MYC, which has been identified as a target of canonical Wnt signaling, may provide insights with regards to future management of mCRPC.

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