FOXA1 prevents nutrients deprivation induced autophagic cell death through inducing loss of imprinting of IGF2 in lung adenocarcinoma

Abstract

Lung cancer remains one of the most common malignancies and the leading cause of cancer-related death worldwide. Forkhead box protein A1 (FOXA1) is a pioneer factor amplified in lung adenocarcinoma (LUAD). However, its role in LUAD remains elusive. In this study, we found that expression of FOXA1 enhanced LUAD cell survival in nutrients deprived conditions through inhibiting autophagic cell death (ACD). FOXA1 bound to the imprinting control region of insulin-like growth factor 2 (IGF2) and interacted with DNA methyltransferase 1 (DNMT1), leading to initiation of DNMT1-mediated loss of imprinting (LOI) of IGF2 and autocrine of IGF2. Blockage of IGF2 and its downstream insulin-like growth factor 1 receptor (IGF1R) abolished the protective effect of FOXA1 on LUAD cells in nutrients deprived conditions. Furthermore, FOXA1 suppressed the expression of the lysosomal enzyme glucocerebrosidase 1 (GBA1), a positive mediator of ACD, through ubiquitination of GBA1 enhanced by IGF2. Notably, FOXA1 expression in A549 cells reduced the efficacy of the anti-angiogenic drug nintedanib to inhibit xenograft tumor growth, whereas a combination of nintedanib with IGF1R inhibitor linsitinib or mTORC1 inhibitor rapamycin enhanced tumor control. Clinically, high expression level of FOXA1 protein was associated with unfavorable prognosis in LUAD patients of advanced stage who received bevacizumab treatment. Our findings uncovered a previously unrecognized role of FOXA1 in mediating loss of imprinting of IGF2, which confer LUAD cells enhanced survival ability against nutrients deprivation through suppressing autophagic cell death.

Fig. 1. FOXA1 expression conferred a survival advantage in LUAD cells in starvation.

A The endogenous mRNA levels of FOXA1 in LUAD cells were determined using RT-PCR. Mean ± SD, n = 3. B The endogenous protein levels of FOXA1 in LUAD cells were determined using western blotting. C The mRNA levels of FOXA1 in loss-of-function or gain-of-function LUAD cell models were measured using RT-PCR. Mean ± SD, n = 3. D The protein levels of FOXA1 in loss-of-function or gain-of-function LUAD cell models were measured using western blotting. E, F, G Cell survival measured using colony formation assays. Mean ± SD, n = 3. *P < 0.05; **P < 0.01; *P < 0.001.

Fig. 2. FOXA1 expression inhibited stress-induced autophagic flux in LUAD cells.

A Autophagic flux in LUAD cells was evaluated using western blotting. CQ (10 μM). B Autophagic flux in LUAD cells was estimated using tandem fluorescent-tagged LC3 (stubRFP-sensGFP-LC3) reporter. The yellow puncta (RFP⁺GFP⁺) represent autophagosomes, and the red puncta (RFP⁺GFP⁻) represent autolysosomes. CQ (10 μM). Mean ± SD, n = 3. *P < 0.05; **P < 0.01. Scale bar: 10 μm.

Fig. 3. Loss of FOXA1 promoted autophagic cell death in LUAD cells under metabolic stress conditions.

A Colony formation assays were used to evaluate cell survival in FOXA1-depleted or FOXA1-silenced PC-9 cells upon treatment with specific cell death inhibitors. CQ (10 μM), 3-MA (2 mM), Z-VAD (20 μM), Nec-1 (20 μM), Fer-1 (10 μM). Mean ± SD, n = 3. B The protein levels of autophagy-related genes were determined by western blot. C Colony formation assays were used to evaluate cell survival in FOXA1-depleted or FOXA1-silenced PC-9 cells upon inhibition of autophagy-related genes. Mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001, NS not significant.

Fig. 4. FOXA1 interacted with DNMT1 and mediated the loss of imprinting of IGF2 in LUAD cells.

A Heat map of differentially expressed genes induced by gain or loss of function of FOXA1 in LUAD cells. B RT-PCR assays. C Western blot assays. D ChIP-seq data visualized using IGV software. E FOXA1 binding to IGF2-ICR measured using ChIP-qPCR assays. Mean ± SD, n = 3. F Methylation status of IGF2-ICR LUAD cells as evaluated by MSP assays. G Co-immunoprecipitation assays. H Co-localization of FOXA1 with DNMT1 visualized using immunofluorescence assays. Scale bar: 10 μm. I, J DNMT1 or CTCF binding to the CpG sites of IGF2-ICR determined by ChIP-qPCR. Mean ± SD, n = 3. K, L Effects of 5-Aza on IGF2 mRNA and protein levels measured by qPCR and western blot assays. Mean ± SD, n = 3. M, N Effects of silencing DNMT1 on IGF2 mRNA and protein levels measured by qPCR and western blot assays. Mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001, NS not significant.

