Polyamine Pathway Inhibitor DENSPM Suppresses Lipid Metabolism in Pheochromocytoma Cell Line

Abstract

Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N¹,N¹¹-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography-mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.

Keywords: DENSPM; SDHB; paraganglioma; pheochromocytoma; plasmanyl.

Figure 1

Functional annotation as “biological process”, “cellular localization” or “molecular function” of genes identified as downregulated by the RNA-seq analysis of DENSPM treated hPheo1 (A) or SDHB_KD (B) cells. Bars were sorted by adjusted p-values; the length of each bar represents the number of genes in each group. The fatty acid metabolism category is indicated by a red frame.

Figure 2

Relative gene expression of lipid-associated genes from the RNA-Seq analysis of hPheo1 (A) and SDHB_KD (B) cells. The means and SEMs for the number of reads corresponding to either untreated (white bars) or DENSPM-treated (grey bars) cells are shown; black squares represent individual observation. All sets are highly significant (p < 0.001).

Figure 3

Relative gene expression of select lipid-associated genes identified by RNA-seq was confirmed by qRT-PCR. Relative gene expression corresponding to either untreated (dark gray bars) or DENSPM-treated (light grey bars) cells is shown. The data are presented as means and SEMs. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01 *, p < 0.05.

Figure 4

Western blot analysis of FADS2, SCD, and SREBP1 protein expression in hPheo1 (left) and SDHB_KD (right) cells untreated (−) or treated (+) with 10 µM DENSPM.

Figure 5

Heatmap of the 9 genes (including 6 genes associated with lipid metabolism, green box) that are differentially expressed between PPGL (underlined), ACC, and Prostate Cancer (PC) tumors. Transcript names are shown along the right axis. Red: increased expression, blue: decreased expression.

Figure 6

(A) A volcano plot of the LC/MS analysis of lipid composition in untreated vs. 10 µM DENSPM-treated hPheo1 Cells (fold change = 2.0; p-value = 0.05, FDR adjusted). (B) A Pie chart shows the significantly altered lipids by (sub)class. Note that the majority of the lipids belong to the plasmanyl class.

Figure 7

(A) A volcano plot of the LC/MS analysis of lipid composition in untreated vs. 10 µM DENSPM-treated SDHB_KD Cells (Fold change = 2.0; p-value = 0.05, FDR adjusted). (B) A Pie chart shows the significantly altered lipids by (sub)class. Red box: significantly increased lipids by (sub)class. Blue box: significantly decreased lipids by (sub)class. Note that the majority of downregulated lipids belong to the plasmanyl class.

Figure 8

Most common saturated and unsaturated fatty acids within the significantly changed lipids (Figure 6 and Figure 7) in hPheo1 (Orange) and SDHB_KD cells (Blue) treated with DENSPM. Fatty acid classes comprising over 10% of the significant lipids are included. No significant changes between hPheo1 and SDHB_KD cells were noted.