The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells

Abstract

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

Keywords: Autophagy; ER stress; UPR; cancer; clinical trial; dihydroceramide.

Conflict of interest statement

PM-G, SS, EM-R; HP-M, MY-V, CD and JA are Ability Pharmaceuticals employees; CD holds shares of the company; JML is advisory member of Ability Pharmaceuticals.

Figures

Figure 1.

ABTL0812 induces ER stress in cancer cell lines. (a, b) ABTL0812 induces dynamic autophagy. Cells were preincubated 3 h with vehicle or lysosomal protease inhibitors E64d (10 µmol/L) and pepstatin A (PA, 10 µg/mL) (a) or with inhibitor (50 nM) of the vacuolar-type ATPase, bafilomycin A1 (BafA) (b) before treatment with ABTL0812 for 24 h. Levels of lipidated and non-lipidated MAP1LC3B proteins were monitored by immunoblotting. (c) ABTL0812 induces autophagy-mediated cancer cell death. Effect of ABTL0812 treatment (48 h) in viability of MiaPaca2 or A459 stable cell lines transfected with control shRNA (shC) or ATG5-selective shRNA (shATG5). Right panels show the corresponding immunoblots. (d) AKT or MTOR inhibition does not result in the induction of autophagy. MiaPaca2 cells were treated with 25 µmol/L AZD5363 (AKT inhibitor), 0.1 µmol/L AZD2014 (MTOR inhibitor), 10 µmol/L everolimus (MTORC1 inhibitor) or 100 µmol/L ABTL0812 for 24 h. Expression of RPS6, p-RPS6, lipidated and non-lipidated MAP1LC3B and ACTB proteins were determined by immunoblotting. Similar results were obtained in four separate experiments. (e–h) ABTL0812 induces ER stress in cancer cells. (e) Cells were treated with 100 µmol/L ABTL0812 or 200 nmol/L Brefeldin-A (BrefA), total RNA isolated and cDNA synthesized by RT-PCR. XBP1 splicing was determined by PCR using primers that amplify both spliced (XBP1 s) and unspliced (XBP1 u) mRNA species. Similar results were obtained in four separate experiments. (f) MiaPaca2 and A549 cells were treated with 100 µmol/L ABTL0812, then lysed. The levels of p-EIF2A were monitored by immunoblotting. Similar results were obtained in three separate experiments. (g) Cells were treated with 100 µmol/L ABTL0812, then lysed. The levels of ER stress markers HSPA5, ATF4, DDIT3 and TRIB3 were monitored by immunoblotting. The marker of autophagy MAP1LC3B was also analyzed. Similar results were obtained in four separate experiments. (h) MiaPaca2 and A549 cells were treated with 100 µmol/L ABTL0812 for 24 h, pelleted, total RNA isolated, and ATF4, DDIT3 and TRIB3 mRNA levels were analyzed by RT-qPCR. Each value is the mean ± SD of three different experiments. **, P < 0.005; ***, P < 0.001, Student’s t-test

Figure 1.

ABTL0812 induces ER stress in cancer cell lines. (a, b) ABTL0812 induces dynamic autophagy. Cells were preincubated 3 h with vehicle or lysosomal protease inhibitors E64d (10 µmol/L) and pepstatin A (PA, 10 µg/mL) (a) or with inhibitor (50 nM) of the vacuolar-type ATPase, bafilomycin A1 (BafA) (b) before treatment with ABTL0812 for 24 h. Levels of lipidated and non-lipidated MAP1LC3B proteins were monitored by immunoblotting. (c) ABTL0812 induces autophagy-mediated cancer cell death. Effect of ABTL0812 treatment (48 h) in viability of MiaPaca2 or A459 stable cell lines transfected with control shRNA (shC) or ATG5-selective shRNA (shATG5). Right panels show the corresponding immunoblots. (d) AKT or MTOR inhibition does not result in the induction of autophagy. MiaPaca2 cells were treated with 25 µmol/L AZD5363 (AKT inhibitor), 0.1 µmol/L AZD2014 (MTOR inhibitor), 10 µmol/L everolimus (MTORC1 inhibitor) or 100 µmol/L ABTL0812 for 24 h. Expression of RPS6, p-RPS6, lipidated and non-lipidated MAP1LC3B and ACTB proteins were determined by immunoblotting. Similar results were obtained in four separate experiments. (e–h) ABTL0812 induces ER stress in cancer cells. (e) Cells were treated with 100 µmol/L ABTL0812 or 200 nmol/L Brefeldin-A (BrefA), total RNA isolated and cDNA synthesized by RT-PCR. XBP1 splicing was determined by PCR using primers that amplify both spliced (XBP1 s) and unspliced (XBP1 u) mRNA species. Similar results were obtained in four separate experiments. (f) MiaPaca2 and A549 cells were treated with 100 µmol/L ABTL0812, then lysed. The levels of p-EIF2A were monitored by immunoblotting. Similar results were obtained in three separate experiments. (g) Cells were treated with 100 µmol/L ABTL0812, then lysed. The levels of ER stress markers HSPA5, ATF4, DDIT3 and TRIB3 were monitored by immunoblotting. The marker of autophagy MAP1LC3B was also analyzed. Similar results were obtained in four separate experiments. (h) MiaPaca2 and A549 cells were treated with 100 µmol/L ABTL0812 for 24 h, pelleted, total RNA isolated, and ATF4, DDIT3 and TRIB3 mRNA levels were analyzed by RT-qPCR. Each value is the mean ± SD of three different experiments. **, P < 0.005; ***, P < 0.001, Student’s t-test

