SEGUIMIENTO AL POA 2014

My findings demonstrate the requirement of Ezh2 for melanoma initiation. I hypothesized whether this

might function through regulation of proliferation, since EZH2 has previously been associated with

proliferation of human melanoma cells in vitro (Fan et al., 2011; Luo et al., 2012a; 2013). Interestingly, I

observed a heterogeneity with respect to EZH2 expression in melanoma cells of both human biopsies as

well as on Tyr::N-Ras

Ink4a

skin tumors and metastases, ranging from high EZH2 expression to

almost background levels (Figure 16). EZH2 highly expressing cells significantly correlated with KI67-

positive cells, implicating EZH2-regulated proliferation of melanoma at primary tumor and metastatic

sites (Figure 25). To address the relevance of these findings for melanoma growth, I first blocked EZH2

activity in human melanoma cell cultures (Zipser et al., 2011), either by RNA interference-mediated

silencing (RNAi) or by chemical inhibition using GSK503. In agreement with my in vivo findings, RNAi

effectively reduced EZH2, while GSK503 application did not affect EZH2 levels. Both approaches,

however, led to a considerable loss of H3K27me3 (Figure 26a, b). Notably, neither RNAi nor prolonged

GSK503 application for 8 days did affect cell survival (Figure 26c, d). However, suppressing EZH2

activity stimulated a G1 cell cycle arrest (Figure 26e, f) and slowed down cell growth in culture (Figure

26g, h).

To functionally study EZH2-regulated proliferation in vivo, I quantified proliferative cells in tumors of

Tyr::N-Ras

Ink4a

mice, either after Ezh2 cKO or GSK503 application. Both approaches led to a

striking reduction of proliferative tumor cells (Figure 27). I next engrafted murine B16-F10 melanoma

cells into C57Bl/6 mice (Figure 28a). Depletion of Ezh2 function using RNAi or GSK503 significantly

reduced global H3K27me3 levels in vivo (Figure 28b - e). Importantly, EZH2 inactivation led to an

inhibition of tumor growth (Figure 28b, f). Finally, I engrafted Tyr::N-Ras

Ink4a

skin melanoma

(RIM)-derived cells into athymic nude Foxn1

mice (Figure 29a, h, k). These RIM allografts were

confirmed to express various melanocytic markers, reassuring their melanoma origin (Figure 30). RIMs

were first allowed to reach a considerable size for 15 to 25 days after transplantation (Figure 29a, f, j, m).

Subsequent TM-induced Ezh2 cKO in growing RIMs completely abolished Ezh2 protein and

H3K27me3, while GSK503 application significantly reduced H3K27me3 (Figure 29b - e). This was

associated with an inhibition of further tumor growth in both cKO and GSK503-treated samples (Figure

29b, f, g, i, j, l, m) highlighting Ezh2 inactivation as an efficient strategy for blocking melanoma growth.

Figure 25 # Heterogeneous high EZH2 expression correlates with KI67 positivity. (a - d) Immunofluorescent staining on human melanoma metastases sections (a) and melanoma sections of Tyr::N-RasQ61K Ink4a-/- mice (b - d) for EZH2 and ΚΙ67 to quantify a correlation between high EZH2 expression and KI67 positivity. White arrowheads, EZH2-high cells considered KI67-positive. Data are mean ± s.e.m. of n = 5 (a, b), mean ± s.e.m. of n = 8 (c), mean ± s.e.m. of n = 4 (d). P- values calculated with unpaired Student’s t-test. Scale bars, 50µm.

Figure 26 # EZH2 inactivation in human melanoma cells interferes with cell growth. (a) Western blot for EZH2 protein and H3K27me3 on whole cell lysates from 2 melanoma cell cultures (M010817, M050829) after EZH2 depletion with 3 different siEs or EZH2 inhibition using GSK503. (b) Immunofluorescent staining on M010817 for H3K27me3 after GSK503 treatment. (c, d) FACS analysis of M010817 and M050829 for Annexin V positivity after EZH2 depletion with siE or EZH2 inhibition using GSK503 to quantify induced apoptosis. (e, f) Cell cycle profiles of M010817 and M050829 using PI after EZH2 depletion with siE or EZH2 inhibition using GSK503 to quantify a G1 / (S + G2M) ratio. (g, h) Growth of M010817 and M050829 after EZH2 depletion with siE. PI, propidium iodide; Ve, vehicle. Data are mean ± s.e.m. of n = 3. P-values calculated with unpaired Student’s t-test (f), ANOVA and Fisher’s LSD-test (g, h). Scale bars, 25µm.

