Development of pheochromocytoma in ceramide synthase 2 null mice

Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in w15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the ceramide synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes ceramides with very-long (C22–C24) chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullary tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by w13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulation of ceroid in the X-zone was observed as early as 2 months of age and as a consequence, older mice displayed elevated levels of lysosomal cathepsins, reduced proteasome activity and reduced activity of mitochondrial complex IV by 6 months of age. Together, these findings implicate an additional pathway that can lead to PCC formation, which involves alterations in the sphingolipid acyl chain length. Analysis of the role of sphingolipids in PCC may lead to further understanding of the mechanism by which PCC develops, and might implicate the sphingolipid pathway as a possible novel therapeutic target for this rare tumor.


Introduction
The adrenal gland contains two distinct hormone-producing zones, the outer adrenal cortex, which produces glucocorticoids and mineralocorticoids, and the adrenal medulla, which is directly innervated from sympathetic nerves and secretes norepinephrine (NE) and epinephrine X-zone apparent long after birth in mice (Sucheston & Cannon 1968). Moreover, in aged mice, accumulation of lipofuscin, also known as aging pigment or ceroid, is detected in the X-zone (Rosol et al. 2001, Seehafer & Pearce 2006 and may extend into the medulla (Greaves 2007). Ceroid, a generic name given to lipopigments that accumulate in association with lipid degeneration or lipid excess, accumulates in lysosomes and consists of advanced lipoxidation end-products (ALEs), which harbor biochemical modifications leading to cross-linking of proteins, lipids and carbohydrates. Ceroid accumulation is associated with vitamin E deficiency, and with neurodegenerative diseases such as the neuronal ceroid lipofuscinoses (NCLs), possibly reflecting chronic oxidative stress (Weglicki et al. 1968, Seehafer & Pearce 2006, Hayashi 2009). Chronic oxidative stress and ceroid formation have been associated with tumor formation in the adrenal gland, and in particular with pheochromocytoma (PCC) (Pacak 2011). In humans, PCC is a rare tumor of which w25% of cases are associated with germline mutations, commonly in subunits of succinate dehydrogenase (SDH), which is part of mitochondrial complex II (Kantorovich & Pacak 2010).
We recently generated a ceramide synthase 2 (CerS2) null mouse (Pewzner-Jung et al. 2010a) and now document a high incidence of bilateral medullary hyperplasia and PCC in mice older than 13 months of age. CerS2 is one of six mammalian CerS (Levy & Futerman 2010, Park et al. 2014, each of which synthesizes ceramide, a key intermediate in the pathway of sphingolipid (SL) biosynthesis (Levy & Futerman 2010, Park et al. 2014 and an important signaling lipid (Hannun & Obeid 2008), particularly with acyl chains of different length. The CerS2 null mouse is unable to synthesize very-long chain (VLC) ceramides (C22-C24ceramides). As a result, the ceramide and SL profile is altered such that the mice contain no VLC-SLs but rather contain elevated levels of long chain (LC) SLs, and in some cases, elevated levels of the CerS substrate, sphinganine (Pewzner-Jung et al. 2010a, Ben-David et al. 2011. These mice develop lesions in several organs including the liver (progressive hepatopathy), lung (emphysema and chronic inflammation) and brain (bilateral and symmetrical demyelination) (Pewzner-Jung et al. 2010b, Ben-David et al. 2011, Petrache et al. 2013, and as we now report, in the adrenal gland. The mechanism of PCC formation in CerS2 null mice may be related to mitochondrial dysfunction due to complex IV deficiency, as has been observed in hepatocytes from these mice (Zigdon et al. 2013).

Generation and maintenance of CerS2 null mice
CerS2 null mice were generated as described (Pewzner-Jung et al. 2010a). All mice were treated according to the Animal Care Guidelines of the Weizmann Institute of Science Animal Care Committee and the National Institutes of Health's Guidelines for Animal Care.

Real time PCR
RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and cDNA was generated using the Verso cDNA Synthesis Kit (Thermo Scientific, Hudson, NH, USA). Real time PCR was performed using a SYBR Green PCR master mix (Finnzyme, Espoo, Finland) and an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Primers for cathepsin were from Vitner et al. (2010).

