Colon cancer, a well-characterized inflammation-driven disease, is the third leading cause of cancer-related deaths worldwide, with more than one million new cases reported each year [1
]. These outcomes are mainly due to its poor prognosis, where long term treatment leads to patients acquiring resistance to therapy. In China, according to the data of a population-based cancer registry, the age-standardized 5-year relative survival rate for colon cancer is less than 50% [2
]. The major cause of colon cancer therapy failure is the emergence of tumor-driving cells or cancer stem cells (CSCs), which possess high plasticity and a mesenchymal-like phenotype [3
]. Among these contributors, leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)+
colon stem cells, existing near the base of intestinal crypts that normally contribute to all cell lineages in the intestinal epithelium, are the most rapidly self-renewing epithelial tissues in mammals [4
] and are known to serve as a cell of origin for colon cancer due to their inherent plasticity and longevity [5
]. Therefore, new therapies based on understanding the molecular mechanisms underlying colon CSC regulation are needed.
There is growing evidence associating colon cancer development with environmental causes, such as a high-fat diet [6
]. Pro-obesity diets enhance the numbers and function of the Lgr5+
stem cells and/or colon tumorigenesis by activating multiple pathways, including peroxisome proliferator-activated receptor delta (PPAR-δ) and Wnt/β-catenin signatures [7
], retinol binding protein 4 (RBP4)-stimulated by the retinoic acid 6 (STRA6) pathway [8
], and fatty acid oxidation [9
]. Similar changes were observed when dietary constituents, such as palmitic acid and oleic acid (OA), were administered, suggesting a critical role for fatty acid in the maintenance of colon CSCs.
Fatty acids function as critical constituents of membrane structure, large energy sources, and serve as signal mediators for signal transduction [10
]. Stearoyl-CoA desaturase-1 (SCD1), a transmembrane protein predominantly located at the endoplasmic reticulum, is a rate-limiting lipid desaturase responsible for generating monounsaturated fatty acids, mainly palmitoleic acid and OA [11
]. We recently showed that SCD1 is upregulated in CSC-like cell populations compared with non-CSC populations in human liver cancer cell lines [12
]. Other studies also shed light on the roles of unsaturated fatty acids in maintaining CSC stemness by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [14
] and Wnt-β-catenin [15
] signaling pathways. These studies indicate that lipid desaturation is a metabolic signature, and SCD1 may be an important therapeutic target in CSCs.
SCD1 has been reported to promote the metastasis of colon cancer by inducing epithelial–mesenchymal transition [16
]. Furthermore, the inhibition of SCD1 suppresses colon cancer cell proliferation [17
]. Although the underlying molecular mechanism has not been fully understood, possible pathways include either induced ceramide biosynthesis [17
] or the depletion of monounsaturated fatty acid [18
]. However, a limitation of this study is that in vitro monolayer cell cultures did not mimic in vivo three-dimensional (3D) tumor growth [19
]. Therefore, here we focus on the effects of SCD1 inhibition on the spheroid growth potential of a human colon cancer cell line. We employed a simple 3D multicellular tumor spheroid system known to be significantly more resistant to chemotherapeutic agents compared with monolayer cultures [20
] and found that a chemical inhibitor of SCD1, CAY10566, efficiently prevented the 3D spheroid formation of colon cancer cells.
Our data show that SCD1 inhibition is an effective strategy for suppressing the spheroid growth of colon cancer cells. Mechanically, data mining in the TCGA database revealed that the master regulator of lipid homeostasis SREBP2 acted as a master transcription factor, whose gene expression positively correlated with SCD1 in human colon cancer. Importantly, siRNA knockdown of SCD1
significantly inhibited SREBP2
expression, suggesting that the anti-tumor effects of SCD1 inhibition are mediated, at least in part, through the downregulation of the SREBP signaling pathway. SREBP2 belongs to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids, cholesterol, triacylglycerol, and phospholipid (Figure 4
]. Recently, the inhibition of SREBP has been shown to suppress tumor growth and initiation in colon cancer through the downregulation of cellular lipid biosynthesis [25
]. Our data provide direct evidence that SREBP signaling is under the control of SCD1 in colon cancer cells.
The pathway and network analyses suggested that the pro-tumorigenic effects of lipid desaturases, such as SCD1, in a human colon cancer cell lines are related to Myc signaling. Myc (c-Myc
), a basic helix–loop–helix–zipper transcription factor, is reported as one of the Yamanaka factors [26
] and plays a critical role in maintaining stem cell pluripotency. Myc also functions as an oncogene, contributing to both tumor initiation and maintenance by controlling cell growth [27
]. A recent study has shown that c-Myc
activation positively regulates cell self-renewing and is indispensable for crypt regeneration and colon tumorigenesis [28
]. Of interest, a domain-based interaction resource suggested a binding between human Myc and SCD1 proteins [29
]. It has been reported that aberrant stabilization and dysregulated activation of Myc reprograms cancer cell metabolism to enhance precursor provisions for phospholipids [30
]. In addition, OA administration partially rescues cell death-induced Myc signaling suppression, highlighting a critical role for lipid biosynthesis in Myc-driven cancer survival [30
]. It seems plausible that there is a positive feedback loop between Myc activation and lipid metabolism reprograming, which is probably regulated by the phosphatase and tensin homolog (PTEN)/the mechanistic target of rapamycin (mTOR) signaling pathways [31
In this study, we observed an increase in SCD1
gene expression in human colon cancer, which was significantly correlated with a poor prognosis. It has been reported that Toll-like receptor 4 (TLR4) deficiency can lead to the reduction of mRNA levels of genes involved in de novo lipogenesis, such as SCD1, but not genes involved in gluconeogenesis and fatty acid oxidation [33
]. These data suggest that SCD1 expression might be regulated by the lipopolysaccharide (LPS)/TLR4 signaling pathways. Studies from the past few years demonstrate a strong impact of environmental factors, such as high-fat diet, on microbial composition and colon cancer risk [34
]. Enhanced TLR4 expression in the intestinal epithelium has been shown to cause the expansion and proliferation of Lgr5+
colon stem cells [35
]. Enhanced TLR4 expression in colon cancer is related to increased metastasis [36
] and poor prognosis [37
]. Therefore, it seems reasonable to hypothesize that increased SCD1
gene expression in colon cancer is controlled by microbial dysbiosis and the activation of TLR4 signaling.
