Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth

Abstract

Uveal melanoma (UM) displays a high frequency of metastasis; however, effective therapies for metastatic UM are limited. Identifying unique metabolic features of UM may provide a potential targeting strategy. A lipid metabolism protein expression signature was induced in a normal choroidal melanocyte (NCM) line transduced with GNAQ (Q209L), a driver in UM growth and development. Consistently, UM cells expressed elevated levels of fatty acid synthase (FASN) compared to NCMs. FASN upregulation was associated with increased mammalian target of rapamycin (mTOR) activation and sterol regulatory element-binding protein 1 (SREBP1) levels. FASN and mTOR inhibitors alone significantly reduced UM cell growth. Concurrent inhibition of FASN and mTOR further reduced UM cell growth by promoting cell cycle arrest and inhibiting glucose utilization, TCA cycle metabolism, and de novo fatty acid biosynthesis. Our findings indicate that FASN is important for UM cell growth and co-inhibition of FASN and mTOR signaling may be considered for treatment of UM.

Keywords: GNAQ; fatty acid synthase; mTOR pathway; metabolic inhibition; uveal melanoma.

Figure 1

Increased FASN expression in UM cells. (A) GNAQ and pERK1/2 levels in UMC026 cells transduced with exogenous GNAQ (Q209L) shown by Western blot. GNAQ antibody detects all forms of GNAQ. (B) An enrichment plot of GSEA results using the weighted enrichment statistic parameters. (C) Expression of major lipogenic enzymes in NCMs (MCN#1459 and MCN#1462) and UM cell lines were evaluated by Western blot. (D) The levels of ALCY, ACC, FASN and SREBP1 in PDX-derived UM cell lines compared to NCMs. (E) FASN levels following GNAQ knockdown by siRNA transfection for 72 h in MP46 cells. β-actin or HSP90 served as loading controls. NCMs; normal choroidal melanocytes, ACLY; ATP-citrate lyase, ACC; acetyl-CoA carboxylase, FASN; fatty acid synthase, SREBP1; sterol regulatory element-binding protein and PDX; patient derived xenografts.

Figure 2

Elevated FASN expression in UM patient sample. (A) Gene expression of FASN in various human cancer types. Data were derived from a TCGA dataset and analyzed through the Firebrowse web resource (http://firebrowse.org, accessed on 24 February 2021). Red box indicates uveal melanoma samples (UM). (B) Dot plot showing the average expression and percent of cells expressing FASN from patient tumor scRNA-seq data. Cells were separated into non-malignant and tumor-specific malignant cell groups. BSSR0022, UMM041L and UMM067L were isolated from metastases whereas the other malignant cases were from primary tumors. (C) FASN expression in liver metastasized UM tumors. S100 was detected as marker for melanoma cells. Scale bar: 10 µm.

Figure 3

Co-targeting FASN and mTOR reduces UM cell growth. (A) 92.1, UM001 and UM004 cells were treated with Fasnall (0, 2.5, 5 and 7.5 μM) for 4 days or GSK 2194069 (0, 20, 40 and 60 μM) for 3 days. Cell viability was measured by crystal violet staining. Quantification of cell growth following treatment with Fasnall and GSK2194069 is shown as fold changes in crystal violet stain compared to controls. (B) FASN knockdown was performed by siRNA transfection. Silencing of FASN was confirmed through Western blot. (C) Activation of mTOR modulates de novo lipogenesis in cancer cells by controlling the expression of transcription factor (SREBP1) and key lipogenic enzymes (e.g., ACLY, ACC and FASN; colored red). (D) Phosphorylated and total mTOR levels in NCMs and UM cell lines were probed by Western blot. β-actin served as a loading control. (E) The effects of co-inhibition of FASN and mTOR in 92.1, UM001 and UM004 cell growth were measured by IncuCyte. The cells were treated with Fasnall (5 μM) or GSK2194069 (40 μM), with or without AZD2014 (200 nM), for 72 h. Percent confluency of the cells was measured on day 0 and day 3. Data are shown as mean ± SEM (n = 4) * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 unpaired t-test. NCMs; normal choroidal melanocytes, mTOR; mammalian target of rapamycin, ACLY; ATP-citrate lyase, ACC; acetyl-CoA carboxylase, FASN; fatty acid synthase and SREBP1; sterol regulatory element-binding protein.

