Cell surface fucosylation does not affect development of colon tumors in mice with germline Smad3 mutation

Abstract

Background/aims: Neoplasia-related alterations in cell surface alpha(1,2)fucosylated glycans have been reported in multiple tumors including colon, pancreas, endometrium, cervix, bladder, lung and choriocarcinoma. Spontaneous colorectal tumors from mice with a germline null mutation of transforming growth factor-beta signaling gene Smad3 (Madh3) were tested for alpha(1,2)fucosylated glycan expression.

Methods: Ulex europaeus agglutinin-I (UEA-I) lectin staining, fucosyltransferase gene Northern blot analysis, and a cross of mutant mice with Fut2 and Smad3 germline mutations were performed.

Results: Spontaneous colorectal tumors from Smad3 (-/-) homozygous null mice were found to express alpha(1,2)fucosylated glycans in an abnormal pattern compared to adjacent nonneoplastic colon. Northern blot analysis of alpha(1,2)fucosyltransferase genes Fut1 and Fut2 revealed that Fut2, but not Fut1, steady-state mRNA levels were significantly increased in tumors relative to adjacent normal colonic mucosa. Mutant mice with a Fut2-inactivating germline mutation were crossed with Smad3-targeted mice. In Smad3 (-/-)/Fut2 (-/-) double knockout mice, UEA-I lectin staining was eliminated from colon and colon tumors; however, the number and size of tumors present by 24 weeks of age did not vary regardless of the Fut2 genotype.

Conclusions: In this model of colorectal cancer, cell surface alpha(1,2)fucosylation does not affect development of colon tumors.

Fig. 1

UEA-I lectin staining of normal colonic mucosa and tumors of 24 week old Smad3 homozygous null mice. The colons of Smad3 (-/-) null mice were prepared for UEA-I lectin immunohistology as described in “Materials and Methods.” Sections are oriented with the luminal surface at the top and submucosa below. A) Non-neoplastic colon crypts with apical staining of goblet cells (bar = 20 μm). B) Higher magnification of goblet cell granules (bar = 20 μm). C) Abnormal crypts with UEA-I binding of apical surfaces and enclosed secretions (bar = 100 μm). D) Adjacent control section stained identically to C except without UEA-I lectin showing little non-specific staining. (bar = 100 μm)

Fig. 2

Expression of Fut1 and Fut2 in distal colon tumors of 5-6 month old Smad3 homozygous null mice. Polyadenylated RNA was isolated from spontaneous colon tumors and normal colon from four Smad3 homozygous null mice. Two μg of polyadenylated RNA per lane were electrophoresed by formaldehyde agarose gel electrophoresis, transferred to nylon membranes, hybridized with gene-specific probes, and quantified by PhosphorImager. The columns display the mean +/- the standard deviation in relative mRNA expression between tumor and control colon samples. Statistically significant elevation of Fut2 steady-state mRNA levels were found in tumors compared to adjacent normal colon (^*p <0.05). Sample autoradiographs for L32 control, Fut1, and Fut2 gene probes are shown below.

Fig. 3

UEA-I lectin staining of colon tumors of 24 week old Smad3 (-/-)/Fut2(-/-) double homozygous null mice. Colon tumors from mice with germline mutations of Smad3 and Fut2 were tested for UEA-I lectin staining. A) Tumors Fut2 (+/-)/Smad 3 (-/-) stained extensively with UEA-I lectin similar to tumors from Smad3 (-/-) mice with wild type Fut2 (+/+) expression (Fig 1). B) Tumors from double null Fut2 (-/-)/Smad 3 (-/-) μιχε λαχκ σπεχιφιχ YEA-I σταινινγ indicating expression of Fut2 is required to generate α(1,2)fucosylated epitopes (bar = 100 μm).