Fig. 5. FOXA1 prevented metabolic stress-induced autophagic cell death via activation of IGF2/IGF1R/mTORC1 signaling.

A, B Status of the IGF1R/AKT/mTORC1 signaling pathway upon loss or gain of expression of FOXA in LUAD cells cultured in complete medium or PBS as evaluated by western blot assays. C Exogenous IGF2 expression levels in A549 cells as determined by RT-PCR or western blot assays. Mean ± SD, n = 3. D Survival of A549 cells with or without IGF2 forced expression as measured using colony formation assays. Mean ± SD, n = 3. E Survival of PC-9 cells in nutrients deprived conditions upon treatment with linsitinib (0.5 μM), anti-IGF2 antibodies (0.5 μg/mL), anti-VEGF antibodies (0.5 μg/mL), or rapamycin (50 nM) were determined using colony formation assays. Mean ± SD, n = 3. F Colony formation assays demonstrated that supplementation with recombinant IGF2 restored survival in FOXA1-deficient PC-9 cells under metabolic stress conditions. Mean ± SD, n = 3. G Colony formation assays demonstrated that stable expression of IGF2 restored survival in FOXA1-deficient PC-9 cells under metabolic stress conditions. Mean ± SD, n = 3. H Colony formation assays indicated that treatment with IGF2 antibody (0.5 μg/mL), VEGF antibody (0.5 μg/mL), linsitinib (0.5 μM) or rapamycin (50 nM) abolished the protective effects of FOXA1 expression on A549 cells in nutrients deprived conditions. Mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001, NS not significant.

Fig. 6. Repression of GBA1 protein by IGF2 signaling contributed to the survival advantage of LUAD cells conferred by FOXA1 under metabolic stress conditions.

A The mRNA levels of GBA1 were evaluated by RT-PCR. B The protein levels of GBA1 in LUAD cells were measured by western blotting. C, D The mRNA and protein levels of GBA1 in siRNA-transfected LUAD cells were measured by RT-PCR and western blot assays. Mean ± SD, n = 3. E Effect of silencing GBA1 on cell growth under nutrient-rich conditions was evaluated by using CCK-8 assays. Mean ± SD, n = 5. F Effects of silencing GBA1 on survival in LUAD cells under nutrient-rich or starvation conditions were measured using colony formation assays. Mean ± SD, n = 3. G, H The mRNA and protein levels of GBA1 in FOXA1-expressing or FOXA1-deficient cells were measured by RT-PCR and western blot assays. I GBA1 protein levels in FOXA1-expressing A549 cells treated with or without MG132 as determined by western blot assays. J Colony formation assays. Mean ± SD, n = 3. K, L Effects of IGF2 on GBA1 mRNA and protein levels were determined by RT-PCR or western blot assays. Mean ± SD, n = 3. M, N Effects of IGF2 on GBA1 mRNA and protein levels in FOXA1-depleted PC-9 cells were determined by RT-PCR or western blot assays. O, P Effects of linsitinib on endogenous GBA1 levels in PC-9 cells were determined by RT-PCR and western blot assays. Q, R Effects of linsitinib and rapamycin on GBA1 levels in FOXA1-expressing A549 cells were determined by RT-PCR or western blot assays. Mean ± SD, n = 3. S Ubiquitination of GBA1 was determined by immunoprecipitation and western blot assay. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. FOXA1 expression reduced the efficacy of nintedanib against xenograft tumors derived from A549 cells.

A Growth curve of xenograft tumors in nude mice. Mean ± SD, n = 10. B Macro view of xenograft tumors. C Weights of xenograft tumors as measured at the endpoint of the experiment. Mean ± SD, n = 10. D H&E staining and immunohistochemistry staining of xenograft tumors. Ni nintedanib (100 mg/kg). li linsitinib (25 mg/kg). ra rapamycin (4 mg/kg).

Fig. 8. High levels of FOXA1 protein corresponded to unfavorable progression-free survival in patients with LUAD who underwent treatment with a combination of EGFR-TKI and bevacizumab.

A Negative staining of FOXA1 protein in lung tissues. B Negative or weak staining of FOXA1 protein in LUAD samples. C Intense nuclear staining of FOXA1 protein in LUAD samples. D Progression-free survival in patients with LUAD patients receiving EGFR-TKI treatment alone or EGFR-TKI combined with bevacizumab stratified according to FOXA1 protein. Scale bar: 100 μm.