Studies On Association Between Fatty Acid Synthase (FASN) Expression And Clinicopathological Outcomes Of Colorectal Cancer And Its Impact On Biology Behavior Of Colon Cancer Cells

Background and ObjectiveFatty acid synthase (FASN) is the key enzyme to grow polymeric chain fatty acid with the small molecule carbon units in process of fatty acid biosynthesis, FASN capable of modulating the synthesis of endogenous fatty acid, palmitic acid which is the main product, and the energy stored in the form of triglycerides, mainly involved in phospholipid synthesis, cell membrane structure, pulmonary surfactant production, intracellular signal transformation, protein acylation and so on. FASN is not expressed in normal tissues or micro expression, and expression is upregulated in a variety of malignant tumors. FASN-mediated metabolic abnormalities in tumors play an important role in the development process of cancer. Further investigations should focus on whether the altered FASN expression correlate with the colorectal tumorigenesis or different biologic properties, including those associated with proliferation, invasion and metastasis. In the present study, we investigated the relationship between FASN and colorectal carcinoma, especially focused on the effects of FASN regulation on the biological behavior of colorectal cancer. This work will contribute to clarifing the relationship between FASN and colorectal cancer and provide laboratorial evidences for prevention and therapy of patients.Methods1.71 clinical paraffin-embedded human colorectal cancer tissue specimens were immunostained with antibodies against FASN. To observe whether the expression of FASN was associated with the colorectal cancer pathological type, clinical staging or prognosis;2.74 plasma samples of colorectal cancer patients were collected and plasma concentration of FASN was tested by the way of enzyme-linked immunosorbent assay (ELISA). To observe whether the expression of FASN was related to the colorectal cancer pathological type, clinical staging or prognosis;3. Quantitative real-time polymerase chain reaction was used to measure the expression of FASN mRNA in Caco-2, HT29, LoVo, and LS174T cell lines. We chosed the cell line with higher FASN expression as target cell in further studies. pcDNATM6.2-GW/EmGFP-miR-FASN RNAi vectors and one negative vector were generated and tested by real-time polymerase chain reaction for their effects on reducing FASNmRNA levels. Established MUC2-RNAi and control-negative target cells, transfected with pcDNA6.2-GW/EmGFP-miR-FASN or control vectors, respectively. Inhibition of FASN expression was detected by real-time PCR and western blot analyses. To examine the possible involvement of FASN in tumor progression, the capacity of target cells for proliferation and invasion/migration was assessed after RNAi treatment using MTT, colony formation and transwell assays.4. We used a nude mouse subcutaneous injection model to clarify the effects of FASN on carcinogenesis in vivo. Xenograft tumors were evaluated by hematoxylin and eosin staining and by immunohistochemistry for the expression of FASN.Results1. Immunohistochemical results showed:56 cases of FASN protein signaling positive, accounting for 79% (38 cases of FASN protein signaling moderate positive,18 cases of FASN protein signaling strong positive); 15 cases FASN protein signaling negative, accounting for 21%. FASN protein moderate positive expression rate and strong positive expression rate were 55% (22/40) and 35%(14/40) in the ulcer type of colorectal cancer tissues and 52%(16/31) and 13% (4/31) in the protrude type of colorectal cancer tissues, a significant difference (P=0.012) between the two groups. FASN protein moderate positive expression rate and strong positive expression rate were 20% (1/5) and 0% (0/5) in well-differentiated carcinoma of colorectal cancer,71% (27/38) and 16% (6/38) in moderate-differentiated carcinoma of colorectal cancer and 36%(10/28) and 43%(12/28) in poor-differentiated carcinoma of colorectal cancer, a significant difference (P=0.000) among the 3 groups. FASN protein moderate positive expression rate and strong positive expression rate were 57% (34/60) and 28% (17/60) in colorectal cancer patients with the range of tumor invasion·T3+T4,36% (4/11) and 9%(1/11) in patients with the range of tumor invasion T1+T2, there is a significant difference between the two groups (P=0.011). The FASN protein moderate positive expression rate and strong positive expression rate in colorectal cancer patients with