Fas/CD95 Deficiency in ApcMin/+ Mice Increases Intestinal Tumor Burden

Background Fas, a member of the tumor necrosis family, is responsible for initiating the apoptotic pathway when bound to its ligand, Fas-L. Defects in the Fas-mediated apoptotic pathway have been reported in colorectal cancer. Methodology/Principal Findings In the present study, a variant of the ApcMin/+ mouse, a model for the human condition, Familial Adenomatous Polyposis (FAP), was generated with an additional deficiency of Fas (ApcMin/+/Faslpr) by cross-breeding ApcMin/+ mice with Fas deficient (Faslpr) mice. One of the main limitations of the ApcMin/+ mouse model is that it only develops benign polyps. However, ApcMin/+/Faslpr mice presented with a dramatic increase in tumor burden relative to ApcMin/+ mice and invasive lesions at advanced ages. Proliferation and apoptosis markers revealed an increase in cellular proliferation, but negligible changes in apoptosis, while p53 increased at early ages. Fas-L was lower in ApcMin/+/Faslpr mice relative to ApcMin/+ cohorts, which resulted in enhanced inflammation. Conclusions/Significance This study demonstrated that imposition of a Fas deletion in an ApcMin/+ background results in a more aggressive phenotype of the ApcMin/+ mouse model, with more rapid development of invasive intestinal tumors and a decrease in Fas-L levels.


Introduction
Apoptosis is a regulated process that eliminates individual cells that are damaged or infected. There are several signals capable of triggering apoptosis, of which the activation of Fas/CD95/Apo-1 receptor (Fas) by Fas ligand (Fas-L) is the most studied [1]. Most information about the Fas pathway is based on the immune system, where Fas is highly expressed in activated T and B cells, thymocytes, and lung and liver cells [2]. Cloning of the Fas receptor (lpr) [3] and the Fas-L (gld) [4] genes led to studies involving their potential roles in the activation of the apoptotic pathway by death receptors. From these studies, a potential mechanism evolved in which interaction of Fas-L with Fas results in a conformational change of the receptor resulting in the assembly of the death-inducing signaling complex, which is able to recruit and cleave procaspase-8 (reviewed in [5]).
During colon cancer, regulation of the Fas system facilitates tumor development. Some studies have shown that resistance of colon carcinoma cells to apoptosis can be attributed to the elevated expression of Fas-associated phosphatase-1 [6], which inhibits Fas signaling by binding to the cytoplasmic tail of Fas. Another factor that may contribute to apoptosis resistance is incomplete Fas surface expression, with appropriate Fas mRNA levels but deficient posttranslational processing, attenuating its cell surface expression and remaining inactivated [7]. Amplification of a decoy receptor for Fas-L in lung and colon cancer has also been reported [8]. A number of investigations have proposed a ''Fas counterattack'', which is thought to be an anti-host tumor-derived response [9], where lymphocyte proliferation is compromised by tumor cells expressing Fas-L. This induces apoptosis in the lymphocytes, sparing tumor cells due to another mechanism, i.e., low Fas surface expression [10,11,12]. This notion however, remains controversial, and has been refuted by some investigators [13] and contradicted by other in vivo studies [14,15,16,17].
The adenomatous polyposis coli multiple intestinal neoplasia (Apc Min/+ ) mouse model presents phenotypes reminiscent of Familial Adenomatous Polyposis (FAP) in humans [18,19]. Patients with FAP develop multiple adenomas in the large intestine, which lead to the development of malignant adenocarcinomas. The relevance of the Apc Min/+ mouse model is that most colorectal cancers also show alterations in expression of the Apc gene [20,21]. Apc is a gatekeeper that regulates the levels of bcatenin [22], a transcription factor that has Matrix metalloproteinase-7 [23] among its target genes. In the current study, Apc Min/+ mice were crossed with Fas deficient (Fas lpr ) mice to generate Apc Min/+ / Fas lpr mice in order to study the effect of a disrupted Fas-mediated apoptotic machinery on tumor development and progression. The results are summarized herein.

