**7. Dihydroceramide Desaturase**

Dihydroceramide Δ4-desaturase (DES) is the member of the desaturase family which converts the dihydrosphingosine backbone within ceramide into a sphingosine backbone [97]. The first step is utilizing molecular oxygen to introduce a hydroxyl group into the C4 position of the dihydrosphingosine backbone, which is then followed by a dehydration reaction producing a double bond in the C4–C5 position of dihydroceramide, with the aid of NADPH [98–100]. The only difference between dihydroceramide and Cer is that Cer has a *trans* double bond at the C4–C5 position. In mammals, two gene isoforms named *DES1* and *DES2* have been identified [101]. The *DES1* gene contains multiple transmembrane domains, and a recent study shows that it requires myristoylation on its N-terminus for full activity [101,102]. DES1 is localized in the ER membrane where it has access to newly synthesized dihydroceramide species [97]. 4-hydroxyceramide is an intermediate reaction product in the conversion of dihydroceramide to ceramide, which is also known as phytoceramide. In plants and yeast, it is the predominant ceramide species. In mammals, DES1 is found in all tissues and only converts dihydroceramide species into fully desaturated Cer, whereas DES2 is capable of creating either phytoceramide or ceramide from dihydroceramide precursors [103,104]. DES2 is highly expressed in skin, intestines, and kidneys [103]. The deletion of *DES1* and *DES2* shifts the SL synthesis pathway toward the SL, lacking the double bonds introduced by DES1 and DES2, such as dhS1P, dhSph, dhsphingomyelin (dhSM), and especially DhCer [105]. In *Des1*−/<sup>−</sup> mice, the inability to form Cer leads to highly elevated dihydroceramide, low levels of Cer, multi-organ dysfunction, and failure to thrive [106]. Cer has signaling properties that are distinct from dihydroceramide and phytoceramide, suggesting that most cells have evolved to recognize Cer as a more significant determinant for initiating a cellular response [107].

#### *7.1. Role of Dihydroceramides in Various Diseases*

#### 7.1.1. DhCer in Brain Diseases

Increased DhCer levels have been observed after hypoxia and subarachnoid hemorrhage [108,109]. Both studies suggest the involvement of DhCer in the mechanisms of disease in oxygen deprivation states such as stroke. Altered DhCer levels have also been noted in studies related to certain neuronal diseases such as leukodystrophia [110], Alzheimer's [111], and Huntington's disease (HD) [112]. The association of DhCer with the progression of degenerative brain diseases and other brain-related diseases makes it a potential target as a biomarker or diagnostic tool.

#### 7.1.2. DhCer in Cardiovascular Disease

DhCers were found to be increased in both human atherosclerotic plaques and rat models of hypercholesterolemia [113,114]. DhCer also correlates with the release of macrophage inflammatory protein 1β (MIP-1β). However, the role of DhCer in plaque stability is debatable, because the extracellular addition of DhCer to human aortic smooth muscle cells did not cause apoptosis, whereas the addition of Cer did [115]. Apart from these studies, increased DhCer levels have been found in patients with rheumatoid arthritis [116] and in doxorubicin-induced cardiac toxicity [117]. All these studies suggest the role of DhCer as a marker for cardiac pathology.