lymph node metastasis were 39%( 11/28) and 43% (12/28), 63% (27/43) and 14% (6/43) in patients without lymph node metastasis, there is a significant difference between the two groups (P=0.023). The FASN protein moderate positive expression rate and strong positive expression rate were 25% (2/8) and 75% (6/8) in colorectal cancer patients with distant metastasis,57% (36/63) and 19% (12/63) in patients without distant metastasis, there is a significant difference between the two groups (P=0.002). The FASN protein moderate positive expression rate and strong positive expression rate were 52% (22/42) and 17% (7/42) in colorectal cancer patients with clinical stage Ⅰ+ Ⅱ,55%(16/29) and 38% (11/29) in patients with clinical stage Ⅲ +Ⅳ, there is a significant difference between the two groups (P=0.020). According carcinoma FASN expression of negative, moderate positive or strong positive, patients were divided into three groups, follow-up of 5 years, disease-free survival time and overall survival time were recorded, and the results of comparative analysis between the three groups showed:Overall survival:survival rate in colorectal cancer patients with FASN-negative expression were 86.7%,79.4% and 68.1% when 1 year,2 years and 5 year, the average survival time was 47.0±5.5 months; survival rate in colorectal cancer patients with FASN-moderate-positive expression were62.2%,48.1% and 48.1% when 1 year,2 years and 5 year, the average survival time was 34.1±4.3 months and median overall survival time was 23.0 months; survival rate in colorectal cancer patients with FASN-strong-positive expression were 50.0%,36.8% and 21.9% when 1 year,2 years and 5 year, the average survival time was 23.1±5.2 months and median overall survival time was 10.0±5.0 months; There were significant differences among the three groups (P=0.025). Disease-free survival:survival rate in colorectal cancer patients with FASN-negative expression were 80.0%,64.6% and 64.6% when 1 year,2 years and 5 year, the average disease free survival time was 42.7±6.3 months; survival rate in colorectal cancer patients with FASN-moderate-positive expression were 47.2%,39.9% and 35.4% when 1 year,2 years and 5 year, the average disease free survival time was 26.9±4.3 months and median disease-free-survival time was 10.0±8.5 months; survival rate in colorectal cancer patients with FASN-strong-positive expression were 33.3%, 20.8% and 13.9% when 1 year,2 years and 5 year, the average disease free survival time was 15.2±4.7 months and median disease-free survival time was 5.0±3.2 months; There were significant differences among the three groups (P= 0.010).2. ELISA test results display:The plasma FASN average concentration in colorectal cancer patients was 0.82±0.56 units/L and 0.38±0.25 units/L in healthy people, there is a significant difference between the two groups (P=0.000). The plasma FASN average concentration in colorectal cancer patients with the range of tumor invasion T3+T4 was 0.87±0.60 units/L and 0.57±0.22 units/L in patients with the range of tumor invasion T1+T2, there is a significant difference between the two groups (P=0.003). The plasma FASN average concentration in colorectal cancer patients with lymph node metastasis was 1.14±0.68 units/L and 0.62±0.35 units/L in patients without lymph node metastasis, there is a significant difference between the two groups (P=0.001). The plasma FASN average concentration in colorectal cancer patients with distant metastasis was 1.36±0.58 units/L and 0.75±0.52 units/L in patients without distant metastasis, there is a significant difference between the two groups (P=0.002). The plasma FASN average concentration in colorectal cancer patients with clinical stage Ⅰ+Ⅱ was 0.62±0.36 units/L and 1.12±0.68 units/L in patients with clinical stage Ⅰ+Ⅳ, there is a significant difference between the two groups (P=0.000). According to the FASN plasma concentrations above or below the critical value (0.82 units/L, the FASN average plasma concentration), all patients were divided into two groups, the follow-up of 5 years, disease-free survival time and overall survival time were recorded, and the results of comparative analysis between the two groups showed:Overall survival rate in colorectal cancer patients with lower FASN plasma concentrations were 76.5%,65.0% and 60.7% when 1 year,2 years and 5 year, the average overall survival time was 41.8±3.8 months; Overall survival rate in colorectal cancer patients with higher FASN plasma concentrations were 51.2%, 39.6% and 23.5% when 1 year,2 years and 5 year, the