Wnt5A promotes an adaptive, senescent-like stress response, while continuing to drive invasion in melanoma cells

Abstract

We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF(V600E) mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence-associated β-galactosidase (SA-β-gal), senescence-associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA-β-gal and SAHF, these Wnt5A-high cells are able to colonize the lungs in in vivo tail vein colony-forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.

Keywords: BRAF; Wnt5A; metastasis; senescence; therapy resistance.

Figure 1. Wnt5A increases markers of senescence in melanoma cells

(A) Wnt5A-high cells were positive for SA-β–galactosidase whereas Wnt5A-low cells were largely dead at 5 days post- treatment with PLX4720. (B) SA-β-galactosidase staining in Wnt5A high and Wnt5A low cells after irradiation. (C) Immunofluorescent analysis of PML bodies following 5 days of treatment with PLX4720. (D) Quantification of changes in PML bodies in response to 5 days of PLX4720 treatment. (E) Immunofluorescent analysis of PML 5 days post irradiation. (F) Quantification of changes in PML bodies in Wnt5A-high and –low cells 5 days following irradiation. (G) Treating Wnt5A-low cells with rWnt5A results in an increase in basal levels of SA-β–galactosidase, as well as an increase after irradiation. (H) Wnt5A high cells express basal levels of SA-β–galactosidase and knockdown of Wnt5A results in a decrease in the number of SA-β–galactosidase positive cells before and after irradiation.

Figure 2. Wnt5A regulates p21 expression in melanoma

(A) Western analysis of cell cycle regulators, p16 and p21 in Wnt5A-high and Wnt5A-low cells. (B) Treatment of Wnt5A-low cells with rWnt5A increases p21 expression. Phospho-CAMKII is shown as a marker of Wnt5A signaling. (C) Knockdown of Wnt5A in Wnt5A-high cells decreases p21 expression by Western analysis. (D) p21 expression increases in Wnt5A high cells following irradiation. (E,F) p21 expression remains high in (E) Wnt5A-high melanoma cells following 5 days of treatment with PLX4720, but not in (F) Wnt5A-low melanoma cells. (G) WM983B parental (PAR) and PLX4720- resistant (RES) WM983B cells were analyzed for Wnt5A and p21. (H) Patient samples with > 30% and <30% response by RECIST were stained for p21 expression by IHC.

Figure 3. Senescent-like cells retain invasive properties

(A) Wnt5A-high cells continue to invade 1, 3, and 5 days post-irradiation, measured using an invasion assay. The fold change in invasion at each time point was calculated compared to invasion on day 0. (B) Knockdown of Wnt5A in highly invasive (FS4) cells results in the inability of the cells to invade pre- and post- irradiation, measured using an invasion assay. (C) Wnt5A-low cells treated with rWnt5A in a 3D spheroid assay showed increased invasion in the absence of irradation, and retain this ability to invade post irradiation. (D,E) BrdU incorporation assays of Wnt5A-high (FS) cells indicate that there is little difference in the percentage of cells in G2/M between invaded and non-invaded cells either before (D) or after (E) irradiation. (F) Five days following irradiation, cells were plated into an invasion assay for 48 h. Invaded (bottom chamber) Wnt5A-high cells stain positive for SA-β–galactosidase. (G) Wound healing assay demonstrates that Wnt5A high cells invading into the scratch wound stain positive for SA-β–galactosidase. Wnt5A low cells neither invade, nor stain positive for SA-β–galactosidase. (H) Wnt5A high cells are invasive following treatment with PLX4720 for five days. (I) Time-lapse imaging of PLX4720 treated Wnt5A high and low cells labeled with the fluorescent marker of SA-β–galactosidase, C₁₂FDG, and subjected to a wound-healing assay (see also supplemental movies).

Figure 4. Wnt5A high cells retain clonogenic properties in vitro and in vivo following stress

(A) FS4 cells were injected via the tail vein into mice to perform in vivo colony forming assays. Shown are two representative lungs from mice four weeks after injection with irradiated or non-irradiated invasive cells, and graphical representation of the severity of metastases in both groups (n=10 mice/group). (B) Frozen sections of mouse lung were stained for SA-β–galactosidase. Nests of tumor cells are indicated by arrows. (C) SAHF were identified and quantitated in sections from mouse lungs. (D) Five days following irradiation, there is still a significant increase in the percentage of irradiated cells in G2/M compared to untreated cells. (E) Highly invasive cells were irradiated, sorted into G0/1 and G2/M, and were seeded into colony forming assays. (F) Colonies from G2/M sorted cells retain higher expression of p21 and Wnt5A. (G) Overlay of SA-β–galactosidase and Ki67 in mouse lungs from mice injected with irradiated cells.