Colonic mucosal fatty acid synthase as an early biomarker for colorectal neoplasia: modulation by obesity and gender

Abstract

Background: We have previously reported that colonic pericryptal microvascular blood flow is augmented in the premalignant colonic epithelium, highlighting the increased metabolic demand of the proliferative epithelium as a marker of field carcinogenesis. However, its molecular basis is unexplored. In this study, we assessed the expression of a regulator of the "lipogenic switch," fatty acid synthase (FASN), in early colon carcinogenesis for its potential biomarker utility for concurrent neoplasia.

Methods: FASN expression (IHC) in the colonic epithelium from azoxymethane and polyposis in rat colon (Pirc) models of colorectal cancer was studied. FASN mRNA expression from endoscopically normal rectal mucosa was evaluated and correlated with colonoscopic findings (pathologic confirmation of neoplasia).

Results: FASN expression progressively increased from premalignant to malignant stage in the azoxymethane model (1.9- to 2.5-fold; P < 0.0001) and was also higher in the adenomas compared with adjacent uninvolved mucosa (1.8- to 3.4-fold; P < 0.001) in the Pirc model. Furthermore, FASN was significantly overexpressed in rectal biopsies from patients harboring adenomas compared with those with no adenomas. These effects were accentuated in male (∼2-fold) and obese patients (1.4-fold compared with those with body mass index < 30). Overall, the performance of rectal FASN was excellent (AUROC of 0.81).

Conclusions: FASN is altered in the premalignant colonic mucosa and may serve as a marker for colonic neoplasia present elsewhere. The enhanced effects in men and obesity may have implications for identifying patient subgroups at risk for early-onset neoplasia.

Impact: These findings support the role of rectal FASN expression as a reliable biomarker of colonic neoplasia.

Figure 1

Increased immunohistochemical expression of FASN in the uninvolved colonic mucosa of the azoxymethane (AOM) treated rat (carcinogen model of CRC). Distal colonic segments were fixed in formalin and sections immunostained for FASN as described in “Materials and Methods”. The composite figure displays a collage of representative images of FASN staining (20× optical and 10× digital zoom) of the uninvolved colonic tissue collected from saline-treated (Panel A; Control; n=8), 20 week AOM-treated (Panel B; premalignant stage; n=8) and 40 week AOM-treated (Panel C; malignant stage; n=8). The inset in Panel C (20× digital magnification) exhibits cross sectional crypts with discrete cytoplasmic localization of FASN in the epithelial cells. Complete longitudinal crypts extending from the muscularis mucosa to colonic lumen were scored for FASN positive epithelial cells (8-10 random crypts/colon; n=8) by two independent observers. As shown in the histogram, FASN expression in the uninvolved mucosa progressively augmented in the AOM-treated rats from pre-malignant (1.9 fold compared to saline-treated group; p<0.0001) to malignant stage (2.5 fold compared to saline-treated group; p<0.0001). FASN was highly expressed in the crypt base and the proliferative zone was greatly extended in the AOM-treated colons.

Figure 2

Increased immunohistochemical expression of FASN in the uninvolved colonic mucosa of the Pirc rat (genetic model of CRC). Sixteen Pirc and 8 wild-type rats were euthanized at 12 weeks of age (a stage when these animals express large number of intestinal polyps). The colons were longitudinally opened in entirety and rolled lengthwise into “Swiss rolls” with polyp-laden mucosa facing inwards. The whole colonic preparation was then carefully placed in a formalin fixative and immunostained for proliferation marker PCNA as well as FASN as described in “Materials and Methods”. The composite figure exhibits representative tissue sections from wild-type (Panels A, C, E and G) and Pirc rats (Panels B. D, F and H). Panels A-B depict H&E staining of the whole colon (Swiss roll; 10× image) showing the presence of adenomas, micro adenomas and adenocarcinomas in the Pirc rat colon. Panels C - D demonstrate the hyper-proliferative state of a section of the mucosa from the pirc rat colon (compared to wild-type). Panels E-F demonstrates the overall staining pattern of FASN in the whole colon (Swiss roll), with higher expression in Pirc rats. Panels G-H demonstrate the high power (40× optical zoom) expression of FASN in the colonic mucosa from wild-type rat (Panel G; n=8) and Pirc-rat colon (Panel H; n=8). Complete longitudinal crypts extending from the muscularis mucosa to colonic lumen were scored for FASN positive epithelial cells (8-10 random crypts/colon; n=8) by two independent observers. FASN was overexpressed in the colonic uninvolved tissue (1.8 fold compared to wildtype rat; ⁺p≤0.05) and in colonic adenomas (3.4 fold compared to wildtype rat; *p≤0.001).

Figure 3

Increased mRNA expression of FASN in the rectal biopsies from patients with presence of colonic adenomas - Gender-Related Implications. For these studies two random rectal biopsies were collected from endoscopically normal appearing rectal mucosa from subjects diagnosed with (24 patients) or without (24 patients) any adenomas detected elsewhere in the colon. The patient samples selected were equally randomized between males and females. Total RNA from these biopsies was isolated using Ribopure RNA kit (Ambion) and reverse transcribed with human FASN specific Taqman probes as described in the “Materials and Methods”. Compared to patients with no detected adenomas, the FASN was significantly overexpressed in patients with adenomas detected elsewhere in the colon in both males (p<0.001) and females (p<0.018) (Panel a). However, the increase was found to be greater in males (~5.5 fold) than females (~2.8 fold). Panel (b) depicts the predictive ability of FASN expression as a biomarker of CRC. The accuracy of the test was higher for males (AUROC=0.87) than for females (AUROC=0.77).

Figure 4

Increased mRNA expression of FASN in rectal biopsies from patients with presence of colonic adenomas - obesity-related implications. For these studies, we collected 2 rectal biopsies from subjects separated on the basis of their BMI of <30 (non-obese) or >30 (obese) with each group equally distributed between subjects with (8) or without adenomas (8). The biopsies were subjected to FASN RT-PCR as described in the “Materials and Methods”. FASN was significantly overexpressed in patients harboring adenomas in both obese (p≤0.0001) and non-obese (p≤0.0002) subjects (Panel a). The extent of overexpression however was greater in adenoma-harboring obese subjects than non-obese subjects with adenomas (~1.4 fold). Panel (b) depicts the predictive ability of FASN expression as a biomarker of CRC.