Macroscopic and microscopic analyses
Mice were removed for necropsy at various ages. The pathological study was based on 49 mice (30 CerS2 null (15 males and 15 females) and 19 WT mice (five males and 14 females)) aged 2-19.5 months. This group includes 21 mice older than 13.5 months (the youngest age at which PCC was observed), 11 CerS2 null mice (six females and five males) and ten WT mice (seven females and three males). The adrenal gland was freshly excised, weighed, photographed and fixed. Samples for histology were fixed in 10% neutral buffered formalin, processed routinely and stained with hematoxylin and eosin (H&E), Nissl, Periodic acid-Schiff (PAS) or Ziehl-Nielsen and Pearl's iron stains. Sections for immunohistochemistry were fixed in 3% paraformaldehyde for 2 days. Paraffin sections (4 mm) were deparaffinized and antigen retrieval was performed using 10 mM citric acid (pH 6.0) prior to blocking in 20% horse serum in PBS containing 0.2% Triton X-100 for 2 h. Antibodies were added overnight at 4 8C and a biotinylated secondary antibody was added for 2 h. Sections were processed with an avidin-biotin HRP kit (ABC kit, Vector Laboratories, Burlingame, CA, USA) for 2 h and developed using diaminobenzidine (DAB) (Sigma-Aldrich).

Stress-induced corticosterone secretion
CerS2 null mice were restrained for 30 min using a 50 ml conical tube with the bottom removed (Chen et al. 2006). Blood was collected before, 30 and 60 min after restraint and serum was isolated. Corticosterone levels were measured using a corticosterone ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). Epinephrine, metanephrine (MN), NE and normetanephrine (NMN) levels in urine were analyzed by the Bio-Rad HPLC method (Peaston et al. 1996).
Five micrograms of protein from the supernatant was diluted in 100 ml proteolysis buffer (50 mM Tris (pH 7.8), 20 mM KCl, 5 mM MgOAc, 0.5 mM dithiothreitol) and incubated with 200 mM Suc-LLVY-MCA at 37 8C for 1 h. The reaction was terminated by adding 1 volume of cold ethanol and measured at 380 nm excitation and 440 nm emission after addition of 0.125 M sodium borate (pH 9.0). Free methylcoumarin (MCA) was used for quantification.

Results
CerS2 is the major CerS transcribed in the adrenal gland ( Fig. 1A) and as expected, no CerS2 mRNA was detected in the adrenal gland of CerS2 null mice, in which a small elevation of CerS1 mRNA was detected (Fig. 1A). VLCceramides were the major ceramide species in the adrenal gland of WT mice and were significantly reduced in CerS2 null mice, while levels of C16:0 and C18:0-ceramides were elevated (Fig. 1B). As a result, total ceramide levels were elevated approximately two fold, similar to that observed in some other tissues (Ben-David et al. 2011, Pewzner-Jung et al. 2014. Sphinganine and sphingosine levels were also elevated ( Fig. 1B). CerS2 was detected both in the cortex and medulla of WT adrenal glands, although at higher levels in the cortex, but was not detected in CerS2 null mice (Fig. 1C). A similar expression pattern was observed by x-gal staining (which detects the LacZ gene inserted in the first intron of the CerS2 gene in the CerS2 null allele) (Fig. 1D). CerS2 was not detected in the X-zone ( Fig. 1C and D).

Ceroid accumulation in CerS2 null mouse adrenal gland
Since CerS2 was the major CerS in the adrenal gland, we examined the effect of ablating CerS2 on adrenal gland pathology. Mild to marked ceroid accumulation was observed in the X-zone of CerS2 null mice as early as 2 months of age, and was often more severe in older mice ( Fig. 2A), although no differences were observed between male and female mice. The material accumulated in, and distended the cytoplasm of cells arranged into variablysized clusters. The clusters were most-commonly concentrated in the X-zone but in some cases were also found in the cortex and medulla ( Fig. 2A). The ceroid was finely granular, light brown by the HE stain, PAS-positive, acid fast negative for iron (Fig. 2B), and autofluorescent ( Fig. 2C), as is typical of ceroid (Greaves 2007). Ceroidladen cells were F4/80-positive (Fig. 2D) and sometimes multinucleated, consistent with macrophages.
We also examined levels of cathepsins since ceroid is known to affect lysosome function (Keller et al. 2004). mRNA levels of a number of cathepsins were significantly elevated (Fig. 3A), as were protein levels of cathepsin D, L and S (Fig. 3B) and activity of cathepsin D (Fig. 3C). Cathepsins D, L and S were localized in ceroid aggregates ( Fig. 3D). Proteasome activity, which can be reduced by lipofuscin or ceroid (Sitte et al. 2000), was decreased between 2 and 6 months of age (Fig. 3E).