Figure 27 # Ezh2 ablation and GSK503 treatment attenuates melanoma cell proliferation in vivo. (a) Mouse genotypes and strategy as in (Figure 23) used to analyze the effect of conditional Ezh2 ablation on melanoma proliferation in Tyr::N-RasQ61K _Ink4a-/- _{mice. (b) Immunofluorescent staining on skin melanoma sections for Sox10 (control) or β-Gal}

(cKO) and Ki67 4 weeks after conditional Ezh2 ablation to quantify a proliferation rate. White arrowheads, Sox10- or β- Gal-positive / Ki67-positive cells. (c) Mouse genotypes and strategy as in (Figure 24) used to analyze the effect of temporary GSK503 treatment on melanoma proliferation in Tyr::N-RasQ61K Ink4a-/- mice. (d) Immunofluorescent staining on skin melanoma sections for Sox10 and Ki67 4 weeks after treatment start with vehicle or GSK503 to quantify a proliferation rate. White arrowheads, Sox10-positive / Ki67-positive cells. Data are mean ± s.e.m. of n = 5 (b), mean ± s.e.m. of n = 6 (Vehicle), n = 5 (GSK503) (d). P-values calculated with unpaired Student’s t-test. Scale bars, 50µm.

Figure 28 # Ezh2 inactivation interferes with murine B16-F10 melanoma growth. (a) Mouse genotypes and strategy used to s.c. engraft B16-F10 cells following Ezh2 silencing with shE to analyze the effect of Ezh2 depletion and GSK503 treatment on melanoma growth. (b) Representative macroscopic pictures of shCo, shE, and GSK503-treated B16-F10 isografts. (c) RT-qPCR for Ezh2 mRNA expression on lysed shCo, shE, and GSK503-treated B16-F10 tumors. (d, e) Western blot for Ezh2 protein and H3K27me3 on lysed shCo, shE, and GSK503-treated B16-F10 tumors to quantify loss of H3K27me3. (f) Growth of shCo, shE, and GSK503-treated B16-F10 isografts. Black arrow, GSK503 treatment start. s.c., subcutaneous. Data are mean ± s.e.m. of n = 3 (shCo), n = 4 (shE, GSK503) (c), mean ± s.e.m. of n = 3 (shCo, shE), n = 4 (GSK503) (d, e), mean ± s.e.m. of n = 5 (f). P-values calculated with ANOVA and Fisher’s LSD-test. Scale bars, 1mm.

Figure 29 # Ezh2 inactivation prevents growth of allografted Tyr::N-RasQ61K

Ink4a-/- melanoma. (a, h, k) Mouse

genotypes and strategy used to s.c. engraft and expand Tyr::N-RasQ61K _Ink4a-/- _{mice-derived melanoma cells in Foxn1}nu/nu

animals to analyze the effect of conditional Ezh2 ablation and GSK503 treatment on melanoma growth. (b) Representative macroscopic pictures of control, cKO, and GSK503-treated RIM-1 allografts. (c - e) Western blot for Ezh2 protein and H3K27me3 on lysed tumors to quantify loss of Ezh2 and H3K27me3. (f, g) Growth of control, cKO, and GSK503-treated RIM-1 allografts (f) and relative tumor volume of RIM-1, RIM-2, and RIM-3 allografts at endpoints (g). (i, j) Representative macroscopic pictures and growth of control, cKO, and GSK503-treated RIM-2 allografts. (l, m) Representative macroscopic pictures and growth of vehicle- and GSK503-treated RIM-3 allografts. Black arrows, time points of TM application / start of GSK503 treatment. RIM, Tyr::N-RasQ61K Ink4a-/- melanoma. Data are mean ± s.e.m. of n = 3 (Control), n = 4 (cKO, GSK503) (c - f), mean ± s.e.m. of n = 9 (Control, GSK503), n = 6 (cKO) (g), mean ± s.e.m. of n = 3 (j, m). P-values calculated with ANOVA and Fisher’s LSD-test. Scale bars, 1mm.

Figure 30 # RIM allografts are of a melanoma origin. (a) RT-qPCR for melanocytic lineage genes on lysed heart tissue, RIM-1, RIM-2, and RIM-3 tumors. (b) Immunofluorescent staining on XB2, Melan-a, and RIM-1 cells for Sox10 and Tyrp1. BF, bright field. Data are mean ± s.e.m. of n = 3. P-values calculated with ANOVA and Fisher’s LSD-test. Scale bars, 25µm.