4. Results
4.5 Localization of MITF, YAP, and β-catenin upon treatment with G007-LK
We established through immunoblot analysis and real-time RT-qPCR analysis that regulation of MITF was not dependent on the presence of β-catenin, suggesting another mechanism involved.
Therefore, we wanted to investigate if treatment with G007-LK for 72 hours could change the localization of MITF, YAP, or β-catenin proteins in murine B16-F10 cells. Previous studies with immunofluorescent staining of B16-F10 cells indicated an accumulation of YAP in the nucleus upon treatment with G007-LK for 24 hours [25]. Immunofluorescent staining analysis and confocal microscopy were implemented to identify the localization of the intended proteins.
Our data implied that YAP protein seems to be predominantly in the nucleus when treated with G007-LK (Figure 26A). In addition, MITF proteins formed vesicles in the treated cells, which is difficult to explain (Figure 26A). β-catenin show indications of re-localization from the cell membrane towards the cytoplasm upon treatment (Figure 26B).
Looking at the composite images, the main impression indicates that MITF protein expression increase upon treatment with G007-LK. However, the experiment is repeated once, and we cannot draw any final conclusions about MITF, YAP or β-catenin protein localization.
Figure 25. Regulation of MITF expression by G007-LK is β-catenin independent.
A. Immunoblot analysis of B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2 cytoplasmic, nuclear, and total fractions of MITF after treatment with G007-LK (1 µM) 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 treatment with G007-LK (1 µM) for 72 hours compared to controls (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. Two-tailed t-test is indicated by ** (P < 0.01). Combined data from two independent experiments with three replicates each are shown.
Absence of depicted statistical comparisons indicates lack of statistical significance.
44 Figure 26. Effect of G007-LK on the localization of MITF, YAP and β-catenin in B16-F10 cells
A. Representative Images of cells treated with control (0.01% DMSO) and G007-LK (1 µM) for 72 hours. Cells were stained with anti-MITF (Fluor 488 nm/green), anti-YAP (Fluor 647 nm/red) and nuclear DAPI (Fluor 358 nm/blue) and analyzed by ZEISS LSM 980 microscope. Scale bar = 20 µm.
45 B. Representative Images of cells treated with control (0.01% DMSO) and G007-LK (1 µM) for 72 hours. Cells were stained with anti-MITF (Fluor 488 nm/green), anti-β-catenin (Fluor 647 nm/red), and nuclear DAPI (Fluor 358 nm/blue) and analyzed by ZEISS LSM 980 microscope. Scale bar = 20 µm.
46
4.6 G007-LK can counteract WNT-induced morphology changes in B16-F10 cells in vitro
A dynamic switch in phenotype occurs in the melanoma cells as they transit from highly proliferative melanoma cells, being less invasive, to de-differentiated cells that gain other properties such as stemness, invasiveness, and motility [64, 70]. WNT signaling is known to drive phenotypic changes and influence the antigen presentation, resulting in resistance to
checkpoint inhibition therapy [70, 103]. Therefore, we wanted to examine the effects of tankyrase inhibition and activating WNT signaling on the morphology of B16-F10 to identify any possible switch in phenotype.
The representative images of the murine B16-F10 cells were taken at treatment start (0 days), after 3 days and 6 days. All images were compared to the control culture. The cells observed 3 days after treatment start appeared to inhabit a more dendritic morphology upon activation of WNT signaling, seemingly connecting with adjacent cells (Figure 27). Further treatment for 6 days with WNT3a also showed a dendritic morphology. In vitro monotherapy with G007-LK did not show any substantial morphology changes.
Figure 27. WNT3a can induce morphology changes in B16-F10 cells.
Changes in morphology for B16-F10 cells treated with control (0,01% DMSO), G007-LK (1 µM), recombinant WNT3a and WNT3a + G007-LK for 3 days and 6 days. Image of cells at treatment start is shown in the top panel (0 days). Scale bar = 100 µM.
47 Switching phenotype can be a reversible process, where melanoma cells can shred the invasive properties and re-gain proliferative and differentiated properties [65, 104]. To investigate this phenomenon, we changed the treatment of B16-F10 cells cultured for 3 days with WNT3a to control, G007-LK and WNT3a + G007-LK. After 3 more days, the inspected cells showed a less dendritic morphology when removing WNT signaling activating ligand WNT3a (Figure 28). In addition, we observed that the changed treatment made the G007-LK containing cells resembled the original control cells in Figure 27 more than the control cells in Figure 28, indicating a reversible property of G007-LK.
