**3. Sphingolipids and colorectal cancer**

## **3.1 Sphingolipids' levels in plasma and tumor tissue**

The last decade was fruitful in the investigation of the metabolic switch during tumorigenesis [75]. Lipids are central in different cellular levels of physiology that go from plasmatic and membrane organization, plasticity, and signaling mechanisms [76–78].

Data from the literature indicate that the equilibrium between ceramides of various chain lengths is crucial for cell fate [35]. As noted before, the S1P/Cer ratio changes remain the best-characterized outcome of the alterations of SL metabolism in cancer.

The amount of new information and knowledge regarding sphingolipids in colorectal cancer can hardly be systematized. The best option is to follow the sphingolipids' metabolic pathways and see which alterations are present in cancer cells.

Ceramides and their proportion are different in plasma of patients with CRC and tumor tissue compared with plasma and tissue control levels. On the other hand, plasma ceramide concentration is not directly related to ceramide concentration in tumor tissue. One must also be aware that different chain lengths can have different actions regarding cell localization and the microenvironment. Chen et al. demonstrated increased levels of C16:0 and C24:0 ceramides and reduced levels of both C18 and C20 ceramides in colorectal tumor tissues [79–81]. Levels of C22:0 ceramide were unchanged [80]. Those results were in line with the protein expression and enzymatic activity of SCD1 (Stearoyl-CoA desaturase-1), a key conversion enzyme that regulates lipogenesis. SCD1 inhibition impairs the proliferation of cancer cells probably by cellular endogenous ceramide signals mediation [80]. Another study showed an increased amount of S1P and C14:0 compared to normal tissue and a significantly lower amount of C18:0 and C20:0, as previously noted [36].

The plasma profile of sphingolipids appears to be different than in tissues with the highest concentration in the plasma for C24:0-ceramide and C24:1-ceramide [36]. The concentration for C22:0, C16:0-ceramides, and S1P is smaller but significant [36]. Another study, however, showed significantly higher concentration levels of C16, C18, C18:1, and C24:1-ceramide than those of controls and lower levels of C24 sphingomyelin; there was a relation between these results and stage IV CRC. These results are limited by the small sample size and retrospective design of the study [82]. Markowski et al. divided the patients into two groups regarding their stage and showed that a higher tumor content of C20:0 and C24:0-ceramide was present in the TNM III + IV group. In plasma, there was a statistically significant relation between CRC patients in TNM stage III + IV and higher levels of C16:0 and C18:1-ceramides. Their data raise the possibility that it could be possible to distinguish patients between early and advanced stages based on this model [36]. Taken together, one must note that plasma ceramide concentration is not directly related to ceramide concentration in tumor tissue.

In another study with patients with pulmonary and hepatic metastasis submitted to radiotherapy, it was observed that although pre-treatment levels of ceramides did not correlate with response to treatment, patients with complete response had higher post-treatment total plasma ceramide levels than non-responders [83].

Lymph node invasion was shown to have a positive correlation with C24 ceramide levels in CRC tumor tissues [79]. It was also demonstrated that Sphingosine 1-phosphate (S1P) signaling pathways were associated with lymphangiogenesis [84].

### **3.2 Sphingolipids enzymes in colorectal cancer**

### *3.2.1 Pro-ceramide metabolic pathways*

As mentioned before, sphingolipids' metabolism is regulated through a complex equilibrium between different enzymes' actions, which will, in the end, change the balance between ceramide and S1P. For example, different enzymes will provide

different ceramides, with different actions depending on the tissue and subcellular localization.

The discovery and cloning of CERS1–6 were crucial for understanding the roles of ceramides with different fatty acyl chain lengths in cancer cell signaling. Hartmann et al. showed that overexpression of CerS4 and CerS6 in HCT-116 human colon cancer cells inhibits cell proliferation by upregulation of long-chain ceramides C16:0, C18:0, and C20:0. In contrast, upregulation of CerS2 and concomitant increase of C24:0 and C24:1 promotes cell proliferation [35].