average overall survival time was 24.6±4.2 months and median overall survival time was 14.0±4.9 months; there were significant differences between the two groups (P=0.003). Disease-free survival rate in colorectal cancer patients with lower FASN plasma concentrations were 65.0%,53.5% and 49.7% when 1 year,2 years and 5 year, the average disease-free survival time was 35.0±4.1 months and median disease-free survival time was 28.0 months; Disease-free survival rate in colorectal cancer patients with higher FASN plasma concentrations were 35.5%,27.9% and 20.0% when 1 year,2 years and 5 year, the average disease-free survival time was 18.3±4.0 months and median disease-free survival time was 6.0±2.4 months; there were significant differences between the two groups (P= 0.003).3. The expression levels of FASN mRNA were lowest in LS174T cells. Real-time quantitative analysis showed that the FASN mRNA expression levels in Caco-2, HT-29 and LoVo cells were 56.11±15.35,28.46±9.56,79.98±11.03-fold than that in LS174T cells, and we chosed Caco-2, HT-29 and LoVo cell lines in further studies. pcDNATM6.2-GW/EmGFP-miR-FASN RNAi vector and one negative vector were successfully obtained and identified by sequencing. The negative vector was transiently transfected into Caco-2, HT-29 and LoVo cells, the results showed high transfection efficiency in the Caco-2 cells and the poor transfection efficiency in LoVo and HT-29 cells, and Caco-2 cells were selected as target cells for subsequent experiments. After Caco-2 cells were stably transfected with pcDNA6.2-GW/EmGFP-miR-FASN vector, the expression levels of FASNmRNA and protein were down-regulated by real-time PCR and western blot assays. After down-regulated FASN expression in Caco-2 cells by FASN-RNAi, the OD value at 96h of FASN-RNAi group was 0.74±0.11, the ratio of colonies formed by FASN-RNAi cells was (5.83±1.93)%, the number of FASN-RNAi Caco-2 cells that invaded for 24h was 95±9.46 cells/site, as assessed by MTT, colony formation and transwell assays, respectively. Compared with control groups, FASN inhibition depressed the proliferation and invasion/migration of Caco-2 cells (P<0.05).4. In vivo tumor xenograft model, subcutaneous tumors visible to the naked eye had formed approximately 10.2±1.9 days in control group and 12.8±1.3 days in experimental group after subcutaneous injection of Caco-2 cells into mice. Tumors were observed in 92% injected mice. On the 18th day after injection, the tumor volumes of different groups were 284.68±115.50mm3 (control-negative) and 94.58±62.69mm3 (FASN-RNAi). Thevolume of control group xenograft tumors was larger than that of FASN-RNAi group (P<0.01). Morphologically, tumor tissues showed more solid cords, sheet-like cells andlittle necrosis was found in tumors. Expression levels of FASN protein were higher in control xenografts than in FASN-RNAi xenografts (P<0.05).Conclusions1. FASN was over expression in colorectal cancer cancer tissue, and plasma FASN concentrations in patients with colorectal cancer were significantly higher than that in healthy human plasma.2. FASN expressions in the ulcer type of colorectal cancer tissues and poor-differentiated carcinoma of colorectal cancer were significantly higher than that in the protrude type of colorectal cancer tissues and well-differentiated carcinoma of colorectal cancer.3. Both the FASN expression of tumor tissue and the FASN concentration of plasma in colorectal cancer patients with the range of tumor invasion T3+T4, lymph node metastasis, distant metastasis and clinical stage Ⅲ+Ⅳ were significantly higher that in patients with the range of tumor invasion T1+T2, without lymph node metastasis, without distant metastasis and clinical stage Ⅰ+Ⅱ stage.4. Colorectal cancer patients with FASN-negative cancer tissue or low serum FASN concentration (<0.82units/L) had significantly longer disease-free survival and overall survival than those colorectal cancer patients with FASN-positive cancer tissue or high serum FASN concentration (>0.82units/L).5. Down-regulated FASN expression in Caco-2 cells by RNAi induced the proliferation and invasion/migration of tumor cells in vitro and in vivo.The expression of FASN was related to the malignant biologic behavior of Caco-2 cells.The results suggest that:FASN positive expression in colorectal carcinoma tissue is closely related to the malignant biological behavior and prognosis of colorectal cancer. FASN could be used as the specificity of diagnosis and treatment of colorectal cancer tumor markers and molecular new targets.