CPT1A-mediated fatty acid oxidation confers cancer cell resistance to immune-mediated cytolytic killing

Abstract

Although tumor-intrinsic fatty acid β-oxidation (FAO) is implicated in multiple aspects of tumorigenesis and progression, the impact of this metabolic pathway on cancer cell susceptibility to immunotherapy remains unknown. Here, we report that cytotoxicity of killer T cells induces activation of FAO and upregulation of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme of FAO in cancer cells. The repression of CPT1A activity or expression renders cancer cells more susceptible to destruction by cytotoxic T lymphocytes. Our mechanistic studies reveal that FAO deficiency abrogates the prosurvival signaling in cancer cells under immune cytolytic stress. Furthermore, we identify T cell-derived IFN-γ as a major factor responsible for induction of CPT1A and FAO in an AMPK-dependent manner, indicating a dynamic interplay between immune effector cells and tumor targets. While cancer growth in the absence of CPT1A remains largely unaffected, established tumors upon FAO inhibition become significantly more responsive to cellular immunotherapies including chimeric antigen receptor-engineered human T cells. Together, these findings uncover a mode of cancer resistance and immune editing that can facilitate immune escape and limit the benefits of immunotherapies.

Keywords: cancer metabolism; carnitine palmitoyltransferase 1A; cellular immunotherapy; fatty acid oxidation; therapeutic resistance.

Fig. 1.

T cell–mediated cytotoxicity induces FAO elevation in cancer cells. B16 melanoma cells were cocultured with Pmel cells for 6 h. The FAO rate in cancer cells was determined by measuring the oxygen consumption rate (OCR) using Seahorse assays following extensive wash to remove T cells (A). The expression of CPT1 enzymes in B16 melanoma (B) or RM1-OVA prostate cancer cells (C) was examined by immunoblotting. Densitometry analysis of immunoblot replicates for CPT1A expression is shown. Data are representative of at least three independent experiments. *P < 0.05.

Fig. 2.

CPT1A deficiency sensitizes cancer cells to T cell killing. (A) Deletion of CPT1A in B16 with CRISPR-Cas9 was confirmed by immunoblotting. (B) Loss of CPT1A impairs FAO in B16 cells, indicated by OCR reduction in B16^{CPT1A KO} cells. (C) LDH assays of B16^WT or B16^{CPT1A KO} cells following coculture with Pmel cells. (D) B16 cells were treated with Etomoxir for 16 h prior to coculture with Pmel T cells and then subjected to cytotoxicity assays. (E) Immunoblotting validation of CPT1A deletion in CRISPR-Cas9-transfected RM1-OVA cells. RM1-OVA^WT or RM1-OVA^{CPT1A KO} cells were exposed to OVA-specific OT-I cells at a ratio of 1:20 for 6 h, followed by immunoblotting analyses of cell death (F) and proapoptotic or prosurvival signaling (G). Cells collected before coculture with OT-I cells were used as controls (0 h). Data are representative of at least three independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig. 3.

Lack of CPT1A renders metastatic melanoma susceptible to T cell therapy. C57BL/6 mice (n = 5) were injected with B16^WT or B16^{CPT1A KO} tumor cells (2 × 10⁵, s.c.) on day 0. Mice received two doses of Pmel T cells on days 4 and 7 (10⁷ cells, i.v.) or left untreated. Tumor growth (A) and animal survival (B) were followed. (C) Tumor-bearing mice were treated with Pmel T cells on day 17 post-tumor inoculation; tumor tissues were collected on day 21 and subjected to TUNEL assays. The frequencies of IFN-γ⁺ or IFN-γ⁺ TNF-α⁺ Pmel cells (D) or IFN-γ⁺ or IFN-γ⁺ TNF-α⁺ endogenous CD8⁺ T cells (E) in tumors were assessed by intracellular cytokine staining and flow cytometry analysis (gating on total cells in tumor tissue). (F) C57BL/6 mice (n = 5) were established with experimental lung metastases by inoculating B16^WT or B16^{CPT1A KO} tumor cells (1 × 10⁵ cells, i.v.) on day 0. Mice received Pmel T cells (10⁷ cells, i.v.) on day 8 or left untreated. Tumor nodules in the lungs were quantified on day 19. (G) Colony-formation assays were performed using lung cell suspensions to examine the frequency of metastatic cells. (H) The frequency of IFN-γ⁺ and IFN-γ⁺TNF-α⁺ CD8⁺ T cells in the lungs was analyzed by flow cytometry analysis (gating on total cells in lung tissues). Data are representative of three independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Fig. 4.