Mitochondrial dysfunction in CerS2 null mouse adrenal gland
Since ceroid can be formed as a result of oxidative stress (Kurz et al. 2007), we analyzed oxidative stress in the adrenal glands of CerS2 null mice. Lipid peroxidation products (identified using the anti-4-hydroxy-2,3-nonenal (HNE)-Michael adducts antibody) co-localized with ceroid aggregates (Fig. 4A), suggesting that chronic oxidative stress may lead to ceroid formation, or vice versa. No difference in the activity of the mitochondrial electron transport chain was detected at 2 months of age (not shown), but a significant reduction in the activities of mitochondrial complexes III and IV was detected in adrenal glands from 6 month-old CerS2 null mice (Fig. 4B), consistent with the notion that mitochondrial dysfunction is secondary to ceroid formation (Terman et al. 2006).

CerS2 null mice develop PCC and secrete elevated levels of epinephrine and NE
Bilateral PCC was found in six out of 11 CerS2 null mice (three males and three females) between the ages of 13-19 months (Fig. 5A). In the remaining five CerS2 null mice, medullary hyperplasia (unilateral or bilateral) was observed (Fig. 5B). Microscopic examination of mice with PCC showed marked enlargement of the adrenal gland in  medullary tissue of heterogeneous appearance, which at times compressed the cortex into a thin peripheral band (Fig. 6A). The neoplastic cells were of variable cellular and nuclear size, variable cytoplasmic staining (dark blue to clear), variable nuclear to cytoplasmic ratio, and were arranged in irregular nodules (Fig. 6B). The mitotic rate was high (Fig. 6B, arrows). Clusters of ceroid-laden cells were common throughout the tumor (Fig. 6C, asterisks), as were irregular areas of fibrillar eosinophlic material, consistent with neuronal processes.
Medullary hyperplasia (Mohr 2001) was seen at an earlier age than PCC in the CerS2 null mouse (Fig. 6D). It was present in three out of six of the 10.5-11 month-old males and in four out of five of the 12 month-old females. In this group, hyperplasia appeared to be in its early stage and the histologic findings were subtle and in some cases equivocal. By definition, in all cases with hyperplasia, the architecture of the adrenal gland was well-preserved and without significant cortical atrophy (Fig. 6D and E). Paragangliomas were not identified. None of the histologic changes (i.e. PCC or hyperplasia) were observed in WT mice (Fig. 6F).
No difference in stress-induced corticosterone secretion was detected between WT and CerS2 null mice (not shown). In contrast, evaluation of medullary function, ascertained by measuring urinary levels of epinephrine (E), NE, MN and NMN demonstrated that NE and NMN were elevated in 3 month-old CerS2 null mouse urine, while MN and E were elevated at 7 and 10 months of age (Fig. 7). This finding correlates with, and may precede, the morphological abnormalities observed in the medulla of CerS2 null mouse adrenal gland at a later age.