In conclusion, these findings illustrate morphology changes that may indicate a possible switch in phenotype in B16-F10 upon treatment with WNT3a. We observed reversible morphology
changes suggesting switching phenotype when removing WNT3a, in addition to a reversible function of G007-LK in morphology context.
Figure 28. Reversible morphology changes in B16-F10 cells.
Changes in morphology in B16-F10 cells treated with recombinant WNT3a for 3 days were treated with control (0,01%
DMSO), G007-LK (1 µM), and WNT3a + G007-LK for 3 days. Scale bar = 100 µM.
48
4.7 G007-LK-induced phenotype-switching in melanoma
A transition that occurs during the transformation between differentiated, not-invasive cells and invasive, de-differentiated cells are the EMT. During EMT, melanoma loses contact with adjacent keratinocytes and interact with fibroblasts and endothelial cells [64]. We wanted to investigate whether the observed morphology changes in murine B16-F10 cells correlated to an EMT in vitro and in vivo. This was accomplished by selecting two hallmarks on each side for analysis, CDH1 is a marker for the epithelial phenotype, and CDH2 is a marker for the mesenchymal phenotype.
To explore the effect of G007-LK and WNT3a in the context of EMT, immunoblot was
implicated to B16-F10 cells, B16-F10Ctnnb1KO1 and B16-F10Ctnnb1KO2. Immunoblot analyzes of the cytoplasmic fractures indicated neither stabilization nor destabilization of CDH1 and CDH2 in all cell lines (Figure 29A).
Real-time RT-qPCR was used to explore translational regulation of Cdh1 or Cdh2 in wild type B16-F10 cells. The data implied a moderate stabilization of both Cdh1 and Cdh2 gene expression (Figure 29B). Cdh2 gene expression was moderately increased upon WNT3a + G007-LK
treatment.
Figure 29. G007-LK stabilizes both Cdh1 and Cdh2 gene in B16-F10 cells in vitro.
A. Immunoblot analysis of cytoplasmic CDH2 (upper) and CDH1 after treatment with G007-LK (1 µM), recombinant WNT3a and WNT3a + G007-LK for 72 hours compared to controls (0.01% DMSO). KO1/KO2 was treated with G007-LK compared to controls. Actin/GAPDH document equal protein loading.
B. Real-time RT-qPCR analysis of Cdh1 and Cdh2 in B16-F10 in vitro. 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 < 0.01). Combined data from five independent experiments with three replicates each are shown.
49 Tumors provide a more realistic model for analyzing changes in the expression of genes and proteins. We utilized previously stored B16-F10 and Clone M-3Z1 tumors for examining the effect of tankyrase inhibitors in vivo [25]. Immunoblot analysis of B16-F10 tumors indicated no statistical significance between control and treated mice at the protein level (Figure 30A/B).
On a transcriptional level, B16-F10 tumors showed no significant regulation of neither Cdh1 nor Cdh2 in response to treatment with G007-LK (Figure 30C). Clone M-3Z1 tumors revealed a substantial reduction of CDH1 expression upon treatment with G007-LK. (Figure 30D).
Figure 30. Effect of G007-LK on EMT target genes in B16-F10 and Clone M-3Z1 cells in vivo.
A. Immunoblot analysis of CDH1 in wild type B16-F10 tumor after treatment with G007-LK (1 µM) (right) for 72 hours compared to control (0.01% DMSO) (left). GAPDH document protein loading.
B. Statistical analysis of immunoblot of B16-F10 in vivo. The y axis represents the relative band intensity of CDH1protein upon treatment with G007-LK (1 µM) for 72 hours compared to controls (0.01% DMSO).
For B, C and D: Absence of depicted statistical comparisons indicates lack of statistical significance.
C. Real-time RT-qPCR analysis of Cdh1 and Cdh2 in B16-F10 tumor. For C and D: 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. Two-tailed t-tests are indicated by *** (P < 0.001). Combined data from one experiment with seven tumors of each treatment are shown.
D. Real-time RT-qPCR analysis of Cdh1 and Cdh2 in Clone M-3Z1 tumor.
50 In conclusion, G007-LK seems to moderately stabilize Cdh1 and Cdh2 genes in B16-F10 cells in vitro, but not in vivo. CDH1 and CDH2 protein did not show any significant regulation upon treatment in vitro or in vivo. The results also suggest that any regulation may be cell-dependent on transcription level based on the Clone M-3Z1 results. However, the experiment needs to be repeated to draw firm conclusions as it was only performed once.