4. Results
4.3 G007-LK regulates YAP signaling on RNA and protein level independent of β-catenin …. 39
Like the WNT signaling pathway, YAP signaling plays a key role in tumorigenesis. WNT and YAP are also closely inter-connected signaling pathways [100]. Previous experiments for 24 hours have shown that tankyrase inhibition can suppress YAP signaling and stabilize AMOT proteins in cell culture and tumors [25]. We wanted to further assess the effect of G007-LK for 72 hours on YAP signaling components in murine F10 cells. β-catenin knockout cells B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2 were utilized to determine if the regulation of YAP signaling by G007-LK is dependent on β-catenin. Notably, regulation of WNT signaling components in B16-F10 cells has previously been analyzed by immunoblot, which indicated G007-LK induced stabilization of AXIN1 protein and destabilization of non-phospho β-catenin and total β-catenin protein [25]. In addition, previous real-time RT-qPCR analysis showed that G007-LK could counteract WNT signaling [25].
Immunoblot analysis was applied to the nuclear fractions of B16-F10 cells to explore the effect of G007-LK on its intended targets, YAP and TAZ. In addition, treatments with WNT3a and
WNT3a + G007-LK were implicated to see if G007-LK could counteract the effect of WNT3a.
The results showed YAP and TAZ protein stabilization in the nucleus upon G007-LK treatment (Figure 22A). In addition, WNT3a treatment decreased both YAP and TAZ protein.
To assess if the regulation of YAP target genes is catenin dependent, murine B16-F10 β-catenin knockout cells B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2 cultured for 72 hours with control and G007-LK were utilized. Immunoblot analysis was applied to nuclear fractions of B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2 cells on its intended target YAP and TAZ. In response to treatment, the results showed a substantial increase in YAP and TAZ protein levels in both β-catenin knockout cells (Figure 22A).
To investigate whether G007-LK and WNT3a impacted gene expression on YAP signaling, the YAP target genes Ctgf, Cyr61and Amotl2 were selected. Ctgf is a direct YAP target gene essential for normal embryonic development and tissue repair [101]. Activation of Ctgf and Cyr61 promotes cell growth, proliferation, and survival. Knockdown of Amotl2 increases YAP
40 signaling, thereby acting as a negative feedback regulator, in addition to inhibiting the WNT signaling pathway [102]. In contrast, real-time RT-qPCR analysis revealed no significant changes in Ctgf and Amotl2 gene expression (Figure 22B). Real-time RT-qPCR analysis of Cyr61
indicated that G007-LK could reduce gene expression in Cyr61 and counteract WNT signaling (Figure 22B). Transcriptional regulation of WNT target gene Axin2 demonstrates treatment efficacy of G007-LK (Figure 22B).
In summary, the results suggest an increase of YAP and TAZ protein in the nucleus independent of the presence of β-catenin upon treatment with G007-LK. YAP target genes showed variable results and no apparent attenuation of Ctgf and Amotl2 upon treatment with G007-LK, whereas Cyr61 was reduced upon treatment with G007-LK. Further research is needed before drawing any firm conclusions.
Figure 22. Effect of G007-LK on YAP target proteins and genes in B16-F10 cells.
A. Immunoblot analysis of nuclear YAP and TAZ in B16-F10 cells after treatment with G007-LK (1 µM), recombinant WNT3a and WNT3a + G007-LK for 72 hours compared to controls (0.01% DMSO). β-catenin knockout 1 and knockout 2 cells were only treated with G007-LK, compared to controls. Lamin B1 documents protein loading. Quantified YAP and TAZ levels, versus loading controls, are depicted.
B. Real-time RT-qPCR analysis of YAP signaling target genes (Ctgf, Cyr61, and AMOTL2) and Axin2. The y axis represents the relative quantification values from the real-time RT-qPCR analysis, normalized to the control. Boxplots show median, first, and third quartiles, and maximum and minimum whiskers. One-way ANOVA on ranks tests (Dunn's method) are indicated by * (P < 0.05), and *** (P < 0.001). Combining data from four independent experiments with three replicates each is shown.
