Claudin-1 overexpression in intestinal epithelial cells enhances susceptibility to adenamatous polyposis coli-mediated colon tumorigenesis

Increased claudin-1 expression has been frequently observed in colon cancer, however the consequences of the in-vivo upregulation in colonic epithelial cells have not been investigated. To determine the role of claudin-1 in colon tumorigenesis, we crossed Villin-Claudin-1-Tg mice with APC^Min mice (APC) to generate APC^Min-Villin-Claudin-1 (APC-Cldn1) mice. We observed robust expression of claudin-1, localized to the membrane, in the colon of APC-Cldn1 mice compared to APC mice (Additional file1: Figure S1). APC mice characteristically develop adenomas in the small intestine with little to no tumor occurrence in the colon[8]. In our studies, APC-Cldn1 (n = 18) mice developed colonic tumors at a significantly higher frequency (p = 0.0003) than APC mice (n = 18) (Figure 1A). Endoscopy of mouse colon at 10 weeks of age showed that APC-Cldn1 mice developed colonic tumor at this early age compared to APC mice (Figure 2A, Day 12, water treated group). Further, the tumors in APC-Cldn1 mice colon appeared larger than the APC mice colon tumors (p = 0.0178; measured using imaging analysis software (Figure 1C,D). The histological analysis further demonstrated that the tumors in APC-Cldn1 mice colon were less differentiated and high grade compared to the APC^Min mice (p = 0.0007) (Figure 1D, Table 1). Notably, it is rare that adenomas of APC^Min mice, originating from the colon or small bowel progress to invasive adenocarcinoma, yet we were able to detect an incident of invasion in the APC-Cldn-1 mice (Figure 1E, Table 1). Through routine care and observation of the mice, we also noticed that APC-Cldn1 mice began showing signs of morbidity much sooner than APC^Min mice. The average life span of an APC^Min mouse is approximately six months. To determine if there was a significant difference in survival, we plotted a Kaplan Meir curve and found that APC-cldn1 mice (n = 40) have a statistically significant reduced survival time to four months (p = 0.0027) compared to APC^Min mice (n = 43) (Figure 1F). As mentioned previously, multiple adenoma formation is restricted to the small intestine in the APC model and is thought to attribute to their limited life span. Therefore we thought it important to assess whether tumors of the small intestine also progress with claudin-1 overexpression. We found no significant difference in the number of intestinal tumors between the APC^Min and APC-Cldn1 mice. However, an increased trend was observed and the intestinal tumors in APC-Cldn1 mice were in general, advanced and displayed high-grade dysplasia (Additional file2: Figure S2 and Additional file3: Table S1). Taken together, these results suggested that increased claudin-1 expression enhances susceptibility to tumor development in the colon of APC^Min mice as well as contributes to the tumor progression.

An increase in tumor size or number usually results from an increase in proliferation and/or an associated decrease in apoptosis. Therefore to assess proliferation in these tumors, we performed immunostaining for Ki67, a well-known marker of cellular proliferation. We quantified a significant increase (p = 0.0125) in the proliferation in APC-Cldn1 mice tumors compared to the APC^Min mice (Figure 3A). Immunostaining using anti-cleaved caspase-3 antibody however suggested no significant differences in the apoptosis (Additional file4: Figure S3). As claudin-1 is a downstream target of Wnt signaling[10], and tumors that arise from the loss of APC have constitutive Wnt activation, we decided to examine the colonic tumors in each of these mice for potential upregulation of the Wnt/β-catenin pathway. We immunostained tumors for β-catenin, the primary effector of Wnt-activation, to assess for nuclear/cytoplasmic staining, which is an indicator of activated β-catenin. APC-Cldn1 tumors showed a noticeable increase in nuclear and cytoplasmic staining compared to that of APC^Min tumors (Figure 3B). To further assess Wnt upregulation, we performed qRT-PCR analysis using total RNA isolated from the colonic tumors of APC^Min and APC-Cldn1 and detected increased mRNA expression of the established Wnt-target genes CD44 (p = 0.0159), Lgr5 (5-fold increase), and Axin2 (3-fold increase) (Figure 3C). Previously, we have shown that IEC-specific claudin-1 expression in mice induces Notch-signaling[5]. Additionally, several studies have shown a role for the Notch pathway in colon tumorigenesis and targeted therapy[11, 12]. Indeed, we did find increased Hes1 and decreased Math1 expression in the tumors in APC-Cldn1 mice colon (p < 0.0001), suggesting upregulation of the Notch-signaling (Figure 3D). Taken together, we concluded that claudin-1 expression aids in colon tumor progression via upregulation of the Notch- and Wnt-signaling pathways.

