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.

62