Figure 4

Effects of FASN and mTOR inhibitors on levels of cell cycle modulators in UM. UM004, 92.1 and UM001 cells were treated with Fasnall (5 μM) or GSK2194069 (40 μM), with or without AZD2014 (200 nM), for 48 h. (A) RPPA data were used to determine proteins/phospho-proteins that were significantly different between control, single treatments of each inhibitor and combo treatments of different cell lines (p-value < 0.05 and a 25% log2 fold change). Comparisons were performed between each group using the two-sample t-test method with 1000 permutations and assumed unequal variance. Hierarchical clustering was performed based on median-centered log2-transformed expression values. Statistical calculations were performed in Matlab^® (v2015b) using the mattest function. (B) Results of RPPA analysis were validated by Western blot in UM001 and UM004 cells. β-actin, HSP90 and S6 served as loading controls. Protein expression was normalized to the average intensity of the loading controls.

Figure 5

Inhibition of FASN and mTOR induce cell cycle arrest in 2D and 3D cell growth. UM004, 92.1 and UM001 cells were treated with Fasnall (5 μM) or GSK2194069 (40 μM), with or without AZD2014 (200 nM), for 48 h. (A) Cells were collected for EdU corporation (upper panel) and annexin/PI staining assays (bottom panel). (B) 3D spheroid cultures of 92.1 cells were treated with Fasnall (5 μM) or GSK2194069 (40 μM), with or without AZD2014 (200 nM), for 48 h. EdU incorporation of 92.1 grown as 3D spheroids is shown. (C) Representative figures of spheroids are shown. Tumor spheroids were treated with Fasnall (5 μM) or GSK2194069 (40 μM), with or without AZD2014 (200 nM), for 48 h. For cell viability, 3D spheroids were stained with Calcein-AM and PI for live cells and necrotic cells, respectively. (D) Quantitation of Calcein-AM and PI. Magnification: 150X, Scale bar: 100 µm. Quantification bar graph is shown. Data are shown as mean ± SEM (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 unpaired t-test.

Figure 6

Suppression of FASN and mTOR decrease de novo FA biosynthesis in UM cells. UM001 and OMM1.3 cells were incubated in the presence of 13C-glucose for 4 and 24 h along with GSK2194069 (40 μM) and with or without AZD2014 (200 nM). (A) A flow of carbon atoms from the glucose to the fatty acid palmitate, including depictions of M + 4 through M + 16 isotopologues. Black circles = carbon-13; white circles = carbon-12. (B) Fractional labeling of myristate (14:0), palmitate (16:0), palmitoleate (16:1) and stearate (18:0) in UM cells at 24 h after the addition of 13C-glucose. Data are shown as mean ± SD (n = 3). ns, not significant, ** p < 0.01, and **** p < 0.0001 Two-way ANOVA with Dunnett’s multiple comparison testing. (C) Isotopologue distribution patterns of palmitate from 13C-glucose in UM001 and OMM1.3 cells at 4 h (left panel) and 24 h (right panel). Data are shown as mean ± SD (n = 3).

Figure 7

The effects of FASN and mTOR inhibitors on glucose utilization in UM cells. UM001 and OMM1.3 cells were incubated in the presence of 13C-glucose for 4 and 24 h along with GSK2194069 (40 μM) and with or without AZD2014 (200 nM). (A) Net flux of glucose consumption and lactate secretion from UM cells calculated at fmol/hour/cell. Data are shown as mean ± SD (n = 3). ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. Two-way ANOVA with Dunnett’s multiple comparison testing. (B) Glycolytic capacity of UM001 and OMM1.3 cells after the treatment (24 h) was measured by ECAR using the Seahorse analyzer. Data were normalized to protein level and analyzed via Agilent Seahorse XF report generators. Data are shown as mean ± SEM (n = 12). ns, not significant, * p < 0.05, and ** p < 0.01.

Figure 8

Inhibition of FASN and mTOR decrease the flux of glucose into the TCA cycle in UM cells. UM001 and OMM1.3 cells were incubated in the presence of 13C-glucose for 4 and 24 h along with GSK2194069 (40 μM) and with or without AZD2014 (200 nM). Fractional labeling of TCA cycle intermediates in UM001 and OMM1.3 cells (4 and 24 h) after the addition of 13C-glucose. Inset numbers indicate percent labeling for each isotopologue (n = 3).