Pharmaceutical inhibition of FAO improves the antitumor efficacy of T cell therapy. Male mice with OVA-expressing RM1^WT or RM1^{CPT1A KO} prostate tumors received OT-I cells on days 2, 4, and 7 after tumor implantation (A). Prostate tumor-bearing mice (n = 5) received OT-I cells on days 9 and 12 following tumor implantation. Tumor tissues were collected 2 d after treatment for TUNEL assays (B). Infiltration and activation of OT-I cells in the tumors were assessed (C). Mice (n = 5) with B16^WT or B16^{CPT1A KO} melanomas were treated with Pmel T cells on days 4 and 8 post-tumor implantation (day 0). Etomoxir (30 mg/kg, i.p.) was administered daily starting from day 3. Tumor growth (D) and animal survival (E) were followed. (F) Melanoma-bearing mice received Pmel T cells when tumor sizes have reached 8 to 10 mm in diameters. Etomoxir treatment was initiated 2 d before T cell therapy and given daily for a total of seven doses. Tumors were collected 4 d after T cell therapy for TUNEL assays. Intracellular staining and flow cytometry analyses were performed to evaluate the activation of adoptively transferred Pmel T cells (G) or endogenous CD8⁺ T cells (H) in the tumors. Data are representative of three independent experiments. *P < 0.05. **P < 0.01. ****P < 0.0001.

Fig. 5.

T cell–derived IFN-γ up-regulates CPT1A-mediated FAO in an AMPK-dependent manner. (A) B16 tumor cells were stimulated with IFN-γ at indicated concentrations for 24 h. The FAO activity was determined by measuring conversion of ³H-palmitic acid to ³H₂O. (B) The expression of CPT1 enzymes in IFN-γ-treated B16^WT or B16^{CPT1A KO} cells was examined by immunoblotting. (C) Analysis of CPT1A, AMPKα, or phosphorylation of AMPKα and ACC in B16 cells after treatment with IFN-γ. (D) B16 cells and Pmel cells were plated into the lower and upper chambers of the transwells, respectively. The FAO activity in B16 cells was determined by diffusion assays after coculture. (E) Immunoblotting analysis of the levels of CPT1A, phosphorylated AMPKα, AMPKα, and PPARα in B16^WT or B16^{CPT1A KO} cells after coculture with T cells in transwells plates. (F) B16 cells were cocultured with activated T cells in transwell assays in the presence or absence of IFN-γ-neutralizing antibodies, followed by immunoblotting analysis of CPT1A, pAMPKα, or HADHA in tumor cells. (G and H) B16 tumor cells were treated with IFN-γ (5 ng/mL) in the presence of the AMPKα inhibitor Compound C (G) or PPAR inhibitor GW6471 (H) for 24 h. Immunoblotting was performed to determine the levels of CPT1 enzymes or pAMPKα. Data are representative of three independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig. 6.

Blockade of FAO improves human breast cancer responsiveness to CAR T therapy. (A) The publicly available data on Oncomine were used to analyze the correlation of CPT1A mRNA expression and survival in patients with breast cancer. Kaplan–Meier survival and Gehan–Breslow–Wilcoxon test of two groups were performed using GraphPad PRISM software. (B) Human breast cancer MDA-MB-231 cells were stimulated with IFN-γ and analyzed for expression of CPT1 enzymes, pAMPKα, PPARα, or HADHA. (C) FAO analysis of MDA-MB-231 cells after IFN-γ stimulation at different concentrations. (D) Knockdown (KD) of CPT1A in MDA-MB-231 cells by short hairpin RNA (shRNA) was confirmed in comparison with those transduced with scramble shRNA. (E) FAO activity in MDA-MB-231^Scram or MDA-MB-231^{CPT1A KD} cells was measured by quantifying the conversion of ³H-palmitic acid to ³H₂O. (F) MDA-MB-231 cells with or without CPT1A KD were cocultured with B7-H3 CAR T cells for 48 h followed by MTT assays. (G) NSG mice (n = 5) with MDA-MB-231 tumors with or without CPT1A KD were treated with B7-H3 CAR T cells (2 × 10⁶ cells, i.v.) 1 wk after tumor implantation. (H) NSG mice with MDA-MB-231 tumors were treated with B7-H3 CAR T cells 5 wk after tumor implantations. Etomoxir treatment was initiated 1 d before CAR T therapy and administered daily during the course of study. Data are representative of three independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. (I) Schematic illustration of tumor resistance to immune cytolysis by IFN-γ-induced FAO in cancer cells. Upon recognition and destruction of tumor targets, IFN-γ-derived from activated T cells results in FAO elevation in cancer cells by inducing CPT1A expression via AMPKα activation. Activation of this metabolic pathway confers cancer cell resistance to immune effector cells by promoting prosurvival signaling, e.g., upregulation of Bcl-2 and Bcl-XL and downregulation of Bax. Targeted inhibition of CPT1A or blockade of FAO (e.g., Etomoxir) can enhance proapoptotic signaling, thereby potentiating tumor sensitivity to immune killing.