Examination of G007-LK-induced interference with YAP signaling in B16-F10 cells

Previous studies have established the attenuating effect on WNT signaling with our tankyrase inhibitor G007-LK in murine B16-F10 melanoma cells [25]. Here, the immunoblot analysis of the WNT signaling components showed stabilization of AXIN1 protein in response to treatment with G007-LK, whereas total and non-phospho β-catenin were both destabilized [25].

Furthermore, Axin2 and transcription factor 7 (Tcf7) are target genes in the WNT signaling pathway, where G007-LK could counteract WNT signaling in both target genes [25]. We did not repeat any of these experiments in this thesis as these results were previously shown.

5.2 Examination of G007-LK-induced interference with YAP signaling in B16-F10 cells

YAP and TAZ can function as downstream effectors of the canonical WNT signaling pathway and mediate cell migration, gene expression, differentiation, and antagonism of alternative WNT signaling [100]. In addition, WNT3a has been identified as a potent activator of YAP and TAZ [100]. Therefore, we wanted to investigate the effect of G007-LK and WNT3a on the YAP signaling components. Tankyrase inhibitors can inactivate YAP/TAZ activity by stabilizing AMOT proteins which functions as negative YAP regulators [111]. At the genome-wide level, the transcription factor TAZ mediated a substantial proportion of WNT transcriptional responses [112]. TAZ is dependent on phosphorylated catenin, which pairs TAZ to its ubiquitin ligase β-TrCP in the destruction complex [112]. When WNT signaling is activated, β-catenin translocate to the nucleus and impairs TAZ from the destruction complex, leading to TAZ accumulation [112]. Hence, we wanted to examine the response of YAP and TAZ proteins and the YAP target genes Ctgf, Cyr61, and Amotl2 upon treatment with G007-LK and WNT3a.

We observed an increase of YAP and TAZ proteins upon treatment with G007-LK and a decrease when treated with WNT3a. These findings do not align with the literature described in this thesis or previous reports showing nuclear reduction of YAP/TAZ upon treatment with tankyrase inhibitors [113, 114]. However, our previous research also revealed increased nuclear YAP protein level upon G007-LK treatment [25, 49]. Real-time RT-qPCR analysis, however, showed a reduction of Cyr61 gene expression upon tankyrase inhibition in B16-F10. Amotl2 and Ctgf showed conflicting regulations that needs further experimentation.

In summary, we show that G007-LK is effective in regulating YAP signaling in vitro. The contradictory results in protein- and gene expression may indicate that YAP regulation is cell lineage-dependent. Further evaluation of tankyrase inhibiton, and the interference with YAP signaling, is necessary to finalize any conclusions.

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5.3 Examination of G007-LK-induced interference with MITF expression in B16-F10 cells

Our previously published research identified MITF as the most statistically significant key regulator separating YAP^high and YAP^low untreated subgroups of human melanoma cell lines, connecting MITF^high with YAP^low expressing cells, and MITF^low with YAP^high expressing cells.

Moreover, the exposure of G007-LK to the cells further decreased MITF expression in the MITF^low subgroup, as well as increased the MITF expression in the MITF^high subgroup. High MITF expression correlates to a less invasive state of melanoma, benefiting melanoma some melanoma patients [50]. Seeing that MITF drives endolysosomal and melanosomal biogenesis and functioning, changes in MITF can affect the composition of antigens presented by MHC on the T cell [50, 115]. In addition, there has been established that phenotype switching and

differenced/de-differentiated states in melanoma correlate to MITF^high or MITF^low states of the cells [115]. Therefore, an upregulation in MITF expression may favor a differentiated phenotype that beneficially influences immune recognition, then sensitizes melanoma to anti-PD-1 treatment [115]. Hence, we wanted to explore the regulation of MITF on the transcriptional level and protein level upon treatment with G007-LK.

