3. Materials and Methods
3.3 Immunoblot analysis
3.3.3 Incubation with antibodies and detection of target protein
After transfer, the membranes were blocked 5% milk in tris-buffered saline, 0,1% tween (TBST)/phosphate-buffered saline, 0,1% tween (PBST) for 1 hour to prevent non-specific
binding of antibodies, before adding primary antibody diluted in TBST/PBST for incubation over 1-2 nights at 4 °C. The primary antibody concentration is critical to get a specific protein signal (see table 4). The primary antibody binds to the target protein on the membrane (Figure 19). The membranes were then washed with TBST/PBST for 5 minutes before adding secondary antibody for 3-4 hours incubation. The secondary antibody is reactive toward the primary antibody and carries the horseradish peroxidase (HRP) enzyme. After incubation of the secondary antibody, the membranes were rewashed thoroughly with TBST/PBST to minimize background and remove unbound antibodies. A 1:1 ratio of ECL™ Prime Western Blot Detection Reagent Solution A and B were prepared, including HRP substrate. These enzymes catalyze the chemical reaction generating a signal that corresponds to the position of the target protein in the form of light [98]. Light is only produced while the enzyme has access to the substrate, making this a time-limiting step. The membranes were placed between two blank sheets after adding the solution mix to the membrane part with the desirable protein. The results were visualized in BioRad Machine. The appropriate exposure time was used to capture a good image before the signal degenerated [99].
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3.4 Immunofluorescent staining of B16-F10 cells
Immunofluorescence is e technique using antibodies to stain proteins, thereby visualizing the location of the antibodies in the cells. We treated the cells with vehicle control (0.01% DMSO), G007-LK, recombinant WNT3a, and combinational treatment with G007-LK and Wnt3a.
3.4.1 Poly-L-lysine coating
Precision cover glasses (VWR) were washed and sterilized with ethanol and carefully placed in a 12-well plate. Coating of the slides was performed to keep the cells attached to the glass. The coating was accomplished using poly-L-lysine diluted 1:10 in sterile PBS, which promotes attachment of endothelial cells to glass, followed by washing with PBS 2-3 times. The plate was left in UV lights for 15 minutes before seeding 10,000 B16-F10 and B16-F10Ctnnb1KO cells, left overnight to attach. Treatment was added to the wells the following day and incubated 37°C 5%
CO2 for 72 hours.
Figure 19. Detection of target proteins through chemiluminescent immunoblotting. Chemiluminescence is a
frequently used method to detect target proteins, using enzymatic reaction with HRP and substrate. The incubated primary antibody binds the target protein on the membrane, and the secondary antibody is reactive towards the primary antibody.
The secondary antibody is labeled with HRP, which produces light when adding chemiluminescent HRP substrate. The method is time-dependent because of the enzymatic reaction. Figure created with Biorender.com, inspired [98].
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3.4.2 Immunostaining of cells
After 72 hours of treatment, the cells were washed twice with PBS before fixing the cells with ice-cold 4% paraformaldehyde (PFA) (Appendix A, Supplementary Table 4) for 15 minutes.
Next, 0,1% Triton X-100 in PBS were added for 15 minutes to permeabilize the cells.
MITF, YAP, and β-catenin primary antibodies were diluted in 4% BSA/PBS and added dropwise to the glasses, creating a “membrane” and left overnight at 4°C (Appendix A, Supplementary Table 5). After incubation in primary antibody, cells were washed twice with PBS, before incubation in secondary antibody diluted in 1:400 4% BSA/PBS for 1 hour at room temperature in the dark. Cells were then washed twice with PBS, before incubation in 1µg/mL DAPI in PBS for 5 minutes. 1.5 (0.17-0.18 mm thick) coverslips were then placed on microscope slides (Marienfield) with fluorescent anti-fading mounting glue and kept in the dark at 4°C until visualizing in a microscope (Appendix A, Supplementary Table 6).
3.4.3 Microscopy
Fluorescent confocal images were acquired by Confocal Laser Scanning Microscope System:
ZEISS LSM 980 with Airyscan 2 using oil objective. Each image was acquired by eight Z planes and three channels. For supplementary fluorescent images, Zeiss Axio vert A1microscope was used together with Zeiss Colibri 7 LED (source of light), Zeiss filterset 90 HE LED (filter), and Zeiss Axiocam 202 mono (camera). All images were processed in ImageJ.
3.5 Statistical analysis
All statistical analysis was performed using Sigma PlotR 12.5 (Systat Software Inc.), and the minimum significance level was defined as P<0,05. Single outlier detections were identified by Dixon’s and/or Grubb’s test (Threshold, P<0,05) using ControlFreak (Contchart software). The two-tailed student’s t-test was used to test for significant differences (*** [P < 0.001], ** [P <
0.01] and * [P < 0.05]) between two samples with normally distributed parameters (Shapiro-Wilk test, P > 0.05). Welch's t-test is indicated by‡ (P< 0.01), and Mann-Whitney rank-sum tests are indicated by †(P < 0.05). Microsoft Excel Software was used to calculate means and standard deviations (SD), and detailed descriptions can be found in the figure legends and figures and the number of events (n).