Mice and Tissue Processing
The animal protocols used in this study were approved by the University of Notre Dame Institutional Animal Care and Use Committee. Male Apc Min/+ and Fas-deficient (Fas lpr ) mice (5 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). All animals were fed a rodent chow diet. For these studies, Apc Min/+ mice were crossed with Fas lpr mice to generate Apc Min/+ /Fas lpr/+ mice, which were then bred to generate Apc Min/+ /Fas lpr mice. Male Apc Min/+ and Apc Min/+ /Fas lpr mice (8,12,16,20, and 30 weeks) in a C57BL/6 background, were used for all analyses. Tumor counting was performed under a dissecting microscope by investigators blinded to the genotype. Intestines were opened longitudinally, cleaned, Swiss-rolled, fixed with periodate-lysine-paraformaldehyde (PLP), and embedded in paraffin.

Blood Analysis
Blood extracted intravenously from individual mice was treated with EDTA and an aliquot (50 ml) was applied to an automated CBC analyzer (Hemavet HV950FS, Drew Scientific, Oxford, CT, USA) in order to determine the number of leukocytes (lymphocytes, neutrophils, monocytes), erythrocytes (red blood cells, hemoglobin and hematocrit), and thrombocytes (platelets).

Stain Quantification and Statistical Analysis
Immunohistochemical stains were quantified using Spectrum Plus (Version 9.0.748.1518) and ImageScope software (Aperio Technologies, Vista, CA). Slides were scanned and loaded into an electronic database at 20X by ScanScope CS (Aperio Technologies). For each stain, algorithms were developed to analyze either the percent positive stained area per tumor area (Fas-L, Akt and pAkt) or the number of positive cells per tumor area (PCNA, caspase-3, p53, CD45, Mac-3 and p-Foxo3a).
Scanned slides were viewed in ImageScope where regions were drawn around each tumor and analyzed with Spectrum software, using algorithms designed for each stain. Stains using percent positive area were analyzed using color deconvolution algorithms. Stains requiring the number of positive cells per tumor area were analyzed using algorithms that separate color by optical density and also separate cells by outer membrane size and roundness. Cells that fit within the parameters set for desired cell size and shape and stain were counted, as was the area of each tumor region. The average number of cells per tumor area was then determined.

Statistical Analysis
For survival studies, the data were analyzed using the Kaplan-Meier treatment and the comparison of survival between both genotypes was performed using the log-rank test with Prism 4 software (GraphPad Software, La Jolla, CA). The Student's t-test was used for comparison of single pairs.