Our project started with a pilot experiment testing how tankyrase inhibitor G007-LK affects MITF expression in B16-F10 tumors. Here, the immunoblot analysis indicated stabilization of MITF upon treatment with G007-LK. We, therefore, continued testing G007-LK on murine B16-F10 cells, also using the β-catenin knockout cells B16-B16-F10^Ctnnb1KO1 and B16-F10^Ctnnb1KO2.

Immunoblot analysis of wild-type B16-F10 and B16-F10^Ctnnb1KO2 cells showed accumulation of MITF in the nucleus. In contrast, no significant stabilization or destabilization of

B16-F10^Ctnnb1KO1 was depicted. However, the loading controls in the nuclear fractions were suboptimal and may have affected the data interpretation. Overall, MITF protein seems to accumulate in the nucleus in wild type B16-F10 cells and both β-catenin knockout cells. Thus, we may speculate that MITF regulation through tankyrase inhibition is connected to YAP signaling rather than WNT signaling. Endoribonuclease-prepared siRNA (esiRNA)-mediated knock-down and CRISPR/Cas9-mediated knock-out of YAP can further elucidate this connection.

Real-time RT-qPCR analysis showed a moderate increase of Mitf expression upon treatment with WNT3a + G007-LK in murine B16-F10 cells. This phenomenon needs further research to

explain. When performing these experiments, we saw contradictory results of transcriptional regulation of Mitf in response to treatment with G007-LK and WNT signaling activation. The MITF-M isoform limited to melanocytes and melanoma cells can be affected by WNT signaling as the MITF-M promoter contains LEF-1/TCF binding site [51]. In addition, the Hippo signaling pathway can regulate MITF-M expression through YAP and TAZ [79]. There was, however, not a substantial increase or decrease of Mitf gene expression, suggesting that regulation of Mitf may occur upon post-translational activities and not on a transcriptional level upon. Notably,

57 transcriptional regulations of Mitf can depend on post-translational modifications and

co-operating partners [82].

We observed no regulation of Mitf in B16-F10^Ctnnb1KO1;however, gene expression of Mitf in B16-F10^Ctnnb1KO2 was only moderately decreased. When creating the B16-F10 β-catenin knockout cells, the CHOPCHOP tools for CRISPR were used, which may lead to different mutations between the clones throughout the process [93]. This mechanism may explain the differences in the response of tankyrase inhibition from B16-F10^Ctnnb1KO1 and B16-F10^Ctnnb1KO2.

We conclude that regulation of MITF in B16-F10 mainly occurs in the nucleus upon tankyrase inhibition, as well as being β-catenin independent. Furthermore, our findings suggest that Mitf is not regulated at the RNA level and through transcriptional regulation.

5.4 Investigation of localization of MITF, YAP and β-catenin proteins upon treatment with G007-LK

Our studies showed that regulation of MITF expression through tankyrase inhibition was not regulated via β-catenin, which may indicate that regulation is YAP signaling dependent. Hence, we wanted to explore changes in the localization of MITF, β-catenin, and YAP proteins in response to treatment with G007-LK.

Firstly, we analyzed our immunofluorescent images with a Zeiss Axio vert A1microscope. The images were not good enough to draw firm conclusions about the localization of any of the intended proteins (Appendix C, Supplementary figures 4 and 5). However, immunofluorescent images with a confocal microscope using oil objectives implied an accumulation of MITF upon treatment. Treatment with G007-LK produced spots of MITF protein, an unclear phenomenon that is difficult to explain but might be lipid-like structures. Treatment for 24 hours with G007-LK has previously showed increase of nuclear YAP protein, which we also could observe in our 72 hour treatment. Our findings do not seem to align with the literature of tankyrase inhibition in the Hippo signaling pathway described in this thesis, where inhibition of tankyrase leads to the accumulation of AMOT proteins which keeps YAP/TAZ proteins in cytoplasmic retention, as several studies has reported before [113, 114]. However, we could not observe any substantial increase of YAP protein, and further research is needed to strengthen our observations. In

addition, the control images of MITF/YAP and MITF/β-catenin showed divergence, which might be a consequence of technical error.