Figure 2.

ABTL0812 induces ER stress in human lung and pancreatic xenografts. Athymic nude mice injected with A549 or MiaPaCa2 cells were treated for 33 d with vehicle or 120 mg/kg ABTL0812 by oral gavage. (a) Tumor xenograft samples were collected and expression of ATF4 or HSPA5 ER stress markers were analyzed by immunoblotting. Left graphs show growth curves of tumors referred to initial values (fold-change ± SEM). Right histograms show the corresponding quantifications referred to ACTB. (b) Expression of DDIT3 protein from MiaPaca2 and A549 tumors was also evaluated by immunohistochemical analysis. Scale bars, 100 µm. Right histogram shows the corresponding quantification. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells, Student’s t-test

Figure 2.

ABTL0812 induces ER stress in human lung and pancreatic xenografts. Athymic nude mice injected with A549 or MiaPaCa2 cells were treated for 33 d with vehicle or 120 mg/kg ABTL0812 by oral gavage. (a) Tumor xenograft samples were collected and expression of ATF4 or HSPA5 ER stress markers were analyzed by immunoblotting. Left graphs show growth curves of tumors referred to initial values (fold-change ± SEM). Right histograms show the corresponding quantifications referred to ACTB. (b) Expression of DDIT3 protein from MiaPaca2 and A549 tumors was also evaluated by immunohistochemical analysis. Scale bars, 100 µm. Right histogram shows the corresponding quantification. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells, Student’s t-test

Figure 3.

ER stress mediates ABTL0812-induced autophagy and cytotoxicity in cancer cells. (a–d) EIF2A-ATF4 axis mediates in ABTL0812-induced autophagy. (a) ABTL0812 does not induce ATF4 in EIF2AS51A knock-in cells. Levels of phosphorylated EIF2A and ATF4 were monitored by immunoblotting. Similar results were obtained in three separate experiments. (b,c) EIF2AK3 silencing, but not EIF2AK4 silencing, prevents ABTL0812-induced ATF4 expression. Cells transfected with control siRNA (siC) or EIF2AK3- or EIF2AK4-selective siRNAs (siEIF2AK3 in (b) or siEIF2AK4 in (c)) were treated with 100 µmol/L ABTL0812 for 4 h, lysed and the indicated proteins were monitored by immunoblotting. The corresponding quantification of MAP1LC3B-II levels referred to ACTB are indicated under the panels. Results representative of three separate experiments. (d) ATF4 silencing prevents ABTL0812-induced autophagy. Cells were transfected with scramble siRNA (siC) or two different ATF4-selective siRNAs, and then treated with 100 µmol/L ABTL0812 for 18 h. Levels of ATF4, TRIB3, MAP1LC3B and ACTB protein expression were analyzed by immunoblotting. The corresponding quantification of MAP1LC3B-II levels referred to ACTB are indicated under the panels. Results representative of four separate experiments. (e,f) Pharmacological inhibition of ER response with molecular chaperones impairs ABTL0812-induced cytotoxicity. Cells were preincubated 3 h with 1 mmol/L 4-BPA (e) or 150 µmol/L sodium tauroursodeoxycholate (TUDC) (f) before treatment with 30 µmol/L ABTL0812 for 48 h. Cell viability was determined by MTT assay. Each value is the mean ± SD of three different experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from ABTL0812-treated cells, one-way ANOVA Bonferroni

Figure 3.

