Paradoxical Role for Wild-Type p53 in Driving Therapy Resistance in Melanoma

Abstract

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

Keywords: Wnt5A; aged microenvironment; melanoma; slow-cycling phenotype; therapy resistance; tumor microenvironment; wild-type 53.

Figure 1.. Wnt5A promotes a slow cycling phenotype through wild type p53.

(A) FACS analysis of slow-cycling cell populations in invasive Wnt5A high (1205Lu, FS4, FS5) and proliferative Wnt5A low (yellow bars) (FS13, FS14, FS12, WM164) melanoma cells. (ANOVA, multiple comparisons) (B) qPCR analysis of slow-cycling and cycling populations for Wnt5A mRNA. (C) FACS analysis of changes in slow-cycling cell populations following knockdown of Wnt5A using shRNA at day 3, 6, and 8 in FS5 invasive melanoma cells. (D) Western blot analysis of markers of therapy resistance, slow-cycling phenotype, and cell cycle regulation in Wnt5A high and low human melanoma cells. Active glycosylated Wnt5A is marked with arrow. β-tubulin used as a loading control. (E) Slow-cycling and cycling populations were sorted by flow cytometry and analyzed by qPCR for p53 mRNA. (F) FACS analysis of slow-cycling cells following p53 KD. (G) FACS analysis of slow-cycling cells following addition of rWnt5A to p53 knock down melanoma cells. Data are represented as mean ± SEM. (H) Expression heatmap for top genes most involved in enriched function/regulators (F,R = number of functions/regulators in which the gene is involved). * genes associated with invasion, stem cells, therapy resistance and proliferation. (I) Enriched functions activated/inhibited in WNT5A/TP53 high vs low (N- number of genes in the function. Z = activation z-score predicted by IPA). Also see Figures S1–S2.

Figure 2.. Wnt5A regulates p53 expression via MDM2 phosphorylation.

(A) Western blot analysis of p53 expression following knockdown of Wnt5A in invasive melanoma cells with shRNA. HSP90 was used as a loading control. (B) Quantification of change in p53 half-life following shRNA knockdown of Wnt5A assessed following treatment with cycloheximide. (C) Changes in half-life of p53 upon Wnt5A knockdown. (D) Western blot of proteins known to regulate p53 expression and function in melanoma. GAPDH used as a loading control. (E) Western blot analysis of MDM2, MDM4, and p53 expression in Wnt5A high (FS4 and FS5) and low (FS13 and FS14) melanoma cells following DNA damage induced by doxorubicin (1μg/mL). HSP90 used as a loading control. (F) Western blot analysis of nuclear and cytoplasmic fractions for p-MDM2ser395, total MDM2, and p53 expression. HSP90 and Histone H3 used as loading control for cytoplasmic and nuclear fractions, respectively. (G) Proximity ligation assay for the interaction of p53 and p-MDM2ser395 in 1205Lu melanoma cells treated with rWnt5A. (H) Quantification of increased p53-pMDM2^ser395 interaction in proximity ligation assay in 1205Lu and WM793 cells +/− rWnt5A (200 ng/mL). Data are represented as mean ± SEM. Also see Figure S3.

Figure 3.. Wild type p53 expression increases following DNA damage and targeted therapy.

(A) Western blot analysis and quantification of basal iASPP expression in Wnt5A high (FS4 and FS5) and low (FS13 and FS14) expressing cells. (B) Western blot analysis of nuclear and cytoplasmic fractions from Wnt5A high (FS4, FS5) and Wnt5A low (FS13, FS14) melanoma cells for iASPP expression. (C) Western blot analysis of iASPP and pPKC in melanoma cells following DNA damage induced by doxorubicin (1μg/mL). (D) Quantification of apoptotic cells using AnnexinV and propidium iodide in Wnt5A high (FS4 and FS5) and Wnt5A low (FS14) cells following doxorubicin 1 ug/mL treatment. Data are represented as mean ± SEM. (E) Schematic of how Wnt5A may be activating iASPP in metastatic melanoma cells. (F) Western blot analysis of iASPP expression in melanoma cells following Wnt5A KD. Also see Figure S3.

Figure 4.. Aged microenvironment promotes a slow cycling phenotype.

(A) Flow cytometry analysis of changes in slow-cycling populations following treatment of WM35 melanoma cells with conditioned media from young (25–35 yrs) and aged (55–65 yrs) fibroblasts (two-way ANOVA sidak’s multiple comparison test). (B) Western blot analysis of Wnt5A and p53 (also see Fig. S4E) expression in Yumm1.7 derived tumors grown in young (8 wks) and aged (>52 wks) C57BL/6 mice (unpaired two-tailed t-test). (C) IHC for p53 in mouse tumors from young and aged mice for p53. (D) Quantification of p53 positive cells using ImageJ in young and aged mouse tumors. (E) Yumm1.7 cells labelled with GFP were stained with a membrane dye, PKH26, and injected into young (8 wks) and aged (>52 wks) C57BL/6 mice (2.5x10⁵cells/mouse). (F) At week 5 post injection the tumors were analyzed for GFP+ cells that were PHK26+ by flow cytometry. (G) Flow cytometry analysis of intensity of PKH26 in GFP⁺ cells in tumors from young and aged tumor bearing mice. (H) Analysis of lungs from young and aged tumor bearing mice for cells that are GFP⁺ and PKH26⁺. Data are represented as mean ± SEM. Also see Figure S4.

Figure 5.. p53 promotes therapy resistance.

(A) Tumor volume of aged (>52 wks) C57BL/6 mice injected with Yumm1.7 cells labeled with mCherry. At day 22, mice were treated with +/− PLX4720 (200mg/kg)/PD0325901(7mg/kg) and on day 23 mice were treated +/− a single intra-tumoral dose of pifithrin-α (2mg/kg). (E) IHC of p53 expression in a patient tumor pre- and post-treatment with BRAF/MEKi (Trametanib/Debrafenib). (B) Western blot analysis of p53 and p21 expression in human melanoma cells treated with 5µM pifithrin-α. (C) FACS analysis of slow cycling populations in human melanoma cell populations day 5 following treatment with 5µM pifithrin-α (one-tailed unpaired t-test analysis) (also see Fig S5C). (D) Western of Wnt5A high melanoma cell lines treated with +/− 5 μM pifithrin-α +/− 1 μg/mL doxorubicin. Quantification of changes in p53 and p21 following treatment with doxorubicin and doxorubicin combined with pifithrin-α. Data are represented as mean ± SEM. Also see Figure S5.

Figure 6.. Wild type p53 is increased following therapy.

(A) p53 was knocked down in Yumm1.7 cells using shRNA. Cyclophilin A used as a loading control. (B) Tumor volume of aged (>52 wks) C57BL/6 mice injected with Yumm1.7 shp53 KD and control cells. Mice were treated with +/− PLX4720 (200mg/kg) /PD0325901(7mg/kg). Data are represented as mean ± SEM. (C) IHC for p53 expression in p53 KD and control tumors from mice treated with +/− BRAF/MEKi (PLX4720/PD0325901). IHC for p53 and p21 expression in WT p53 PDX tumors WM4351 (D) and WM4298 (E) from mice treated with BRAF/MEKi therapy compared to control. (F) IHC for p53 in patient tumor before and after BRAF/MEKi therapy. Also see Figure S6 and Table S1.