Discussion
The main finding of the current study is that CerS2 null mice display a high incidence of PCC, which is preceded by medullary hyperplasia. A number of genes have been associated with the development of PCC in humans, such as the SDH gene (mitochondrial complex II subunits), RET proto-oncogene, von Hippel-Lindau (VHL) tumor suppressor gene or neurofibromin 1 (NF1) gene (Fishbein & Nathanson 2012, Shah et al. 2012. Mutations in VHL and SDH result in accumulation of succinate which directly inhibits prolyl hydroxylase (PHD), thereby inducing Hif-1a accumulation (Favier & Gimenez-Roqueplo 2010). Hif-1a causes PCC tumorogenesis by increasing angiogenesis, proliferation, invasion and metastasis (Favier &  Gimenez-Roqueplo 2010). Mutations in the RET protooncogene and in NF1 activate RAS/RAF/MAPK and PI3K/Akt/mTOR signaling pathways, which play critical roles in tumorogenesis (Shah et al. 2012).
The mechanism responsible for PCC in CerS2 null mice is not known, but is clearly related to the changes in the SL profile. Three major changes in SL levels occur in CerS2 null mice; reduction in levels of VLC SLs, elevation of long-chain SLs and elevation of sphinganine (and sphingosine), although the pathways by which the latter two are altered is not known. Transcriptional up-regulation of either CerS1 or CerS5/6 cannot account for increased C18-and C16-ceramide. Rather, it is likely that changes in levels of these lipids are related to other changes in their metabolism (such as altered rates of synthesis vs degradation), although little information is currently available to assess this possibility. However, some of the pathology can be explained by changes in levels of specific lipids. For instance, C16-ceramide and sphinganine both directly inhibit complex IV activity in the liver (Zigdon et al. 2013), which results in oxidative stress. Altered levels of sphingosine, which is known to permeabilize lysosomes and cause relocation of lysosomal enzymes to the cytosol (Kågedal et al. 2001), may be related to the altered cathepsin levels, which together with oxidative stress might reduce lysosome function and thus accelerate lipofuscin accumulation.
Lipofuscin accumulates in CerS2 null mice as young as 2 months of age. This is somewhat similar to the events that occur in ABCD2-deficient mice (Lu et al. 2007) in which spontaneous and premature deposition of ceroid, an end-product of oxidative damage, occurs predominantly in adrenal medullary cells. Lipofuscin is normally found in senescent post-mitotic cells such as cardiac myocytes, neurons, and in the area between the zona reticularis and the medulla in adrenal glands in old age (Kurz et al. 2007). Thus, the CerS2 null mouse provides additional indications that oxidative stress is a key player in PCC formation, although the mechanism of oxidative stress is somewhat different inasmuch as SDH function was normal, the Hif-1a protein was not up-regulated, and Akt and mTOR phosphorylation was not detected in the PCC (data not shown). Moreover, ceroid accumulation is not observed in PCC caused by SDH mutations, suggesting that the mechanism of PCC formation differs between SDH mutations and that observed in the CerS null mouse. We conclude that defects in cytochrome c oxidase (mitochondrial complex IV) (Lee & Wei 2007) may be the main mechanism by which oxidative stress in generated in the CerS2 null mouse.
The relevance of our findings on altered SL levels to human PCC is currently unclear, with only one study relating to this issue, in which SMPD1, the gene which encodes for acid sphingomyelinase, was elevated in benign compared to malignant PCC (Thouënnon et al. 2007). There is one report of a human patient with a mutation in CerS2, in which a 27 kb de novo deletion in one allele of chromosome 1q21 was detected (Mosbech et al. 2014), although there is no evidence that this patient displays either mitochondrial dysfunction or PCC. There is, however, a relationship between altered CerS expression and cancer. Thus, increased expression of CerS2 occurs in breast cancer (Erez-Roman et al. 2010, Hartmann et al. 2012, Fan et al. 2013 and CerS2 may play a role in prostate cancer (Wang et al. 2012, Xu et al. 2012. Moreover, w15% of CerS2 null mice develop hepatocellular carcinoma (HCC) and most mice develop proliferative nodules (Pewzner-Jung et al. 2010b).
The enhanced levels of secretion of NE and MNE into the urine is consistent with data showing that these two hormones increase in PCC (Pacak 2011). Several factors are known to enhance catecholamine secretion from the medulla of the adrenal gland, including chronic hypoglycemia and increased leptin levels (Takekoshi et al. 2001, Shibuya et al. 2002, both of which are observed in CerS2 null mice (Park et al. 2012). Leptin stimulates the hypothalamus and increases sympathetic nerve tone (Rosol et al. 2001). Whether these factors are also involved in PCC formation in the CerS2 null mouse is unknown, but irrespective of the precise details of the molecular mechanism, interrogating the SL pathway might pave the way for understanding the etiology of PCC formation and for development of novel potential therapeutic approaches for the treatment of PCC.

Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding
This work was supported by the Israel Science Foundation (0888/11) and by National Institutes of Health Grant GM076217. W-J Park was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A1076013).