ER stress mediates ABTL0812-induced autophagy and cytotoxicity in cancer cells. (a–d) EIF2A-ATF4 axis mediates in ABTL0812-induced autophagy. (a) ABTL0812 does not induce ATF4 in EIF2AS51A knock-in cells. Levels of phosphorylated EIF2A and ATF4 were monitored by immunoblotting. Similar results were obtained in three separate experiments. (b,c) EIF2AK3 silencing, but not EIF2AK4 silencing, prevents ABTL0812-induced ATF4 expression. Cells transfected with control siRNA (siC) or EIF2AK3- or EIF2AK4-selective siRNAs (siEIF2AK3 in (b) or siEIF2AK4 in (c)) were treated with 100 µmol/L ABTL0812 for 4 h, lysed and the indicated proteins were monitored by immunoblotting. The corresponding quantification of MAP1LC3B-II levels referred to ACTB are indicated under the panels. Results representative of three separate experiments. (d) ATF4 silencing prevents ABTL0812-induced autophagy. Cells were transfected with scramble siRNA (siC) or two different ATF4-selective siRNAs, and then treated with 100 µmol/L ABTL0812 for 18 h. Levels of ATF4, TRIB3, MAP1LC3B and ACTB protein expression were analyzed by immunoblotting. The corresponding quantification of MAP1LC3B-II levels referred to ACTB are indicated under the panels. Results representative of four separate experiments. (e,f) Pharmacological inhibition of ER response with molecular chaperones impairs ABTL0812-induced cytotoxicity. Cells were preincubated 3 h with 1 mmol/L 4-BPA (e) or 150 µmol/L sodium tauroursodeoxycholate (TUDC) (f) before treatment with 30 µmol/L ABTL0812 for 48 h. Cell viability was determined by MTT assay. Each value is the mean ± SD of three different experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from ABTL0812-treated cells, one-way ANOVA Bonferroni

Figure 4.

ABTL0812 treatment induces TRIB3 and DDIT3 mRNA levels in blood from patients enrolled in phase 2 clinical trial (NCT03366480). (a) Blood TRIB3 and DDIT3 mRNA levels from patients enrolled in phase 2 clinical trial (N = 14 for TRIB3; N = 12 for DDIT3). Values represented in the scatter plot correspond to the mean ± SEM of 2−△△Ct values. Values show fold-changes of mRNA levels, referred to as “0” value. (b) Whole blood TRIB3 and DDIT3 mRNA levels of nine patients before (0) or after 1,300 mg t.i.d. ABTL0812 oral daily treatment as monotherapy (8 h and 7 d) or in combination with chemotherapy (28 d). mRNA levels were evaluated by RT-qPCR. Each value is the mean ± SD of three technical replicates. Patients 6 and 7 showed increased TRIB3 mRNA levels only, whereas patients 8 and 9 showed increased DDIT3 mRNA levels only. Values show fold-changes of mRNA levels, referred to as “0” value. Statistical analyses were performed using the △△Ct values. A one-way ANOVA Tukey test was applied. *, P < 0.05; **, P < 0.005; ***, P < 0.001 compared to day 0 sample

Figure 4.

ABTL0812 treatment induces TRIB3 and DDIT3 mRNA levels in blood from patients enrolled in phase 2 clinical trial (NCT03366480). (a) Blood TRIB3 and DDIT3 mRNA levels from patients enrolled in phase 2 clinical trial (N = 14 for TRIB3; N = 12 for DDIT3). Values represented in the scatter plot correspond to the mean ± SEM of 2−△△Ct values. Values show fold-changes of mRNA levels, referred to as “0” value. (b) Whole blood TRIB3 and DDIT3 mRNA levels of nine patients before (0) or after 1,300 mg t.i.d. ABTL0812 oral daily treatment as monotherapy (8 h and 7 d) or in combination with chemotherapy (28 d). mRNA levels were evaluated by RT-qPCR. Each value is the mean ± SD of three technical replicates. Patients 6 and 7 showed increased TRIB3 mRNA levels only, whereas patients 8 and 9 showed increased DDIT3 mRNA levels only. Values show fold-changes of mRNA levels, referred to as “0” value. Statistical analyses were performed using the △△Ct values. A one-way ANOVA Tukey test was applied. *, P < 0.05; **, P < 0.005; ***, P < 0.001 compared to day 0 sample

Figure 5.