Our findings indicate that G007-LK is effective in regulating MITF, YAP and β-catenin.

However, our observations in based on one experiment, and further investigation needs to be done. In addition, it would be interesting to explore the regulation of TNKS1/2 and AMOTL2 upon treatment with G007-LK for 72 hours.

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5.5 Evaluation of whether G007-LK can affect phenotype switching in melanoma

There has been established a correlation between invasive/non-invasive melanoma, switch in phenotype, and WNT signaling activity [63] [70]. A well-known transition is the epithelial to mesenchymal switch, where cancer cells acquire different properties for either side of the transition [93]. Seeing that changes in WNT signaling are associated with EMT and switch in phenotype, we firstly wanted to investigate whether we could observe a morphology change in murine B16-F10 cells upon activation of WNT signaling pathway [70]. Additionally, we wanted to see whether tankyrase inhibition could counteract the effect of activated WNT signaling. An effect of WNT signaling through tankyrase inhibitors could help drive invasive and

de-differentiated cells towards proliferative, non-invasive, and generally less malignant cells.

5.5.1. Tankyrase inhibition and phenotype switching in vitro

We observed morphology changes upon activated WNT signaling through microscopy, as well as a WNT signaling counteracting effect by G007-LK in cell culture. Only three photos of each culture were taken, which may be too few to get representative pictures. The morphology changes observed in cell culture do not necessarily correlate to an EMT. We chose the EMT hallmarks CDH1 and CDH2 to investigate if the morphology changes corresponded to EMT.

Immunoblot analysis of B16-F10 cells did not show any significant regulation of either CDH1 or CDH2, indicating that regulation of these markers is not regulated on protein level.

Real-time RT-qPCR analysis revealed that Cdh1 gene expression in B16-F10 cells was reduced in response to activated WNT signaling, which is in line with the literature described in 1.6 Phenotype switching. Cdh1 gene specifies E-cadherin, which is an adhering molecule between epithelial sheets. Loss of E-cadherin drives melanoma cells towards mesenchymal phenotype.

G007-LK could moderately counteract the effect of WNT3a on Cdh1 expression. Cdh2 gene (that specifies N-cadherin) showed a moderately synergic effect with WNT3a + G007-LK treatment.

The data show similarities to the data of Mitf in B16-F10 cells, and further research is needed to explain this synergic effect. Real-time RT-qPCR data of Cdh1 and Cdh2 gene expression in B16-F10^Ctnnb1KO1 demonstrated no significant regulation, whereas Cdh2 gene expression was

moderately reduced in B16-F10^Ctnnb1KO2 (Appendix C, Supplementary Fig. 1).

59 To summarize, we could observe a morphology change but cannot prove that the phenomenon is truly regulation of EMT. In addition, the results indicate a cell-type dependent regulation of Cdh1 and Cdh2 and not being β-catenin dependent.

5.5.2. Tankyrase inhibition and phenotype switching in vivo

Immunoblot analysis was applied to previously stored B16-F10 tumors to explore the effect of G007-LK on EMT marker CDH1. A statistical analysis of the immunoblot showed no significant decrease in CDH1 protein expression after treatment.

On the transcriptional level, we observed a definite downregulation in the transcriptional activity of Cdh1 in Clone M-3^Z1 tumor, but not any significant regulation of either Cdh1 or Cdh2 in B16-F10 tumor. During EMT, alternative splicing of mRNA, different growth factors, and kinase activity affect the expression of EMT markers, which may explain the variation between B16-F10 and Clone M-3^Z1 in vivo [116]. For the future, it would be interesting to try different markers of EMT such as vimentin, fibronectin, snail 1 (snail), and snail2 (slug) to explore a possible EMT and how to affect this process to our benefit [65, 117]

5.6 Evaluation of the effect of G007-LK on a panel of human melanoma cells

The C57BL/6-derived B16 melanoma used in this thesis is a widely used and well-established tumor model [118]. B16-F10 cells share several melanocyte differentiation antigens that can be recognized of cytotoxic T-lymphocytes from human melanoma patients. In addition, human melanoma and B16-F10 can express MHC class II upon interferon γ (IFN- γ) treatment.