Fas Deletion in Apc Min/+ Mice Increases the Size and Number of Intestinal Adenomas
Based on preliminary transcriptional profiling results of human colon cancer samples, where a strong downregulation of a FASrelated receptor was observed (data not shown), along with published reports of compromised Fas-mediated apoptosis in colon cancer, Apc Min/+ /Fas lpr mice were generated to determine the effects of a Fas deficiency in the Apc Min/+ mouse model. Intestines harvested at various timepoints revealed that Apc Min/+ /Fas lpr mice developed dramatically more intestinal adenomas than Apc Min/+ mice, increasing rapidly with time (p,0.00001 at 8 weeks, p = 0.0072 at 12 weeks, p = 0.0012 at 16 weeks, p = 0.00019 at 20 weeks and p,0.00001 at 30 wks, n = 4-10 for Apc Min/+ /Fas lpr and 5-21 for Apc Min/+ /Fas lpr ), and reaching a plateau of slightly more than 70 adenomas after 16 weeks of age (Fig. 1A). The largest difference was observed at 20 weeks, where Apc Min/+ /Fas lpr mice had on average 87 intestinal adenomas per mouse compared to 13 in the Apc Min/+ mice. The Fas lpr mice, alone, did not generate any intestinal polyps. For most time points, Apc Min/+ mice had slightly larger adenomas, reaching significance at 16 weeks (p,0.05) (Fig. 1B). At 30 weeks however, adenomas from Apc Min/+ /Fas lpr mice were significantly larger than adenomas from Apc Min/+ mice (p,0.01). Unlike Apc Min/+ mice, where the location of adenomas is almost exclusively in the small intestine, Apc Min/+ / Fas lpr mice after 16 weeks presented with large adenomas in the large intestine, additional to the ones located at the small intestine. Although there was significant tumor burden in Apc Min/+ mice, it was much lower than in Apc Min/+ /Fas lpr mice. Additionally, Apc Min/+ / Fas lpr mice had a significantly reduced survival rate relative to Apc Min/+ mice (Fig. 1C). The survival curves were compared using a log-rank test showing a significantly lower survival (p,0.001) in Apc Min/+ /Fas lpr mice (n = 35) compared to Apc Min/+ mice (n = 89). The percent survival of Apc Min/+ /Fas lpr mice strongly decreased between 25 and 30 weeks and reached 44% at 30 weeks. This is about 12 weeks earlier than that observed in Fas lpr mice [24]. Furthermore, unlike Apc Min/+ mice, Apc Min/+ /Fas lpr mice presented with invasive lesions in all animals at 30 weeks (Fig. 2). The invasive lesions are not to be confused with herniation, a common occurrence in Apc Min/+ mice. All specimens were analyzed by a veterinary pathologist utilizing standardized guidelines [25]. The percentage of invasive lesions ranged from 3.0 to 15.4% (Table 1). However, metastasis to other organs was not detected.

Adenomas from Apc Min/+ /Fas lpr Mice Show Increased Proliferation
To determine if increased polyps in the Apc Min/+ /Fas lpr mice were due to an increase in cellular proliferation and/or a decrease in apoptosis, cells positive for proliferating cell nuclear antigen (PCNA) (Fig. 3) and caspase-3 (data not shown) were determined by immunohistochemistry. Several  polyps from at least 4 mice were used for each time point and genotype. Apc Min/+ /Fas lpr mice had significantly more PCNA+ cells per tumor area than Apc Min/+ mice at most time points (p = 0.0014 at 8 weeks, p = 0.0038 at 16 weeks, p,0.0001 at 20 weeks and 30 weeks, n = 24-205 tumors for Apc Min/+ /Fas lpr and 18-58 tumors for Apc Min/+ in 4-5 mice per genotype) (Fig. 3). Apoptosis activity was determined by measuring cleaved caspase-3 levels. Surprisingly, Apc Min/+ /Fas lpr did not show a significant change in caspase-3+ cells per tumor area at any time point, when compared to Apc Min/+ mice. As another marker of apoptosis, p53 was also measured by immunohistochemistry. An increase in p53 levels was observed in Apc Min/+ /Fas lpr mice compared to Apc Min/+ mice

Fas-L Is Decreased in Apc Min/+ Mice Lacking Fas
Another study has reported an increase in intestinal tumorigenesis once functional Fas-L is lost in Apc Min/+ mice [17]. To determine if, in our Fas-deficient model, the levels of Fas-L within the adenomas were altered, immunostains of Fas-L were performed on intestinal sections of Apc Min/+ and Apc Min/+ /Fas lpr mice (Fig. 5). Adenomas from the Apc Min/+ /Fas lpr mice had significantly less Fas-L per tumor area than Apc Min/+ mice. This observation was consistent for all time points (p = 0.039 at 8 weeks, p,0.0001 at 12, 16, 20 and 30 weeks, n = 28-198 tumors for Apc Min/+ /Fas lpr and 17-93 tumors for Apc Min/+ in 4-5 mice per genotype).