ABTL0812 induces accumulation of long-chain dihydroceramides in cancer cells and tumors. (a) Scheme representing the pathway of de novo sphingolipid biosynthesis. Serine palmitoyltransferase (SPT) catalyzes the condensation of palmitoyl-CoA and serine to produce 3-ketosdihydrosphingosine. KDSR (3-ketodihydrosphingosine reductase) reduces 3-ketodihydrosphingosine to dihydrosphingosine. Then, ceramide synthases (CERSs) convert dihydrosphingosine into the different molecular species of dihydroceramides, which are transformed to ceramides by the insertion of a 4,5-trans double bond catalyzed by the enzyme DEGS1. (b) ABTL0812 effect on levels of total cellular ceramides and dihydroceramides. MiaPaca2 cells were treated with 100 µmol/L ABTL0812 for the times indicated, pelleted and lipid content extracted and analyzed by UPLC-TOF-Ms. Data are expressed in pmol of sphingolipid per 106 cells. Each value is the mean ± SD of three different determinations. (c) Levels of molecular species of dihydroceramides at 6 h (left histogram) and 24 h of ABTL0812 treatment (right histogram) in MiaPaca2 cells. Each value is the mean ± SD of three different determinations. (d) Levels of dihydroceramides in endometrial cancer Ishikawa cells after 6 h of ABTL0812 treatment. Total dihydroceramides and molecular species are shown in the left and right panels, respectively. Each value is the mean ± SD of three different determinations. (e,f) ABTL0812 effect on levels of dihydroceramides in tumors. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells

Figure 5.

ABTL0812 induces accumulation of long-chain dihydroceramides in cancer cells and tumors. (a) Scheme representing the pathway of de novo sphingolipid biosynthesis. Serine palmitoyltransferase (SPT) catalyzes the condensation of palmitoyl-CoA and serine to produce 3-ketosdihydrosphingosine. KDSR (3-ketodihydrosphingosine reductase) reduces 3-ketodihydrosphingosine to dihydrosphingosine. Then, ceramide synthases (CERSs) convert dihydrosphingosine into the different molecular species of dihydroceramides, which are transformed to ceramides by the insertion of a 4,5-trans double bond catalyzed by the enzyme DEGS1. (b) ABTL0812 effect on levels of total cellular ceramides and dihydroceramides. MiaPaca2 cells were treated with 100 µmol/L ABTL0812 for the times indicated, pelleted and lipid content extracted and analyzed by UPLC-TOF-Ms. Data are expressed in pmol of sphingolipid per 106 cells. Each value is the mean ± SD of three different determinations. (c) Levels of molecular species of dihydroceramides at 6 h (left histogram) and 24 h of ABTL0812 treatment (right histogram) in MiaPaca2 cells. Each value is the mean ± SD of three different determinations. (d) Levels of dihydroceramides in endometrial cancer Ishikawa cells after 6 h of ABTL0812 treatment. Total dihydroceramides and molecular species are shown in the left and right panels, respectively. Each value is the mean ± SD of three different determinations. (e,f) ABTL0812 effect on levels of dihydroceramides in tumors. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells

Figure 6.

