Fig 1.
Necroptosis is induced in tumors in vivo and RIPK3 expression is progressively lost during tumorigenesis.
(A) RIPK3 mRNA levels are decreased during progressive stages of colorectal, gastric, ovarian, prostate, adrenocortical, and breast cancers. The results shown here are in part based upon data generated by the TCGA Research Network. See S6 Table for details about the studies. (B) RIPK3 expression is lost during progressive in vivo passages of mouse tumor xenografts of indicated cancer cell lines. (C) As in (B), except data from 47 different cell lines are presented as a heatmap. (D) Necroptosis is induced in tumors in vivo and RIPK3 expression is progressively lost during tumorigenesis. Necroptosis induction is determined by the MLKL p-S358. Ovarian PDX lysates obtained at the indicated in vivo passages were immunoblotted with the indicated antibodies. (E) Quantification of the RIPK3 and p-MLKL levels shown in (D) and their normalization to actin. (F) Ovarian PDX cells at indicated in vivo passages were cultured and treated with TSZ to induce necroptosis. Cell survival was determined 24 hours after treatment, using CellTiterGlo. The underlying data can be found in S1 Data. PDX, patient-derived xenograft; p-MLKL, phospho-MLKL S358; TSZ, TNFα+SM-164+zVAD.fmk.
Fig 2.
Necroptosis sensitivity screen in 941 cancer cell lines identifies drivers of necroptosis resistance.
(A) Outline of the high-throughput screening for differential necroptosis sensitivity in 941 human cancer cell lines. (B) Differential sensitivity of 941 cancer cell lines to TSZ-induced necroptosis across 28 tissues of origin. (C) Numbers and percentages of necroptosis-resistant/sensitive cell lines. (D) Low-throughput confirmation of the screen observations regarding loss of RIPK3 expression and necroptosis resistance, as judged by lack of p-MLKL induction. Indicated cancer cell lines were treated with TSZ for 6 hours and cell lysates were immunoblotted with indicated antibodies. Note that RIPK1, RIPK3, and MLKL levels decrease in lane 2 because of induction of necroptosis, formation of amyloid-like necrosome structure, and translocation of these proteins into a detergent-insoluble fraction. (E) Low-throughput confirmation of the screen observations regarding necroptosis resistance. Indicated cells were treated with indicated treatments and cell survival was measured 16 hours later using CellTiterGlo. Means ± SEM are shown. (F) Genome-wide Pearson correlation analysis of TSZ-IC50 values versus gene expression values across 941 cell lines identifies genes, the expression of which negatively (e.g., RIPK3) and positively (e.g., AXL) correlates with necroptosis resistance. Top genes, the expression of which positively correlates with necroptosis resistance (red box), are listed. The underlying data can be found in S1 Data. p-MLKL, phospho-MLKL S358; TSZ TNFα+SM-164+zVAD.fmk; UT, untreated.
Fig 3.
AXL overexpression in cancer cell lines correlates with loss of RIPK3 expression and gain of necroptosis resistance.
(A) High AXL expression levels are enriched in cancer cell lines fully resistant to necroptosis. GDSC database was employed for the analysis. Means, 10–90 percentile data points ± SEM are shown with t test p-values. (B) High TYRO3 expression levels are enriched in cancer cell lines fully resistant to necroptosis. GDSC database was employed for the analysis. Means, 10–90 percentile data points ± SEM are shown with t test p-values. (C) High AXL expression predicts low RIPK3 expression levels. GDSC database was employed for the Pearson and Spearman correlation analyses. (D) High AXL/TYRO3 expression positively correlates with low RIPK3 expression and high TSZ-IC50 levels (resistant to necroptosis). Heatmap showing clustering of z-score values for TSZ-IC50 versus AXL, TYRO3, and RIPK3 expression levels across 941 cell lines. Numbers indicate clusters described in the text. (E) Inhibition of AXL in cancer cell lines can rescue loss of RIPK3 expression. qRT-PCR and western blotting analysis of RIPK3 expression in A375 and SkMel28 cell lines following 4 days of AXL inhibition by 1 μM of BMS-777607. These cell lines were selected because they did not show significant cell death following treatment with this inhibitor. (F) Inhibition of AXL in cancer cell lines can rescue loss of necroptosis sensitivity. A375 and SkMel28 cells were treated with indicated concentrations of BMS-777607 for 4 days. Drugs were washed out and necroptosis was induced by 24 h treatment with 25 ng/mL TNFα + 0.5 μM SM-164 + 30 μM zVAD.fmk. Cell survival was determined using CellTiterGlo assay. The underlying data can be found in S1 Data. GDSC, Genomics of Drug Sensitivity in Cancer; qRT-PCR, quantitative real-time PCR; TSZ, TNFα+SM-164+zVAD.fmk; UT, untreated.
Fig 4.
Necroptosis sensitivity screen in 941 cancer cell lines identifies BRAF as a mutational driver of RIPK3 expression loss and gain of necroptosis resistance.
(A) Mutational drivers of necroptosis resistance in cancer. z-score analysis of the fold mutation enrichment in NR versus NS cancer cell lines. BRAF is a top oncogene among genes, the mutation of which is enriched in NR cells. The x-axis depicts the different types of mutations (e.g., amplification, deletion, missense) found per gene. Top hits are indicated. (B) Volcano plot showing the results of the Fisher’s exact test analysis for the mutational enrichment data shown in (A). Top hits are indicated. (C) BRAF-activating mutations (BRAF-MUT, e.g., V600E mutation) predict loss of RIPK3 expression in cancer. GDSC database was employed in the analysis. All BRAF-activating mutations were pooled into one group (BRAF-MUT). (D) Inhibition of BRAF in melanoma patients can rescue loss of RIPK3 expression. RIPK3 mRNA levels are increased in 58.3% of melanoma patient tumor biopsies following treatment with BRAF inhibitors Dabrafenib or Vemurafenib. Inset shows percentages of patients with significant changes in RIPK3 expression (Dataset GEO ID: GSE50509). (E) Inhibition of BRAF in cancer cell lines can rescue loss of RIPK3 expression. qRT-PCR and western blotting analysis of RIPK3 expression in ES2 and SkMel28 cell lines following 4 days of BRAF inhibition by 1 μM of TAK-632. These cell lines were selected because they did not show significant cell death following treatment with this inhibitor. The experiments were repeated two times. Bar graphs show means ± SEM with t test p-values. (F) Inhibition of BRAF in cancer cell lines can rescue loss of necroptosis sensitivity. A375 and SkMel28 cells were treated with indicated concentrations of Vemurafenib or TAK-632 for 4 days. Drugs were washed out and necroptosis was induced by 24-hour treatment with 25 ng/mL TNFα + 0.5 μM SM-164 + 30 μM zVAD.fmk. Cell survival was determined using CellTiterGlo assay. The underlying data can be found in S1 Data. BRAF-MUT, BRAF-activating mutation; BRAF-WT, BRAF wild-type; GDSC, Genomics of Drug Sensitivity in Cancer; NR, necroptosis-resistant; NS, necroptosis-sensitive; qRT-PCR, quantitative real-time PCR; TSZ, TNFα+SM-164+zVAD.fmk; UT, untreated
Fig 5.
Role of oncogenes in the escape from necroptosis in cancer.
Oncogenic BRAF (e.g., V600E mutation), AXL overexpression, and other oncogenic factors may promote escape from necroptosis via suppression of RIPK3 expression during tumorigenesis.