Akt, Foxo3a, and Inflammatory Markers in Intestinal
Tumors from Apc Min/+ /Fas lpr and Apc Min/+ Mice Fas-L, aside from triggering Fas dependent apoptosis, is considered to be an inducer of inflammation. To analyze inflammation within tumors, stains for CD45 and Mac-3 were performed on the intestines of Apc Min/+ /Fas lpr and Apc Min/+ mice  (Fig. 6B). Mac-3, an antigen present on macrophages, had the same trend with lower levels in tumors of Apc Min/+ mice (6236166 +cells/mm 2 , n = 21 in 4 mice) than in Apc Min/+ /Fas lpr mice (58046175 +cells/mm 2 , n = 151 in 5 mice) (p,0.0001).

Hematology Profile
Prior to perfusion, blood was collected from all animals for hematological profiling. Measurements of leukocyte (neutrophils, lymphocytes, and monocytes) levels revealed that the averages remained within the normal ranges (data not shown).
Apc Min/+ /Fas lpr mice, at most time points, were anemic as determined by red blood cell counts, hemoglobin, and percent hematocrit. However, Apc Min/+ mice, as well as Fas lpr mice were also anemic. Platelets, in all cases, were within normal ranges (data not shown).

Discussion
This study demonstrated that Apc Min/+ mice develop more intestinal adenomas when Fas is absent. An increase in proliferation (measured by PCNA) was evident, especially at later time points where an increase in Akt and pAkt was also observed. Additionally, there was a decrease in Fas-L in Apc Min/+ /Fas lpr mice. A significant increase in inflammation and Mac-3 was also observed in tumors of Apc Min/+ /Fas lpr mice.
In the present study it was observed that at all time points (8, 12, 16, 20 and 30 weeks), Apc Min/+ mice developed substantially more intestinal adenomas when Fas was eliminated. These adenomas also proved to be more aggressive. Incidences of intestinal prolapse were common in the Apc Min/+ /Fas lpr mice, especially at the later time points. Overall, the poor health of these animals was evidenced by their diminished survival rate as they approached 30 weeks of age. These findings were not surprising and were in accord with expectations from disruption of the apoptotic pathway  [28]. However, the levels of the apoptotic markers, activated caspase-3 and p53, were unexpected. There were no differences in activated caspase-3 levels, but an increase in p53 levels was observed in Apc Min/+ /Fas lpr mice compared to Apc Min/+ mice. Changes in cleaved caspase-3 were also absent in previous studies using Apc Min/+ mice lacking Fas-L [17]. On the other hand, p53, also an apoptotic marker, was higher in tumors of Apc Min/+ /Fas lpr mice compared to Apc Min/+ mice at early time points. However, this difference was attenuated at 30 weeks, when Akt and pAkt were observed to be higher in Apc Min/+ /Fas lpr mice compared to Apc Min/+ mice. Akt is a protein kinase activated by a variety of growth factors [29,30] that in turn triggers activation of several cancerrelevant downstream effector molecules, resulting in an environment that promotes proliferation and cell survival [31]. It has previously been reported that Akt is capable of downregulating p53 through phosphorylation of Mdm2 that results in a translocation to the nucleus [32,33]. The increase of p53 might also be a compensatory mechanism for a lack of Fas pathway. The interdependence between Fas and p53 pathways has been demonstrated by others [34,35].
In accordance with reports indicating that Akt regulation in Fas-L expression is due to phosphorylation and, therefore, cytoplasmic retention of the forkhead transcription factor FKHRL1/Foxo3a [26,27], intestinal adenomas of the Apc Min/+ /Fas lpr mice at later time points presented with fewer Fas-L than tumors found in the Apc Min/+ mice. This observation indicates that in the Apc Min/+ mouse model, a Fas deficiency does not compromise tumor growth. On the contrary, by disrupting the Fas machinery, these tumors developed faster, and in greater numbers. These results are consistent with previous reports highlighting the anti-tumor effects of Fas-L [14,36], as well as with recent findings of increased tumor burden in Apc Min/+ mice deficient for Fas-L [17]. However, there are a number of studies that report opposite findings where Fas-L confers more rapid tumor formation in murine melanoma