Dihydroceramide accumulation by ABTL0812 treatment induces ER stress, autophagy and selective cancer cell death. (a) Cells were treated with 25 µmol/L of the dideuterated dihydroceramide analog d2C8dhCer for 48 h, then lysed. The levels of ER stress markers ATF4 and TRIB3, as well as MAP1LC3B lipidation, were monitored by immunoblotting. Histograms show quantification of ATF4, TRIB3 and MAP1LC3B-II levels normalized to ACTB levels and represent the mean fold-change relative to vehicle-treated cells. (b) MiaPaca2 and A549 cells were treated 48 h with 25 µmol/L of d2c8dhCer, and cell viability was determined by MTT assay. Each value is the mean ± SD of two different experiments performed in triplicates. (c,d) ABTL0812 treatment induces DEGS1 inhibition. (c) A549 cell lysates were treated with vehicle or 100 µmol/L ABTL0812 for 4 h. (d) Cultured MiaPaca2 (pancreatic), H157 (SNSCLC) and Ishikawa (endometrial) cancer cells were treated 6 h with vehicle or 100 µmol/L ABTL0812. DEGS1 activity was determined as described in the Materials and Methods Section using DHCerC6NBD as substrate. Data expressed in percentage of the CerC6NBD pick area. Each value is the mean ± SD of three different determinations. (e) DEGS1 levels visualized by immunoblotting. (f) Selectivity of ABTL0812 cytotoxic effect for cancer cells. Human lung fibroblast MRC5 and squamous NSCLC H157 cells were treated with ABTL0812 for 48 h, and cell viability determined by MTT assay. Results representative of three separate experiments. (g,h) ABTL0812 does not induce accumulation of dihydroceramides in a non-tumoral cell line. (g) Levels of total dihydroceramides in MRC5 fibroblasts and H157 cancer cells, after 6 h of ABTL0812 treatment. (h) Levels of molecular dihydroceramide species. Each value is the mean ± SD of three different determinations. (i) DEGS1 protein levels (immunoblot) in MRC5 and H157 cells. (j) Human endometrial tumors express higher levels of DEGS1 protein than adjacent non-tumoral tissue. Immunoblot analysis of samples (tumor area [T] and non-tumor area [N]) from 4 different patients with endometrial cancer. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells

Figure 6.

Dihydroceramide accumulation by ABTL0812 treatment induces ER stress, autophagy and selective cancer cell death. (a) Cells were treated with 25 µmol/L of the dideuterated dihydroceramide analog d2C8dhCer for 48 h, then lysed. The levels of ER stress markers ATF4 and TRIB3, as well as MAP1LC3B lipidation, were monitored by immunoblotting. Histograms show quantification of ATF4, TRIB3 and MAP1LC3B-II levels normalized to ACTB levels and represent the mean fold-change relative to vehicle-treated cells. (b) MiaPaca2 and A549 cells were treated 48 h with 25 µmol/L of d2c8dhCer, and cell viability was determined by MTT assay. Each value is the mean ± SD of two different experiments performed in triplicates. (c,d) ABTL0812 treatment induces DEGS1 inhibition. (c) A549 cell lysates were treated with vehicle or 100 µmol/L ABTL0812 for 4 h. (d) Cultured MiaPaca2 (pancreatic), H157 (SNSCLC) and Ishikawa (endometrial) cancer cells were treated 6 h with vehicle or 100 µmol/L ABTL0812. DEGS1 activity was determined as described in the Materials and Methods Section using DHCerC6NBD as substrate. Data expressed in percentage of the CerC6NBD pick area. Each value is the mean ± SD of three different determinations. (e) DEGS1 levels visualized by immunoblotting. (f) Selectivity of ABTL0812 cytotoxic effect for cancer cells. Human lung fibroblast MRC5 and squamous NSCLC H157 cells were treated with ABTL0812 for 48 h, and cell viability determined by MTT assay. Results representative of three separate experiments. (g,h) ABTL0812 does not induce accumulation of dihydroceramides in a non-tumoral cell line. (g) Levels of total dihydroceramides in MRC5 fibroblasts and H157 cancer cells, after 6 h of ABTL0812 treatment. (h) Levels of molecular dihydroceramide species. Each value is the mean ± SD of three different determinations. (i) DEGS1 protein levels (immunoblot) in MRC5 and H157 cells. (j) Human endometrial tumors express higher levels of DEGS1 protein than adjacent non-tumoral tissue. Immunoblot analysis of samples (tumor area [T] and non-tumor area [N]) from 4 different patients with endometrial cancer. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells

Figure 7.