4. Materials and Methods
4.1. Cell Culture
WiDr cell, derived from human colon adenocarcinoma cell line HT-29 [38
], was maintained at 37 °C and 5% CO2
, in Dulbecco’s modified Eagle medium (DMEM, Wako Industries, Osaka, Japan) containing 10% fetal bovine serum (FBS; Mediatech, Herndon, VA, USA), 100 U/mL penicillin/streptomycin, and 2 mM l
4.2. 3D Spheroid Culture and Chemical Treatment
The cells were seeded onto non-adherent 96-well round-bottomed Sumilon PrimeSurface™ plates (MS-9096U, Sumitomo Bakelite, Tokyo, Japan) [20
], in 100 µL of media (DMEM plus 10% FBS), at a concentration of 5000 cells per well. The spheroids were grown for 4 days, and then, 50 µL of media were exchanged with 50 µL fresh serum-free media containing 40 µM CAY10566 (ab144421, Abcam, Cambridge, MA, USA), a SCD1 inhibitor, and/or 200 µM OA (O1008, Sigma, Louis, MO, USA). The spheroids were further cultured for 3 days, and photos were taken using an optical microscope (DS-Fi1, NIKON, Tokyo, Japan). Dimethyl sulfoxide (DMSO, Sigma) was used as the primary solvent for all chemicals.
4.3. Cell Viability Assay
Cell viability was measured using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan). The absorbance was measured using a plate reader (ARVO MX, Perkin Elmer Inc., Waltham, MA, USA) at 450 nm.
4.4. In vitro RNA Interference
A pool of 3 target-specific siRNAs targeting human SCD1 (sc-36464; siSCD1) and a control siRNA (sc-37007; siCtl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). WiDr cells were plated in 48-well plates for 24 h and then transfected with 100 nM siRNAs using Lipofectamine 2000 transfection reagent (Life Technologies, Gaithersburg, MD, USA). On the following day, cells were collected for RNA isolation and further analysis.
4.5. RNA Isolation and Real-time (RT)-PCR
Total RNA was isolated using a FastGene RNA Basic Kit (FG-80250, NIPPON Genetics, Tokyo, Japan) and quantified using a NanoDrop spectrophotometer (NanoDrop products, Wilmington, DE, USA). cDNA was synthesized using a PrimeScript RT Master Mix Kit (TaKaRa Bio, Otsu, Japan). Primer sequences were as follows (5’ to 3’): glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward (CAATGACCCCTTCATTGACC) and reverse (GACAAGCTTCCCGTTCTCAG), SCD1 forward (GTACCGCTGGCACATCAACTT) and reverse (TTGGAGACTTTCTTCCGGTCAT), and SREBP2 forward (ATCGACCTAGGCAGTCTGGT) and reverse (ATAGAGGGCTTCCTGGCTCA). PCR reactions were performed using a Roche LightCycler 96 Real-Time PCR System (Roche Diagnostic Co., Ltd., Mannheim, Germany) and the SYBR Premix ExTaq II (TaKaRa Bio).
4.6. Data Mining
expression from a clinical dataset, which contains transcriptome profiling of 566 colon cancer samples and 19 adjacent non-tumor samples, was downloaded from the GEO database (www.ncbi.nlm.nih.gov/geo
, accession no. GSE40967) [21
]. A PrognoScan database (http://www.abren.net/PrognoScan/
) analysis, which contains a large collection of publicly available cancer microarray datasets with clinical annotations, was conducted to investigate the clinical significance of SCD1
expression in predicting cancer prognosis [22
4.7. Knowledge-Based Pathway Analysis
Genes correlated with SCD1
gene expression in colon cancer were selected through an analysis of the TCGA database using cBioPortal software [39
]. With a threshold of Spearman’s correlation coefficient of >0.2, 806 genes were selected and input into the knowledge-based functional analysis software IPA (Ingenuity Systems) as previously described [40
]. The generated biological networks were ranked by score, which is the likelihood of a set of genes being found in the networks owing to random chance, identified by a Fisher’s exact test. The generated diseases or functions annotations were ranked by the activation z
-score, which can be used to find likely regulating molecules based on a statistically significant pattern match of up- and downregulation and also to predict the activation state (either activated or inhibited) of a putative regulator. An absolute z
-score of more than 2 was considered to be significant.
4.8. Statistical Analysis
Quantitative data were expressed as the mean ± SD of at least three replicates. The significance of differences between the values was assessed using Student’s t-test or Mann–Whitney U test. p-values of <0.05 were considered significant.