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4.4 G007-LK induces nuclear accumulation of MITF protein
The transcription factor MITF was identified as the most statistically significant expressed gene separating YAPhigh and YAPlow groups in human melanoma cells [25]. Previous reports have shown that the subgroup showing elevated baseline YAP signaling activity upon tankyrase inhibition also reduced MITF expression [25]. However, little was known regarding the underlying mechanisms controlling MITF expression when melanoma cells are exposed to tankyrase inhibition followed by counteracted WNT or YAP signaling. Therefore, we wanted to examine MITF expression in B16-F10 cells treated with G007-LK and WNT3a to evaluate whether WNT signaling functions in regulating MITF expression.
A pilot experiment on B16-F10 tumors had previously shown stabilization of MITF upon treatment with G007-LK for 72 hours through immunoblot analysis (Figure 23). We wanted to further evaluate treatment with G007-LK for 72 hours could regulate MITF protein.
To study the regulation of MITF protein, we prepared cytoplasmic, nuclear, and total fractions of murine B16-F10 cells for immunoblot analysis. In addition to treatment with G007-LK, WNT signaling activating ligand WNT3a and WNT3a + G007-LK treatment was included to evaluate if WNT regulates MITF and if G007-LK could counteract this regulation. The overall result pattern of the immunoblot analysis revealed a tankyrase inhibitor-mediated stabilization of MITF in the nucleus but not in the cytoplasm (Figure 24A).
We also wanted to investigate whether treatment with G007-LK for 72 hours could impact Mitf at a transcriptional level. Therefore, real-time RT-qPCR analysis was implemented to evaluate the transcriptional regulation of Mitf. In vitro monotherapy with G007-LK could not induce
activation of Mitf transcript as well as monotherapy with WNT3a (Figure 24B). However, the results indicated a synergic effect of G007-LK + WNT3a.
Figure 23. MITF expression increase upon treatment with G007-LK in vivo.
Immunoblot analysis of MITF in B16-F10 mice treated with G007-LK diet for four days (right) compared to mice treated with control diet (left). Control diet (n=7) and G007-LK diet (n=7). Actin documents protein loading.
42 Furthermore, we wanted to investigate whether the G007-LK-mediated regulation of MITF is β-catenin dependent. Therefore, we used murine F10 β-β-catenin knockout cells
B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2 cultured for 72 hours with control and G007-LK.
Immunoblot analysis was applied to B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2 cells to explore the effect of G007-LK on MITF. The data revealed G007-LK mediated accumulation of MITF in B16-F10Ctnnb1KO2 in the nucleus, but not in B16-F10Ctnnb1KO1 (Figure 25A).
Real-time RT-qPCR data suggested no regulation of Mitf on the transcriptional level through tankyrase inhibition in B16-F10Ctnnb1KO but revealed moderately decreased gene expression of Mitf in B16-F10Ctnnb1KO2 (Figure 25B).
In summary, the main impression of the results is that the accumulation of MITF protein occurs independently of the presence of β-catenin in the nucleus. Furthermore, our findings suggest that regulation of MITF does not occur on the RNA level in any substantial level.
Figure 24. Tankyrase inhibitor G007-LK can increase MITF expression in murine B16-F10 cells.
A. Immunoblot analysis of MITF in wild type B16-F10 cells in cytoplasmic, nuclear and total fractions after treatment with G007-LK (1 µM), recombinant WNT3a (activator of WNT/β-catenin signaling) and WNT3a + G007-LK for 72 hours compared to controls (0.01% DMSO). Actin (cytoplasmic) and lamin B1 (nuclear) document protein loading. Quantified MITF levels, versus loading controls, are depicted.
B. Real-time RT-qPCR analysis of Mitf upon 72hour treatment with G007-LK (1 µM), recombinant WNT3a and WNT3a + G007-LK compared to control (0.01% DMSO). The y axis represents the relative quantification values from the real-time RT-qPCR analysis, normalized to the control. Boxplots show median, first and third quartiles and maximum and minimum whiskers. One-way ANOVA on ranks tests (Dunn's method) are indicated by ** (P < 0.01). Combined data from five independent experiments with three replicates each are shown. Absence of depicted statistical comparisons indicates lack of statistical significance.
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