API5 confers tumoral immune escape through FGF2-dependent cell survival pathway

Abstract

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5(low) cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies.

Figure 1. Identification of API5 as a candidate gene conferring immune resistance in TC-1 tumor cells in vitro and in vivo

(a) Left: TC-1/P3 (A17) cells were treated with siGFP (control) or siApi5 and the levels of Api5 protein analyzed. Right: The percentages of active caspase-3⁺ A17 cells were treated with siGFP or siApi5, generated during CTL assay in response to E7-specific CTL. Data presented are the representative of three independent experiments. (b) Left: TC-1/P0/no insert or TC-1/P0/Api5 were analyzed for expression of Api5, E7, or β-Actin. Right: The percentages of active caspase-3⁺ TC-1/P0/no insert or TC-1/P0/Api5 cells, generated during CTL assay. Data presented are the representative of three independent experiments (Means ± S.D). (c) Schematic representation illustrating in vivo challenge of A17 tumors and subsequent treatment protocol. Left: Western blot analysis of Api5 and β-Actin in siGFP- or siApi5-CNP treated A17 tumor mass. Right: The tumor volumes measured at day 25 in mice treated with either siGFP or siApi5-CNP in the presence or absence of adoptive transfer of E7-specific CTL. Each group contained 5 mice and all data shown are representative of 3 independent experiments. (d) Adoptive transfer protocol for E7-specific CTL following in vivo challenge of TC-1/P0/no insert or TC-1/P0/Api5 tumors. Tumor volume was measured over the course of 25 days after tumor challenge. Each group contained 5 mice and all data shown are representative of 3 independent experiments.

Figure 2. Identification of BIM as a pro-apoptotic molecule downregulated by API5

(a) Western blot analysis characterizing the expression of pro- and anti-apoptotic molecules in TC-1/P0/no insert and TC-1/P0/Api5 cells. (b) Proteasomal degradation of Bim in TC-1/P0/no insert or TC-1/P0/Api5 cells was assessed by Western blot in cells treated with or without MG132. (c) TC-1/P0 cells were transfected with siGFP or siBim and exposed to E7-specific CTL. Fractions of apoptotic tumor cells induced by CTL killing are represented by % activated caspase-3⁺ cells. (d) Putative MEK kinase intermediates upstream of Bim were analyzed by Western blot analysis. Expression of pAkt, Akt pp38 MAP kinase, p38 MAP kinase, pErk, Erk and β-Actin in the TC-1/P0/no insert and TC-1/P0/Api5 tumor cells are shown. (e) TC-1/P0/Api5 tumor cells were incubated with either DMSO or PD98059 for 12 hours and the level of pErk, total Erk, Bim and β-Actin was analyzed by Western blots. (f) TC-1/P0/Api5 tumor cells were incubated with DMSO, SB203580, API-2, LY294002, or PD98059 for 18 hours and the percentage of apoptotic TC-1/P0/Api5 tumor cells was measured following exposure to E7-CTL. (g) C57BL/6 mice were inoculated subcutaneously with 1 × 10⁵ TC-1/P0/Api5 cells/mouse and chitosan hydrogel loaded with either PD98059 or DMSO were intratumorally administered at day 7. One day later, mice were adoptively transferred with E7-CTL. Bar graphs represent tumor volumes at day 18 from TC-1/P0/Api5-challenged mice treated with or without PD98059-loaded chitosan hydrogel in the presence or absence of E7 CTL. The data are representative of three separate experiments and bar graphs represent the tumor volume of 5 mice in each group (Means ± S.D).

Figure 3. API5 controls immune resistance in human cancer cells

(a) API5 expression was determined in HeLa, CaSki, MCF-7, MDA-MB-231, DU145, PC-3, SNU-C4, SNU-368, HCT116, HepG2, A549, A375 and 526mel. (b) pERK, ERK, BIM, and β-ACTIN expression was assessed by Western blot in HeLa, PC-3, HCT116, and 526mel cells silenced with either siGFP or siAPI5. (c) HeLa cells stably transfected with SCT-E7 or 526mel tumor cells transfected with siGFP or siAPI5 were subject to CTL assays using murine E7-specific or human MART-1-specific CD8⁺ T cells, respectively. (d) HeLa/SCT-E7 or 526mel tumor cells treated with DMSO or PD98059 were subject to CTL assay using murine E7-specific or human MART-1-specific CD8⁺ T cells. (e) Expression of API5, pERK, ERK, BIM, and β-ACTIN in HEK293D^b, and A375 cells. (f) Left: HEK293D^b/no insert and HEK293D^b/API5 cells were subjected to CTL assays using E7 CTL. Right: A375 tumor cells transfected with empty vector or API5 were subject to CTL assays, (g) HEK293D^b/API5 or A375 tumor cells treated with DMSO or PD98059 were subject to CTL assays. The data are representative of three separate experiments (Means ± S.D).