To further assess global changes that occur as a result of claudin-1 upregulation, we performed transcriptome analysis of colonic tumors from APC^Min and APC-Cldn1 mice to identify differentially expressed genes (DEGs). Table 2 shows the list of the selected genes that were upregulated or downregulated by a factor of at least 1.5 fold or greater in APC-Cld1 mice (versus APC^Min mice). In accordance with the data shown above, tumors of APC-Cldn1 mice showed altered expression of genes correlative with increased Wnt and Notch activities.

Additionally, Notch target genes known to regulate mucosal defense were downregulated in APC-Cldn1 tumors. To validate mRNA expression of these genes, we performed qRT-PCR analysis (Figure 4A). Mucin-2, a primary component of the goblet cells and previously shown by us to be downregulated with claudin-1 overexpression[5], was also significantly down-regulated (p = 0.0022) in APC-Cldn1 tumors. Kruppel-like factor 4 (Klf4) and Trefoil factor 3 (Tff3), regulators of the goblet cell development and mucosal defense, were also decreased in APC-Cldn1 tumors (p = 0.0031, 0.0653). Studies assessing the effect of the loss of Muc2 or/and Klf4 in the regulation of APC mediated tumorigenesis have shown an increase in colon tumorigenesis as a result of increased mucosal inflammation[13, 14]. Additionally, we observed several genes (Ccl6, Clc28, Pla2g5, Il-23, and Il-1b) corresponding to activated immune response and increased inflammation in our microarray analysis. These observations suggested inflammation as a possible mechanism for observed tumor progression in the colon of APC-Cld1 mice. We performed qRT-PCR analysis for common cytokines upregulated during inflammation and observed a significant increase in IL-10 mRNA expression (p = 0.0139), with an accompanied increase in IP-10 and TNFα (2- and 3-fold increase, respectively) (Figure 4B). These immune regulators have been shown to be upregulated in response to systemic inflammation, most notably observed in diseases such as colitis.

Colon tumorigenesis can be induced in APC^Min mice by the administration of the colitis-inducing DSS[15]. This chemical activates inflammation, which drives the development of colonic tumors in these mice. We have shown previously that Cld-1Tg mice are susceptible to DSS-colitis[5]. Therefore, we reasoned that inflammation-driven colon tumor growth would also be upregulated in APC-Cldn-1 mice. To test, we treated APC^Min and APC-Cldn1 mice with 2% DSS drinking water for 5 days. Subsequently, mice were given normal drinking water and allowed to recover for 2 weeks until sacrifice. Endoscopic imaging was performed on each mouse on days 5, 8, and 12 following DSS administration, which showed increased inflammation and tumor development as early as day-5 post DSS-administration in APC-Cldn1 mice (Figure 2A) compared to APC^Min mice where colon tumor appeared around day-8. The total number of colonic tumors at the time of sacrifice showed that APC-Cldn1 mice developed more (p = 0.022) tumors than APC^Min mice (Figure 2B and C). Also, the tumors appeared larger in APC-Cld1 mice as compared to APC^Min mice. Combined together, we concluded that claudin-1 expression decreased tumor latency to increase tumor growth in the DSS-APC model of colon tumorigenesis.

Recently, it was demonstrated that the barrier defect plays an important role in colon tumor progression by promoting inflammation[9]. Of note, APC mice between the ages of 12 to 14 weeks have increased permeability compared to age-matched wild type mice which is attributed to adenoma development[16]. Importantly, we have previously demonstrated that mucus barrier is compromised in Cld-1TG mice due to suppressed goblet cell differentiation[5]. Considering the advanced and increased colonic tumor burden in APC-Cld-1 mice (versus APC^Min mice), we also decided to assess mucosal permeability in these mice by measuring the permeability to FITC-labeled dextran. We observed an increased FITC-dextran permeability as early as approximately 8 weeks of age in APC-Cldn1 mice, with a significant increase (3-fold) observed at 14-16 weeks (Figure 5A,B), the age at which tumors were found to be present in the colon, compared to the APC^Min littermates. Increased gut permeability has been frequently linked to the increased microbial translocation[17, 18]. Therefore, using genomic DNA isolated from 16 week old mice, we also measured potential microbial translocation into the mucosa of APC-Cldn1 mice via PCR analysis using primers for bacterial specific 16S rDNA, which demonstrated a significant increase (p = 0.0129) in APC-Cldn1 mice (Figure 5C).