#### 7.1.3. DhCer in Cancer Therapy

As Cer has been studied for its apoptotic property, in most of these studies, DhCer has been considered as a precursor to Cer [114,118,119]. Some studies have focused on DhCer's potential role in cancer cell autophagy [120–122] in cancer-induced bone pain [123], and in cell cytotoxicity [124]. The fluctuation in the DhCer and Cer levels in cancer cells seems to differ according to the site of origin of the cancer. For example, in cancerous tissue of human endometrial cells, the level of DhCer was increased 3- to 4.6-fold, and Cer and S1P were increased 1.6- to 1.9-fold [125]; whereas in melanoma cells, DhCers and Cers were significantly lowered compared with non-malignant melanocytes [33]. Recent studies have focused on the gatekeeper enzyme dihydroceramide desaturase 1 (DES1), a new target for cancer therapy, for a better understanding of the pathological effects of DhCer in cancer. DES1 performs desaturation resulting in olefinated functionality in Cer. 4-HPR-fenretinide, a DES1 inhibitor, is currently being studied for different types of cancers including peripheral T-cell lymphomas and solid tumors. In SMS-KCNR neuroblastoma cells, 4-HPR-fenretinide directly inhibits DES1 with an IC50 of 2.32 μM. Inhibition of SK sensitizes cells to 4-HPR-fenretinide's cytotoxic effects due to an increased level of DhCers [126]. The possible interaction between 4-HPR-fenretinide's inhibition of DES1 and SK activity has been supported by a few other studies [122,127–129]. In cancer cell lines like HEK293, MCF 7, A549, and SMS-KCNR cells, oxidative stress can also inhibit DES1, which is followed by an increased level of DhCers [130]. Increasing the exogenous DhCer levels induced autophagy in T98G, U87MG glioblastoma cells [121], and DU145 cells [120] and reduced proliferation in castration-resistant PC cells [131]. In the human gastric cancer cell line, HGC-27, DhCers exerted autophagic effects when DES1 was inhibited by XM462 and resveratrol, resulting in higher levels of DhCer [122]. DhCer only induced autophagy when the de novo SL biosynthesis pathway was altered; in studies where both DhCers and Cers levels were increased, apoptosis occurred instead of autophagy [132]. DES1 assisted with the advancement of metastasis in PC cells [133] and esophageal carcinoma, possibly through increased cyclin D1 expression as a result of NF-кB activation [134]. These studies suggest the potential of increasing DhCer levels to increase autophagy and inhibiting metastasis through DES1 inhibition as promising targets for cancer therapy. A list of DES1 inhibitors that consist of natural products and related molecules that resemble SL structures, are shown in Figures 8 and 9, respectively [103].

**Figure 8.** Natural products and small molecules of non-Sphingolipid analogs reported as dihydroceramide desaturase inhibitors.

**Figure 9.** Chemical structure of sphingolipid analogs reported as dihydroceramide desaturase inhibitors.

#### **8. (Dihydro)ceramide Synthase**

Dihydrosphingosine (DHSph) is further acylated by six different (dihydro)ceramide synthases. In mammals, six distinct (dihydro)ceramide synthases, abbreviated as CerS1-6, have been identified and are encoded by six distinct genes [135,136]. In SL metabolism, no other step has as many genes devoted to it as (dihydro)ceramide synthesis, suggesting that each different CerS has distinct functions.

#### **9. Ceramide Synthases**

Ceramide synthases are a group of enzymes which play a central role in SL metabolism by catalyzing the formation of Cers from sphingoid bases and acyl-CoA substrates. So far, six CerSs (CerS1–CerS6) have been identified and each of them has a unique characteristic which will be discussed below.

#### *9.1. Ceramide Synthase 1*

Studies have shown Ceramide Synthase 1 (CerS1) to prefer stearoyl CoA as a substrate for producing the long-chain C18-ceramide [137]. In humans, CerS1 expression has been detected in glioblastoma cells [138], lung cells [138], and brain tissue [137]. Studies have shown an upregulated expression of CerS1 in the anterior cingulate cortex in post-mortem brain tissue from Parkinson's disease patients [139]. In Parkinson's disease patients, the C16:0-, C18:0-, C20:0-, C22:0-, and C24:1-ceramides concentration level is elevated in plasma, indicating the involvement of other CerS isoforms in the development of the disease [140]. CerS1 is also linked to the autoimmune disorder multiple sclerosis, a neuronal disease characterized by the demyelination of neurons. In the spontaneous relapse-remitting EAE (experimental autoimmune encephalomyelitis), CerS1 expression in the lumber spinal cord is decreased [141]. CerS1 is also associated with the development of obesity. In

liver microsomes of high-fat, diet-induced obese (DIO) mice, an upregulation of CerS1 expression is shown due to a high-fat diet [142].