Figure 4. API5 activates ERK through FGF2/FGFR1 pathway

(a) FGFR1 signaling following API5 expression is evaluated by Western blot analysis of pFGFR1, FGFR1, pPKCδ, PKCδ, pERK, and ERK expression in HEK293D^b/no insert and HEK293D^b/API5 cells. (b) mRNA expression analysis of FGF2, (c) the protein expression of API5, internal FGF2, and secreted FGF2 in the HEK293D^b/API5 cells versus HEK293D^b/no insert. (d) The amount of FGF2 secreted into the media was measured by flow cytomix. (e) Western blot results were shown in siGFP- or siFGF2-transfected HEK293D^b/API5 cells. (f) Western blot analysis of expression in IgG isotype controls or FGF2 antibody treated HEK293D^b/API5 cells. (g) IgG antibody- or FGF2 antibody-treated HEK293D^b/API5 cells were subject to CTL assays with E7-CTL. (h) Scatter plot graph show the linear relationship between expressing API5 (x-axis) and FGF2 (y-axis) in all tumor cell lines tested in Fig. 3a. (i) mRNA expression of FGF2,(j) Protein expression of API5 and internal FGF2 (as surrogates for all FGF2) and (k) secretion of FGF2 was monitored in A375 cells transfected with no insert or API5 as well as HeLa and 526mel tumor cells silenced with either siGFP- or siAPI5. (l) Top, The percent killing of A375/API5, HeLa, or 526mel cells, treated with either IgG antibody- or FGF2 antibody, was measured in CTL assays. Bottom, Western blot results in IgG- or FGF2 antibody treated A375/API5, HeLa, and 526mel cells are shown.

Figure 5. Identification of PKCδ as an immediate target gene for API5 controlling CTL resistance

(a) The protein expression in HeLa/SCT-E7 or 526mel tumor cells was analyzed in the treatment of 0, 10, 100, or 1000 nM of rottlerin. (b) The percentage of caspase-3⁺ apoptotic cells in siGFP- or siAPI5-transfected HeLa/SCT-E7 (Top) and 526mel cells (bottom) following exposure to antigen-specific CTL was shown in the presence of increasing doses of rottlerin. (c) Left, protein expression was assessed in HeLa/SCT-E7 cells silenced with siGFP or siPKCδ. Right, levels of caspase-3⁺ cells in HeLa/SCT-E7 cells silenced with siGFP or siPKCδ following exposure to E7-CTL. (d) Left, protein expression was assessed in 526mel cells silenced with siGFP or siPKCδ. Right, levels of caspase-3⁺ cells in 526mel cells silenced with siGFP or siPKCδ following exposure to MART-1-specific CD8⁺ T cells. (e) Schematic representation illustrating in vivo xenogeneic challenge of 526mel tumors and subsequent treatment protocol. (f) tumor volume was analyzed at day 31. The data are representative of three separate experiments.

Figure 6. Inhibition of API5 renders tumor susceptible to immune-mediated control

(a) Schematic of the therapy regimen in mice implanted with 526mel melanoma cancer cells. NOD/SCID mice were inoculated subcutaneously with 1 × 10⁶ 526mel cells/mouse. Seven days following tumor challenge, siRNA CNP targeting either GFP or API5 (5 μg/mouse) was injected intravenously for three consecutive days. Approaching the tenth day, mice received adoptive transfer with 1 × 10⁷ MART-1-specific CTL. This treatment regimen was repeated for 6 cycles. (b) Tumor growth of mice inoculated with 526mel. (c) Tumor weight of mice at 49 days after challenge. (d) Western blot analysis of expression in tumor mass. (e) Flow cytometry analysis of the frequency of CFSE-labeled, MART-1-specific CTL in the tumors of mice that received adoptive transfer. (f) the frequency of apoptotic cells in the tumors of siGFP- or siAPI5-treated mice, with or without adoptive transfer of MART-1-specific CTL.

Figure 7. Schematic interpretation of API5-mediated immune resistance

Overexpression of API5 upregulates FGF2 and FGFR1 signaling. Subsequent activation of PKCδ leads to ERK phosphorylation and facilitates ubiquitin-dependent degradation of the pro-apoptotic molecule, BIM.