Nevertheless, there are differences between B16-F10 cells and human melanoma, such as varying levels of MHC class I, differences in survival upon melanoma growth and possible problems when culturing cells. Collectively, B16-F10 melanoma is a reasonable model for human

melanoma. We, therefore, wanted to explore further the effect of G007-LK observed in B16-F10 cells in a panel of human melanoma.

5.6.1 G007-LK-induced interference with WNT signaling in human melanoma

We set up a pilot experiment to investigate WNT target genes in the human melanoma cell lines.

We only produced immunoblots of only a subset of the human melanoma cell lines. In line with our studies on murine B16-F10 cells, the AXIN1 protein was stabilized in all analyzed cell lines upon treatment with G007-LK in the cytoplasmic fractions. In addition, all analyzed cell lines showed stabilization of TNKS1/2 proteins upon treatment; however, the loading control was

60 missing for Mel 1, Mel 4, WM1366, and LOX-IMVI, which means that we cannot fully trust the results of TNKS1/2 or non-phospho β-catenin.

When analyzing the WNT signaling target gene AXIN2, we observed a substantial upregulation in WM1366, WM938B, and WM451Lu upon treatment with LK. This treatment with G007-LK should not affect the AXIN2 target gene at any definite level. When analyzing the real-time RT-qPCR data, the instrument picked up some discrepancies in all triplets of all samples for AXIN2. There might have been contamination in the AXIN2 probe or technical contamination, which can explain the regulations. Noteworthy, the experiment has only been performed once, and further investigations are needed to conclude.

Collectively, our findings demonstrate the counteracting effect of G007-LK in WNT target genes.

Furthermore, we show the stabilization of AXIN1 and TNKS1/2 in all melanoma cells analyzed.

5.6.2 G007-LK-induced interference with YAP signaling in human melanoma

We prepared nuclear fractions for immunoblot analysis of WM938B, WM9, and FEMX-V to analyze YAP and TAZ proteins. As demonstrated by recent studies, YAP protein was not affected by treatment with tankyrase inhibitor in any cell line tested [25, 119]. However, TAZ protein level had a substantial reduction in WM938B and FEMX-V in response to G007-LK treatment, which is the opposite effect of what we observed in our in vitro experiments. YAP and TAZ share sequence similarities of 60%, but they still distinguish in interaction sites with

LATS1/2 and AMOT, TEAD binding, mRNA processing, and phosphorylation mechanisms [120]. This may explain the differences in the two paralog’s protein levels. Notably, the experiment has only been performed once, and we cannot draw any firm conclusions.

Additionally, Lamin B1is not equal for WM938B and FEMX-V, which may affect the results.

On the transcriptional level, the YAP target genes AMOTL2 and CTGF were both up-and-down regulated in the panel of human melanoma cell lines. Further investigation is necessary to find a correlation between melanoma cell lines that display the different gene expression regulations.

All cell lines except WM451Lu had a definite regulation of CTGF levels. For AMOTL2, all cell lines except WM852 had a significant regulation upon tankyrase inhibition.

Our findings demonstrate that G007-LK can regulate YAP signaling in a subset of human melanoma cell lines. For a deeper understanding, the regulation of AMOT proteins should be analyzed and more target genes for transcriptional regulation.

5.6.3 G007-LK-induced interference on MITF expression in human melanoma

In murine B16-F10, we observed an increase in nuclear MITF protein level in response to treatment with G007-LK. In contrast, we could not observe the same substantial increase in

61 nuclar MITF protein in either of the cell lines analyzed (WM793B, WM852, WM9, and FEMX-I) except from WM2239A. The cell lines inhabit different levels of YAP and MITF expression, in addition to other BRAF mutations (Appendix C, Supplementary Table 7), which may interfere with the results. MITF did not show any substantial regulation in the cytoplasm upon treatment.