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4. Results
4.1 Tankyrase inhibitor G007-LK and PD-1 inhibition shows synergistic anti-tumor efficacy in mouse melanoma
Previous reports on syngeneic murine B16-F10 melanoma models have shown promising results of overcoming β-catenin-mediated resistance to immune checkpoint blockade in melanoma through tankyrase inhibition [25]. Active tankyrase contributes to the activation of WNT and YAP signaling, whereas specific small-molecule tankyrase inhibitor G007-LK decreases both pathways [25, 53]. No therapeutic drugs targeting WNT signaling are available in clinical practice today. Recently, we showed that in vivo monotherapy with G007-LK or anti-PD-1 did not reduce tumor size in isogenic B16-F10 and Clone M-3Z1.However, the combined anti-PD-1/G007-LK treatment showed synergistic anti-tumor efficacy (Figure 20A, B) [25].
Figure 20. Combinational therapy with G007-LK and PD-1 shows synergic anti-tumor effect [25].
A. B16-F10 tumor (subcutaneous [s.c.]) end volume upon anti-PD-1/G007-LK treatment (−83% when compared to control) in C57BL/6 N mice treated from day 10 through 21. Control diet (n = 10), G007-LK diet (n = 10), anti-PD-1 (n = 11), and anti-PD-1/G007-LK (n = 8). Mann-Whitney rank-sum tests are indicated by †(P < 0.05).
For A, B. Mean values are indicated by grey lines. The absence of depicted statistical comparisons suggests a lack of statistical significance.
B. Clone M-3Z1 tumor (s.c.) end volume reduction upon anti-PD-1/G007-LK treatment (−53% when compared to control) in DBA/2 N mice treated from day 8–18. Control diet (n = 8), G007-LK diet (n = 8), anti-PD-1 (n = 11), and anti-PD-1/G007-LK (n = 10). Two-tailed t-test is indicated by *(P < 0.05).
In summary, the data indicated an increased susceptibility for checkpoint inhibition in B16-F10 and Clone M-3Z1 tumors when combined with a tankyrase inhibitor on the WNT signaling pathway.
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4.2 RNA sequencing reveals a subpopulation transcriptional response profile
Modification of WNT and YAP signaling pathways, immune cells in the tumor
microenvironment, epigenetic and genetic alterations can impact the efficacy of the synergistic anti-PD-1/G007-LK treatment [78]. In addition, inhibition of tankyrase in WNT and YAP signaling pathways can be cell lineage-dependent [25]. Unlike most human melanoma cells, the B16-F10 murine melanoma cells also lack the BRAFV600E mutation [25]. Therefore, to further investigate the beneficial effect on murine mouse models applied to human cell lines, a panel of 18 human melanoma cell lines was treated with G007-LK.
RNA sequencing and bioinformatic analyses classified the control group into YAPhigh and YAPlow expressing melanoma cells (Figure 21A) [25]. Ingenuity Pathway Analysis (IPA) and bioinformatics analysis identified MITF as the most statistically significant key regulator
separating YAPhigh and YAPlow expressing cells [25]. MITF had an overall low relative response profile for the YAPhigh group and vice versa [25]. Upon treatment for 24 hours, the YAPhigh subgroup showed susceptibility to reduced MITF expression, while the YAPlow subgroup showed susceptibility to increased MITF expression (Figure 21B).
Figure 21. High activity of YAP signaling correlates with low baseline MITF expression and potential for decreased MITF transcription upon tankyrase inhibition [25].
A. Expression of YAP signaling target transcripts (Ccn1 [Cyr61], Ccn2 [Ctgf], and Amotl2) for untreated human and murine B16-F10 melanoma cell lines. Seven of 19 samples displayed higher relative transcription of YAP signaling target genes (YAPhigh) than samples with less transcription (YAPlow). YAPhigh is highlighted by orange branches in the dendrogram and arrows. Scale bar indicates differences in Z-score (standard deviations < or > mean) values for log2 transcripts per millions (TPMs) within each row. Cancer stages: Metastatic in blue, radial growth phase (RGP) in pink, and vertical growth phase (VGP) in green.
B. Baseline MITF expression in untreated samples (grey bars, log2-transformed TPMs × 10−1) and change upon treatment with G007-LK (1 µM) for 24 hours (black dots sorted descending from left to right, log2 values from treated versus untreated TPMs). Samples with increased (MITF increased, left) or decreased (MITF decreased, right)
39 expression of MITF upon tankyrase inhibitor treatment are separated by a scattered vertical line. *Indicates that no value for untreated sample is inserted. B16-F10 WNT3a = WNT3a + G007-LK relative to WNT3a-stimulated control. YAPhigh cell lines are highlighted by orange arrows.
In conclusion, the results identified MITF as the most statistical gene separating the two un-treated groups YAPhigh and YAPlow. The MITFlow/YAPhigh subgroup displayed decreased MITF expression upon treatment (MITFdecreased), whereas the MITFhigh/YAPlow subgroup displayed increased MITF expression upon treatment (MITFincreased) with G007-LK.
4.3 G007-LK regulates YAP signaling on RNA and protein level independent of β-catenin
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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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.
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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
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