CerS1 has been identified to play a role in the pathogenesis of head- and neck squamous cell carcinoma (HNSCC). Studies have substantiated correlations between reduced C18-ceramide in HNSCC tumors and increased lymphovascular invasion, nodal metastasis, and higher tumor stages [143]. In A549 human lung adenocarcinoma cells, C18-Cer that is generated by overexpressed CerS1 represses the promoter activity of human telomerase reverse transcriptase (hTERT) [143]. Human breast tumors exhibit increased CerS1 mRNA levels when compared with normal breast tissue, and this was correlated with poor prognosis of the patients [144]. In human colorectal cancer (CRC) tissue compared with nontumor colonic tissue, elevated CerS1 mRNA levels were observed; however, this was accompanied by a reduction in C18-ceramide levels [145].

In neuroblastoma cells, CerS1 downregulation results in ER stress and proapoptotic signaling [146]. In human glioma tissue, C18-ceramide levels are lower than in control tissue, and overexpression of CerS1 or exogenous C18-ceramide triggers ER stress, lethal autophagy, and cell death in glioma cell lines [147]. These studies stipulate that CerS1 and its product C18-ceramide can exhibit antiproliferative effects in different cancer cell lines and tissues.

#### *9.2. Ceramide Synthase 2*

Ceramide Synthase 2 (CerS2) utilizes C20–C26 acyl CoA species and is responsible for long-chain ceramide species [148]. CerS2 has a substrate specificity towards C20:0-, C22:0-, C24:0-, C24:1-, and C26:0-acyl-CoAs. Its KM towards sphinganine is 4.8 ± 0.4 μM. In humans, CerS2 is expressed in the kidneys, liver, brain, heart, placenta, and lungs, and in breast tissue, skeletal muscle, testis, intestines, and adipose tissue [148–151]. This broad and quantitatively strong tissue distribution of CerS2 indicates its prominent role among the CerS isoforms and the importance of CerS2-derived long-chain Cers for basal cellular SL metabolism. Due to its wide distribution and distinct genomic features, the *CERS2* gene has been described as a potential housekeeping gene in mammalian cells. The CerS2 protein is localized in the ER.

CerS2 is strongly associated with the development of multiple sclerosis. In the experimental autoimmune encephalomyelitis (EAE) model, *CerS2* knockdown has a protective effect, possibly due to an impaired migration of neutrophils into the CNS [152]. In the spontaneous relapse-remitting EAE mouse model, *CerS2* expression decreased in the lumbar spinal cord [141]. CerS2 is also linked to the chronic neurodegenerative Alzheimer's disease. In an Alzheimer's disease model, there was increased expression of CerS2 in brain tissue, which led to apoptosis in glial cells [153]. In progressive myoclonic epilepsy (PME) patients, heterozygous deletions of *CerS2* in fibroblasts have been observed, which suggests that a reduced *CerS2* level led to PME development [154]. CerS2 plays a significant role in CNS development and pathological conditions.

Several studies have supported the role of CerS2 as a tumor suppressor protein and in maintaining cell- and tissue integrity. In human HCC tissue, a low expression of CerS2 correlates with tumor progression and poor prognosis [155]. In breast cancer patients, inverse relationships between CerS2 expression and tumor progression, lymph node metastasis, and HER2 expression were discovered [156]. CerS2 overexpression inhibits proliferation and triggers cell cycle arrest and apoptosis in a p21/p53-dependent manner in papillary thyroid cancer cells [157]. A decreased level of CerS2 inhibits tumor growth and metastasis in meningioma, [158] bladder cancer [159–161], and PC [162,163].

#### *9.3. Ceramide Synthase 3*

CerS3 prefers middle- and long-chain acyl CoAs and generates C18:0-ceramide and longer-chain Cers [164,165]. In human tissue, CerS3 is expressed in keratinocytes, and shows high expression in the kidneys and liver with moderate expression in the brain, heart, skeletal muscle, placenta, and lungs [166,167].

Mutation of *CerS3* has been reported as a reason for autosomal recessive congenital ichthyosis (ARCI), a keratinization disorder [168]. CerS3 mRNA is reduced to 70% in these patients' skin. Another study supported this cause by showing a splice mutation in CerS3 leading to a reduced number of very long chain Cers in the skin, which are essential for epidermal differentiation, an essential process for the maintenance of epidermal barrier function [164,169]. There are a lack of data on CerS3 expression in cancer, possibly due to a restricted expression of CerS3 in the mammalian body, the limited availability of specific antibodies, and the lethality of *CerS3* knockout mice. One study reported decreased *CerS3* mRNA levels in human breast tumors compared with normal breast tissue. CerS3 is the only ceramide synthase which is downregulated compared with significantly upregulated CerS2, 4, 5, and 6 [144].