Recent studies have shown that increased inflammation, specifically IL-23/IL-17 signaling, contributes to the colon tumor development due to increased permeability and bacterial translocation[9]. Additionally, presence of IL-23 facilitates bacterial induced colitis[19]. Therefore, we assessed our model for potential upregulation of IL-23 signaling, and indeed, microarray analysis showed a significant increase in IL23a cytokine (Table 2). Validation using qRT-PCR also showed a significant increase (p = 0.0022) in IL23 specific p19 subunit (Figure 6). The downstream targets IL17A (p = 0.0442) and IL6 (4-fold induction) were also upregulated in APC-Cldn1 mice. Taken together, these results suggested that increased claudin-1 expression causes an increase in intestinal permeability in APC^Min mice. The increased gut permeability allows increased bacterial translocation, which in turn upregulates the IL23/IL-17 signaling to promote colon tumorigenesis.

To obtain APC^Min/+-Villin-Cldn1Tg mice (APC-Cldn1), APC^Min males purchased from Jackson Laboratories (Bar Harbor, Maine) were bred with Villin-Claudin-1-Tg females. Claudin-1 overexpression was assessed by PCR as described previously[5] on genomic DNA isolated from tail snips using DNA isolation buffer (Viagen Biotech; Los Angeles, CA). Identification of the mutated APC allele was performed using a modified protocol from Jackson Laboratories. APC and APC-Cldn1 littermates were monitored for signs of morbidity including hunched posture, anemia, and body weight loss and sacrificed according to the guidelines of Vanderbilt University Institutional Animal Care and Use Committee (IACUC). Accelerated tumorigenesis was induced by administering 10-week-old mice dextran sulphate sodium (DSS 2%) in their drinking water. Mice were monitored throughout duration of the experiment as described previously[5], and examined intermittently via colon endoscopy.

As described previously, the colon and the small intestine were dissected and flushed with PBS, opened flat and formalin fixed using the Swiss roll method. Further processing was performed by the Vanderbilt Translational Pathology Shared Resource Core. Distal and proximal sections of the colon were snap-frozen and stored at -80°C for further analysis. Where applicable, colonic tumors were quantified. Tumors were either isolated and frozen for further analysis, or left in the colon to be processed for embedding and H&E staining.

Tumor area was calculated using Axiovision 4 digital imaging processing software (Release 4.8.1, Carl Zeiss Imaging) by outlining tumors of three to four mice each of APC and APC-Cldn1 mice.

Immunostaining of the paraffin-embedded tissues was performed as described previously[5] using VectaStain ABC kit (Vector Laboratories) and the indicated antibodies. Images were obtained using a Zeiss light microscope.

Total RNA was isolated from tumors excised from the colon of APC and APC-Claudin-1 mice using Qiagen RNAeasy Mini kit with DNAse digestion step performed. The integrity of the RNA was determined by performing formaldehyde gel electrophoresis. Samples displaying two bands, corresponding to the 18S and 28S subunits, and having an A260/A280 of ~1.8 were used for experiments. Total RNA was isolated from snap-frozen tumors, as described above, and RNA integrity was measured. Samples were submitted to the Vanderbilt Microarray Shared Resource.

Total RNA (1ug) of each sample was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). Each qRT-PCR reaction contained SYBR Green Master Mix, the indicated primer sets and 25 ng of cDNA. Samples were loaded in triplicates on 96 well plates and run on a Bio-Rad iCycler. Ct values were utilized to calculate fold change and normalization was performed using beta-actin.

Assessment of intestinal permeability to 4 kDa FITC-dextran was performed as described previously by rectal administration[5].

Translocation of bacteria was assessed by detecting the amount of bacterial 16S rDNA using qRT-PCR and specific primers for conserved regions of the bacterial 16S rDNA[33]. Genomic DNA was isolated from the distal colon of three mice per group and 25 ng was used per reaction. The mouse Selp gene was utilized as normalization gene.

Statistical analyses were performed using Graphpad Prism software (San Diego, CA) for t-test analysis where comparisons between two groups were involved. SPSS software (College Station, TX) was utilized for analyses of Logistic regression (for binary outcomes) and Chi² (for categorical). P values less than 0.05 were considered significant.