MITF gene expression increased upon treatment with G007-LK in WM1366, WM938B, and WM9, and moderately decreased in WM793B. Since the real-time RT-qPCR data and

immunoblotting were pilot experiments, more analysis is needed to verify and strengthen these observations. The goal would be to organize the cell lines further on these characteristics and their response to tankyrase inhibition. Notably, the different cell lines represent different states of melanoma. WM1366 was initially classified as a primary melanoma, yet some research indicates that this cell line is becoming metastatic. LOX-IMVI, WM852, and WM9 were classified as invasive, WM266.4 as intermediate, WM1341 and WM2239A, and WM451Lu as proliferative melanoma cell lines. [121]. Hence, these melanoma cell lines should inhabit different gene expressions of MITF and, therefore, different responses to treatment.

It would be interesting to investigate any possible correlation between the target gene expressions of the cell lines and YAP expression, MITF expression, and BRAF mutations. One major

obstacle with immunotherapy is the “cold tumor” phenotype, meaning an inadequate response to treatment. BRAF inhibitors have been shown to increase T cell infiltration and recognition in melanoma, and combining BRAF inhibitors and immunotherapy is, therefore, an exciting approach [122]. Furthermore, research has demonstrated the role of MITF in immune cell

migration. Hence, MITF regulation should be taken into further consideration for immunotherapy in melanoma [123].

More studies are necessary to conclude, but we have not observed the nuclear stabilization of MITF in human melanoma cells (except for WM2239A) when exposed to tankyrase inhibition, as we did in the studies of murine B16-F10 cells.

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6. Conclusion

In this study, we investigated the effects of tankyrase inhibitor G007-LK on several distinct melanoma cells in vitro. Collectively, our results represented here show that G007-LK can (i) counteract WNT and YAP signaling in melanoma cells in vitro, (ii) accumulate MITF protein in the nucleus in vitro, independent of β-catenin, and (iii) affect MITF expression

lineage-dependently in human melanoma. Noteworthy, some of the analyses were pilot experiments and basic research, and further research is needed to strengthen our observations. We investigated the effect of tankyrase inhibition on YAP signaling, which displayed contradictory results between mouse and human melanoma cells regarding nuclear YAP protein. Also, we explored the possibilities of an EMT and MET when melanoma cells were exposed to G007-LK and WNT signaling-inducing ligand WNT3a. Here, we only saw significant regulations of E-cadherin and N-cadherin (the hallmarks of EMT) in Clone-M3^Z1 and not in B16-F10, suggesting a cell-type dependent regulation.

Altogether, tankyrase inhibition can suppress WNT and YAP signaling pathways, in addition, to driving melanoma cells towards a differentiated, non-invasive state. Our data suggest that tankyrase inhibition-mediated regulation of MITF should be further investigated as its

stabilization may help sensitize some melanoma patients to immune checkpoint blockade [2].

Collectively, this can provide pathway-directed anti-tumor effects combined with checkpoint inhibitor treatment in melanoma.

6.1 Future perspective

Some of the results in this thesis are basic research, making room for many interesting experiments for future investigation.

We speculate whether MITF is a direct target for tankyrase inhibition or if MITF and tankyrase are connected. To better understand how the G007-LK mechanism affects MITF and tankyrase protein level, it would be interesting to use immunoprecipitation (IP) to isolate MITF and

tankyrase antigen from a mixture, followed by immunoblot analysis for detection. It could also be helpful to utilize mass spectrometry (MS) analysis to assess whether MITF and tankyrase are connected.

Knockdown and over-expression experiments of MITF siRNA and treatment with tankyrase inhibitor and WNT activating ligands could be interesting to explore if this affects YAP expression and vice versa. Furthermore, we are curious to investigate the effect of G007-LK,

Knockdown and over-expression experiments of MITF siRNA and treatment with tankyrase inhibitor and WNT activating ligands could be interesting to explore if this affects YAP expression and vice versa. Furthermore, we are curious to investigate the effect of G007-LK,

In document Tankyrase inhibition regulates MITF expression in melanoma (sider 70-0)