Jang et al. revealed that all four CerS genes were significantly upregulated in CRC tissues compared with corresponding normal tissues [85]. CERS6 overexpression reduced the proliferation of CRC cells and induced apoptosis, whereas CERS2 overexpression increased the proliferation of CRC cells [35]. Regardless of the mechanism, overexpression of CERS2 and CERS6 decreased the viability of CRC cell lines tested [85]. CerS6-generated C16 ceramide was shown to increase apoptosis in colon cancer cells [46].

CERS5-ko mice showed significantly larger colon tumors than CERS5-wt mice [86]. Another study showed that strong CERS5 staining correlated with poor prognosis in patients with CRC [87]. CERS4 and CERS5 were also found to be upregulated in colon cancer prior to apoptosis induction and down-regulated after apoptosis induction in colon cell lines [88].

The importance of ceramide levels in cancer cells was also demonstrated in studies with ceramide analogs such as LCL-30, the cationic water-soluble analog of C16 ceramide. LCL-30 accumulates in cells' mitochondria and induces mitochondrial swelling, decreases membrane potential, caspase activation, and ultimately cell death [89, 90]. The same group also tested its actions in colon carcinoma cell line CT-26 as an *in vivo* model of colorectal cancer, demonstrating that LCL-30 was cytotoxic to CT-26 cells [90].

Adiseshaiah et al. also showed that injection of nanoliposomal C6-ceramide, an autophagy inducer, in combination with vinblastine, decreased tumor growth in comparison to the individual treatments [75]. The authors used the colon cancer xenograft model (LS174T) and showed that the combination treatment resulted in statistically significant suppression of tumor growth compared to a single treatment. The rationale behind the study was that cancer cells might evade anticancer therapy by inducing autophagy, so blocking it should improve therapeutic response.

It is undoubtedly that microenvironment will largely influence cancer cells' fate during their life cycle. Cancer cell progression is associated with tumorigenic M2 macrophages. Ceramide-treated macrophages were shown to induce the switching of macrophage polarization toward the pro-inflammatory M1-phenotype. Ceramide also abolished macrophage-induced epithelial-mesenchymal transition and migration of colorectal cancer cells [91]. Other studies have demonstrated that M1 and M2 macrophages can switch phenotypes and lipids have the potential to modulate their function and phenotypes [92, 93]. Ceramides act as an intracellular second messenger and membrane component [94]. Araujo Junior et al. have demonstrated that ceramide can reduce M2 phenotype and block migration of cancer cells, suggesting that targeting ceramide in the tumor microenvironment could, in theory, reduce tumor progression and potential for metastasis of colon cancer cells [91].

Ceramide is also generated by the hydrolysis of sphingomyelin by SMases—acid, neutral, and alkaline—based on their pH-dependent optimal activity.

The activities of neutral and alkaline SMase were highest in the ascending colon and decreased in the sigmoid colon and rectum, whereas no significant difference

### *Understanding Sphingolipids Metabolism in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.105465*

was found for acidic SMase activity at all locations [48]. Markowski et al. also examined the relationship of sphingolipids levels in CRC tissue on tumor localization and documented that, albeit complex and ambiguous, the number of total ceramides was lowest in sigmoid and cecum tumors and the largest in rectal tumors [36]. SMase activity was found to be decreased in colorectal carcinomas, mainly alkaline SMase activity, which results in lowered cellular levels of ceramide. In comparison to surrounding normal tissue, SMase activity in colorectal cancer is reduced by 75%, 50%, and 30% for alkSMase, nSMase, and aSMase, respectively [48].

### *3.2.2 Pro-S1P metabolic pathways*

So, on one side of the balance, we can identify the mechanisms responsible for ceramide raised levels; however, on the other side, we should pay attention to the antagonist mechanisms leading to the degradation of ceramide in detriment to S1P and their transitory metabolites.

Among the five ceramidases identified to date [95], neutral CDase is predominantly expressed in the colon and is involved in the metabolism of dietary sphingolipids [96]. It was shown that inhibition of NCDase induces an increase of ceramide in colon cancer cells, decreasing cell growth and increasing apoptosis [50, 81]. Coant et al. also showed that deletion of NCDase protected mice from the onset and progression of colorectal cancer C16:0 ceramide levels were increased. The inhibition of NCDase leads to inhibition of the WNT/β-catenin pathway [81]. HT 29 colon cancer cells treated with NCDase inhibition were accompanied by decreased survival, increased apoptosis, and autophagy [50]. Animal studies also showed that inhibition of NCDase delayed tumor growth, with increased ceramide and reduced tumor cell proliferation [50]. Taken together, NCDase appears to be an important target for new therapeutic strategies.