cells [37]. Higher Fas-L expression has also been reported in liver metastasis of colon cancer compared to the primary tumor [38]. Those findings might represent a compensatory mechanism for a disrupted Fas-mediated apoptotic pathway. Despite the general assumption that high levels of Fas-L would induce inflammation within the tumors, in the present study it was observed that Apc Min/+ /Fas lpr mice, which had far lower levels of Fas-L than Apc Min/+ mice, showed an increase in inflammation at 30 weeks of age. The mechanism behind the increased level of inflammation in tumors of Apc Min/+ /Fas lpr mice is likely related to a balance between membrane Fas-L (mFas-L) and soluble Fas-L (sFas-L) (Fig. 7). mFas-L is a well known inducer of inflammation while sFas-L has the opposite effect [39]. Mmp7 is capable of cleaving mFas-L to yield sFas-L [40]. The Apc Min/+ mouse model and colon cancer in general show elevated levels of matrilysin [41,42]. Therefore, it is not unlikely to assume that in this context the balance between mFas-L and sFas-L would favor its soluble form and therefore a direct correlation between Fas-L and inflammation levels would be expected, as was observed in the present study. Fas-L expression is regulated by Foxo3a, a member of the forkhead family of transcription factors. Akt has been reported to decrease Fas-L expression by phosphorylation and therefore inactivation of Foxo3a. In accord with this mechanism, Apc Min/+ / Fas lpr mice showed higher levels of Akt, p-Akt, and p-Foxo3a, and lower levels of Fas-L.
A relationship between inflammation and cancer has been identified in a number of studies, and it is widely accepted that inflammation creates conditions that promote tumor development (reviewed in [43]). Furthermore, Mac-3, an antigen present on macrophages, was present at higher levels in tumors of Apc Min/+ / Fas lpr mice and their presence within tumors promoted tumor growth, invasion, and metastasis [44,45].
The results of the present study correlate to those of Fingleton et al. [17] with differences that are worthy of further consideration. Fingleton et al. used 17 week Apc Min/+ mice lacking Fas-L instead of its receptor, Fas. Both studies detected high levels of Fas-L in Apc Min/+ mice. While deficiencies of either Fas or FasL resulted in an increase in tumor burden, in the current study, the increases in tumor number ranged from ,300%, at early time points, to over 500%, at 16 weeks, whereas Fingleton et al. observed an increase nearly 100% at 17 weeks. These differences in tumor burden are most likely associated with difference in the inflammatory response observed in these studies. Fingleton et al. did not observe any significant changes in macrophage or lymphocyte infiltration, but did see a 3-fold decrease in neutrophils in Apc Min/+ mice lacking Fas-L. In contrast, the current study demonstrated an obvious increase in the inflammatory response in Apc Min/+ /Fas lpr mice. This supports the premise that the role played by in inflammation is more relevant in tumor development than it is in tumor evasion of the immune system. The current study, however, suggests that in intestinal adenomas, the balance between mFas-L and sFas-L levels, resulting from Mmp7 proteolysis, may regulate the pro-or anti-inflammatory properties of Fas-L.
Among the main objectives for generating the Apc Min/+ /Fas lpr mice was to determine if a more aggressive variant of the Apc Min/+ mouse model would evolve. At 30 weeks of age, all of the Apc Min/+ / Fas lpr mice had invasive lesions, which was not observed in the Apc Min/+ mouse model. It is likely that invasive lesions arise, at least in part, as a result of an increase in Akt and its active form, since this kinase has been shown to be involved in tumor invasion and metastasis [46,47,48].
In summary, this study demonstrated that an additional Fas deficiency in Apc Min/+ mice causes a dramatic increase in the number of intestinal tumors. The increase in the incidence of adenoma development and the invasiveness of these adenomas, paralleling a decrease in Fas-L in these mice, does not support the Fas counterattack notion in this model. The increase in Mac-3 and CD45 suggests a tumor permissive environment caused by a Fas-L modulated inflammation.