#### *9.4. Ceramide Synthase 4*

Ceramide Synthase 4 (CerS4) exhibits a substrate specificity towards C18:0- and C20:0 acyl-CoAs. In humans, it is expressed in kidney tissue (renal papillae, medulla, and cortex) [170] and breast tissue [151].

In an Alzheimer's disease mouse model, upregulation of CerS4 expression and increased C20:0- and C24:0-ceramide in the hippocampal brain tissue was observed [148,153]. In human liver cancer tissue, CerS4 is upregulated at the mRNA- and protein level and promotes liver cancer cell proliferation associated with NF-κB signaling [166,171]. In human breast cancer tissue, there is higher *CerS4* mRNA expression compared with healthy breast tissue [151]. *CerS4* expression is higher in estrogen receptor (EsR)-positive tumors than in EsR-negative tumors [172]. It is possible that the increase in the ceramide synthesis by CerS and other CerSs might promote breast and colorectal cancer cell growth through a disturbed cellular SL homeostasis. Moreover, breast cancer patients with higher mRNA expression of *CerS4*, along with *CerS1* and *CerS5*, show a worse prognosis than those with low *CerS* expression levels [144].

### *9.5. Ceramide Synthase 5*

Ceramide Synthase 5 (Cers5) prefers palmitoyl CoA as substrate, generates predominantly C16-ceramide species [173], and is expressed in human lung, [150] kidney (renal papillae, medulla, and cortex) [170], and breast tissue [151]. The KM towards sphinganine is 1.8 ± 0.4 μM and is expressed in the moth ER and nucleus. A study has shown a mild upregulation of *CerS5* in the lumber spinal cord in the spontaneous relapse-remitting EAE mice model [141]. In addition, *CerS5* mRNA expression is elevated in patient-derived colorectal cancer (CRC) tissue in comparison to normal colonic mucosa [174–176], and *CerS5* can be used as a marker for CRC [176]. Another study where data from a reversephase protein microarray using epithelium-enriched, human CRC tissue samples were used revealed that high CerS5 protein expression is associated with the autophagy-regulating protein signaling network, in contrast to low CerS5 levels that are associated with an apoptosis-related proteomic network [177]. In human neuroglioma tissue, elevated expressions of *CerS5* mRNA and protein levels were observed when compared with normal nervous ganglion tissue [178]. These studies suggest the correlation between high CerS5 expression and tumor cell proliferation and cancer progression in CRC, breast cancer, and other malignancies.

#### *9.6. Ceramide Synthase 6*

Ceramide Synthase 6 (CerS6) has a substrate specificity for C14:0- and C16:0 acyl-CoAs and its KM towards sphinganine is about 2.0 ± 0.6 μM. It is mainly localized at the ER. In humans, it is expressed in kidney (renal papillae, medulla, and cortex) [170] and breast tissue [151].

Increased *CerS6* expression is observed in neutrophils isolated from blood [152] and in macrophages and astroglia in the lumbar spinal cord [179] in a progressive, chronic experimental autoimmune encephalomyelitis (EAE) mouse model. In spontaneous relapseremitting EAE, overexpression of *CerS6* in macrophages was observed [141].

When comparing with corresponding healthy tissues, an abnormal higher CerS6 level is observed in colorectal cancer (CRC) [174,175] and breast cancer [144,151,180,181]. Also, CerS6 expression is higher in estrogen receptor (EsR)-positive breast tumors than in EsRnegative tumors [151,172,182]. A similar kind of pattern was observed in gastric cancer. CerS6 overexpression corelates with poor patient survival and *CerS6* knockdown decreases proliferation, migration, and invasion of gastric cancer cells. The proposed mechanism is the downregulation of the suppressor of cytokine signaling 2 (SOCS2) by overexpressed *CerS6*, leading to the activation of JAK-STAT signaling, followed by enhanced expression of genes involved in cell cycle progression (cyclins A and B) and metastasis (MMP-2 and -9) [183,184].