Studies in mice have demonstrated that oral administration of plant-type sphingolipids increased colonic Sphingosine-1-phosphate lyase (SPL) levels and reduced S1P levels, cytokine levels, and tumorigenesis, indicating that SPL can prevent transformation and carcinogenesis [53]. These studies suggest that dietary sphingolipids can have a role in colon cancer prevention in opposition to high-fat diets that possibly increase the risk of colorectal cancer. SPL is highly expressed in normal intestinal and colonic epithelium, however, it is downregulated in CRC cells and in early adenomatous lesions of Min mice [54]. SPL expression promotes apoptosis through a cascading mechanism that involves p53, p38, PIDD, and caspase-2; however, it is not clear how this interaction occurs [54]. SPL activity provides an exit route from sphingolipid metabolism via the rapid hydrolysis of S1P. SPL appears to be downregulated at the protein level in colon cancer tissues, and SPL silencing promoted colon carcinogenesis, which occurred via S1P accumulation and/or S1PR signaling [53]. On the contrary, SPL overexpression leads to increased apoptosis through reduced S1P signaling in colon cancer cells [54].

The two isoforms of sphingosine kinase, SPHK1, and SPHK2, utilize sphingosine and generate S1P but have significant differences in subcellular localization and function [51]. Sphingosine kinases (SPHK1 and 2) are overexpressed in many cancers, including colorectal cancer, compared with normal mucosa [97]. The expression levels of SPHK1 and 2 were also high in liver metastases compared with matched normal colon tissues. SPHK1 and SPHK2 are observed in different places within the cell; SPHK1 in the cytosol while SPHK2 was detected in both cytosol and nucleus [97]. SPHKs seem to have a role in promoting the metastatic potential of colorectal cancer

cells [97]. FTY-720, an S1P receptor antagonist, reduces cell migration and invasion and significantly decreases cellular proliferation in all cell lines tested [97].

### **3.3 Sphingolipids, treatment resistance, and new strategies**

5-Fluorouracil (5-FU) is one of the first-line chemotherapy agents' in colorectal cancer and despite its efficacy, drug resistance is still an important limitation. Jung et al. conducted a lipidomic analysis showing that resistance to 5-FU is associated with the up-regulation of sphingomyelin and the down-regulation of CERS [98].

SPHK1 contribution to cetuximab resistance in colorectal cancer was investigated. The authors found overexpressed and overactivated SPHK1 in colorectal cancer cells with intrinsic or acquired resistance to cetuximab [24]. It was also documented that treatment of resistant cells with FTY-720 resulted in resensitization to cetuximab both *in vitro* and *in vivo* [24]. This association could be a new therapeutic strategy to overcome chemotherapy resistance and also a biomarker of interest for cetuximab resistance.

In another study involving SPHK2, the authors found that using ABC294649, a novel SPHK2 inhibitor, resulted in growth inhibition and apoptosis of CRC cells, with S1P depletion and ceramide incrementation. Also, exogenously-added S1P inhibited ABC294640 cell effects. The authors also described that ABC294649 sensitized 5-FU and cisplatin-mediated anti-HT-29 cell activity. This agent could be an important anti-CRC weapon, and it is also available in an oral formulation [58]. Xun et al. demonstrated in HT-29 cell lines that SphK2 inhibition (ABC294640) resulted in S1P depletion and ceramide incensement with consequent cell lethality. Oral administration dramatically inhibited H-29 xenograft growth in nude mice [58].

SphK inactivation induces the accumulation of S1P precursors, including sphingosine and ceramide, causing cell apoptosis and growth arrest [99].

Activity in primary cancer cells was also tested. SphK2 expression was different between patients, however, ABC294640 activity was negatively associated with SphK2 expression level [58].

Glucosylceramide synthase (GCS), a ceramide-metabolizing enzyme, has been demonstrated to be overexpressed in CRC tissues compared with non-CRC tissues. Wang et al. documented that high-expression GCS patients were associated with significantly higher lymph node metastasis than the low CGS expression group [63].