DEGS1 specific inhibitor GT11 induces accumulation of dihydroceramides, ER stress, dynamic autophagy and selective cancer cell death. (a) MiaPaca2 cells were treated for 6 h with vehicle, 6 µmol/L GT11, or 100 µmol/L ABTL0812, and total ceramides, dihydroceramides and molecular species of dihydroceramides were quantified. Each value is the mean ± SD of three different experiments. (b–e) DEGS1 inhibition induces ER stress-mediated dynamic autophagy. (b) cCells were treated 48 h with 5 µmol/L GT11 and levels of ER stress markers ATF4 and TRIB3 and MAP1LC3B lipidation analyzed by immunoblotting. Similar results were obtained in three separate experiments. (c,d) Cells were preincubated 3 h with vehicle or lysosomal protease inhibitors E64d (10 µmol/L) and pepstatin-A (PA, 10 µg/mL) (c), or 50 nm bafilomycin-A (BafA) (d) before treatment with 6 µmol/L GT11 for 24 h. Cells were lysed and MAP1LC3B lipidation was visualized by immunoblotting. Similar results were obtained in three different experiments. (e) ATF4 silencing prevents GT11-induced autophagy. Cells were transfected with scramble siRNA (siC) or two different ATF4-selective siRNAs, and then treated with 100 µmol/L ABTL0812 for 18 h. Levels of ATF4, TRIB3, MAP1LC3B and ACTB protein expression were analyzed by immunoblotting. The corresponding quantification of MAP1LC3B-II levels referred to ACTB are indicated under the panels. Results representative of two separate experiments. (f) Non-tumoral cells show lower sensitivity to DEGS1 inhibitor GT11. Human lung fibroblast MRC5 and squamous NSCLC H157 cells were treated with the 30 µM ABTL0812 or with the indicated concentration of GT11 for 48 h, and cell viability was determined by MTT assay. Results representative of three separate experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells

Figure 7.

DEGS1 specific inhibitor GT11 induces accumulation of dihydroceramides, ER stress, dynamic autophagy and selective cancer cell death. (a) MiaPaca2 cells were treated for 6 h with vehicle, 6 µmol/L GT11, or 100 µmol/L ABTL0812, and total ceramides, dihydroceramides and molecular species of dihydroceramides were quantified. Each value is the mean ± SD of three different experiments. (b–e) DEGS1 inhibition induces ER stress-mediated dynamic autophagy. (b) cCells were treated 48 h with 5 µmol/L GT11 and levels of ER stress markers ATF4 and TRIB3 and MAP1LC3B lipidation analyzed by immunoblotting. Similar results were obtained in three separate experiments. (c,d) Cells were preincubated 3 h with vehicle or lysosomal protease inhibitors E64d (10 µmol/L) and pepstatin-A (PA, 10 µg/mL) (c), or 50 nm bafilomycin-A (BafA) (d) before treatment with 6 µmol/L GT11 for 24 h. Cells were lysed and MAP1LC3B lipidation was visualized by immunoblotting. Similar results were obtained in three different experiments. (e) ATF4 silencing prevents GT11-induced autophagy. Cells were transfected with scramble siRNA (siC) or two different ATF4-selective siRNAs, and then treated with 100 µmol/L ABTL0812 for 18 h. Levels of ATF4, TRIB3, MAP1LC3B and ACTB protein expression were analyzed by immunoblotting. The corresponding quantification of MAP1LC3B-II levels referred to ACTB are indicated under the panels. Results representative of two separate experiments. (f) Non-tumoral cells show lower sensitivity to DEGS1 inhibitor GT11. Human lung fibroblast MRC5 and squamous NSCLC H157 cells were treated with the 30 µM ABTL0812 or with the indicated concentration of GT11 for 48 h, and cell viability was determined by MTT assay. Results representative of three separate experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001 from vehicle-treated cells

Figure 8.

DEGS1 inhibition collaborates with MTORC1 inhibition to promote autophagy and cancer cell death. (a) MiaPaca2 (left histogram) and A549 (right histogram) cells were treated with a combination of the DEGS1 inhibitor GT11 and the MTORC1 inhibitor everolimus for 24 h. Cell viability was determined by MTT assay. Each value is the mean ± SD of three different experiments. Lower tables show the combination index (CI) analysis for different concentrations of GT11 and everolimus. CIs were obtained using the indicated doses of inhibitors and the Compusyn software (see Methods Section) (CI > 1, antagonism; CI = 1, summary effect; CI P < 0.005; ***, P < 0.001 from everolimus and GT11-treated cells, one-way ANOVA

Figure 8.

DEGS1 inhibition collaborates with MTORC1 inhibition to promote autophagy and cancer cell death. (a) MiaPaca2 (left histogram) and A549 (right histogram) cells were treated with a combination of the DEGS1 inhibitor GT11 and the MTORC1 inhibitor everolimus for 24 h. Cell viability was determined by MTT assay. Each value is the mean ± SD of three different experiments. Lower tables show the combination index (CI) analysis for different concentrations of GT11 and everolimus. CIs were obtained using the indicated doses of inhibitors and the Compusyn software (see Methods Section) (CI > 1, antagonism; CI = 1, summary effect; CI P < 0.005; ***, P < 0.001 from everolimus and GT11-treated cells, one-way ANOVA