GCS has been associated with several studies that documented its role in chemotherapy resistance [63, 100]. Oxaliplatin-resistant cells demonstrated increased expression of GCS protein compared to the parental cell line, with increased levels of glucosylceramide (GlcCer) [100]. Madigan et al. also showed that inhibition of GCS expression resulted in the reduction of ClcCer levels with restored sensitivity to oxaliplatin. Oxaliplatin-resistant CRC cells also expressed lower ceramide levels compared to parental cells. In fact, the conversion of ceramide to glucosylceramide by GCS represents an essential mechanism for limiting ceramide accumulation [101]. It was also shown that the rate of GCS was higher in patients receiving neoadjuvant chemotherapy than in non-CRC tissues, raising the possibility that chemotherapy drugs might induce the high expression of GCS and increase the risk of MDR [63]. The authors hypothesized that oxaliplatin treatment might result in reduced ceramide levels compared to oxaliplatin-sensitive cells. C16-ceramide was the only species to differ significantly between the two cell lines. Higher sphingomyelin levels were found in the positive nodes of colorectal cancer patients compared to the negative lymph nodes [102].

*Understanding Sphingolipids Metabolism in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.105465*

In recent years, a few new pharmacologic strategies have been used in laboratory and clinical trials. Fenretinide (preclinical; reduces de novo synthesis with dihydroceramide accumulation), Safingol (association with irinotecan, preclinical; SPHK1 inhibitor), Ceramide nanoliposomes (association with tamoxifen, preclinical; apoptosis promoter by ceramide accumulation), α-GalCer (preclinical; α- galactosylceramide-pulsed antigen-presenting cells), and Fingolimod (association with sphingosine and cetuximab, preclinical; functional antagonist of the sphingosine-1-phosphate receptor (S1PR) and structural analog of sphingosine) [60, 103] are the most important in colorectal cancer with exciting and promising results.

### **4. In summary**

Sphingolipids are structural molecules of cell membranes with an essential role in barrier and fluidity functions. They have been implicated in many physiologic and pathologic processes, such as cell growth, cell death, adhesion, proliferation, stress, inflammatory responses, differentiation, migration, invasion, and/or metastasis.

The sphingolipids play an essential role in cancer biology and influence treatment response and aggressiveness. It also happens in colorectal cancer and may be interesting in developing an individualized treatment plan for LARC.

Nevertheless, the molecule's action interpretation is complicated, given the complexity of sphingolipid's metabolism with several activations and counter-regulation pathways. In addition, there are isoforms whose action is different depending on the location in the cell and the type of tissues in which they occur. Finally, the balance among ceramides has also essential for the activity response.

However, we can state that in general terms, there are two central bioactive lipids, ceramides and sphingosine-1-phosphate (S1P), which have opposing roles in regulating cancer cell death and survival. Ceramides have been shown to mediate cell cycle arrest and cell death in response to cell stress. Also, the equilibrium between ceramides of various chain lengths is crucial for cell fate. On the other hand, S1P has been shown to promote cell survival and proliferation.

Thus, the increase in specific ceramides in the tumor may correspond to a lower aggressiveness or effective response to the therapy instituted. In comparison, the rise in S1P in the tumor will correspond to a greater aggressiveness of tumor resistance to the treatment.

In this perspective, the measurement of ceramides and S1P may be of interest to assess the aggressiveness of a particular tumor. Nevertheless, on the other hand, we can try to interfere with the amount of these elements present in the tumor to modify tumor resistance to conventional therapy.

From published studies, it appears that sphingolipids' metabolism in tumor tissue is unsettled in colorectal cancer.

Ceramides and their proportion are different in plasma of patients with CRC and tumor tissue compared with plasma and tissue control levels. On the other hand, plasma ceramide concentration is not directly related to ceramide concentration in tumor tissue.

The knowledge gathered in the past decade can lead us to new ways of treating CCR patients, trying to overcome treatment resistance, and, in the end, achieving higher response rates and improved global life expectancy.

In conclusion, the knowledge of tumor sphingolipids metabolism may be essential in colorectal cancer treatment. Unfortunately, the studies about this issue are small and few. Therefore, investigation in this area is needed.
