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- Materials and Methods
- Disclosure Statement
The incidence of colorectal cancer has been increasing and is associated with obesity and diabetes. We have found that type 2 diabetes model KK-Ay/TaJcl (KK-Ay) mice develop tumors within a short period after treatment with azoxymethane (AOM). However, factors that contribute to the promotion of carcinogenesis have not been clarified. Therefore, we looked at the genetic background of KK-Ay, including two genetic characteristics of KK/TaJcl (KK) mice and C57BL/6J-Ham-Ay/+ (Ay) mice, compared with other non-obese and non-diabetic mouse strains C57BL/6J and ICR, and induced colorectal premalignant lesions, aberrant crypt foci (ACF), and tumors using AOM (150 μg/mouse/week for 4 weeks and 200 μg/mouse/week for 6 weeks, respectively). The mice with a diabetes feature, KK-Ay and KK, developed significantly more ACF, 67 and 61 per mouse, respectively, whereas ICR, Ay, and C57BL/6J mice developed 42, 24, and 18 ACF/mouse, respectively, at 17 weeks of age. Serum insulin and triglyceride levels in KK-Ay and KK mice were quite high compared with other non-diabetic mouse strains. Interestingly, KK-Ay mice developed more colorectal tumors (2.7 ± 2.3 tumor/mouse) than KK mice (1.2 ± 1.1 tumor/mouse) at 25 weeks of age, in spite of similar diabetic conditions. The colon cancers that developed in both KK-Ay and KK mice showed similar activation of β-catenin signaling. However, mRNA levels of inflammatory factors related to the activation of macrophages were significantly higher in colorectal cancer of KK-Ay mice than in KK. These data indicate that factors such as insulin resistance and dyslipidemia observed in obese and diabetic patients could be involved in susceptibility to colorectal carcinogenesis. In addition, increase of tumor-associated macrophages may play important roles in the stages of promotion of colorectal cancer.
Excessive accumulation of visceral adipose tissue can induce many disorders, such as type 2 diabetes mellitus (elevated fasting glucose, insulin, and insulin-like growth factor levels), and dyslipidemia (elevated triglyceride or low high-density lipoprotein cholesterol levels). Obesity is common in Western countries, and is currently increasing almost ubiquitously across the globe. Recently, obesity has attracted much interest as a risk factor for colorectal cancer. The World Cancer Research Fund and American Institute for Cancer Research have evaluated causal relationships between accumulation of visceral adipose tissue and cancer, and concluded “confident evidence” for colorectal cancers. In males in Japan, an overweight condition and obesity (body mass index ≥25) are reported to be associated with colorectal cancer.[2, 3]
We have reported that obese KK-Ay mice are highly susceptible to induction of colorectal premalignant lesions, ACF, and to development of colorectal cancers by AOM treatment. The KK-Ay mice feature severe hyperinsulinemia, severe hypertriglyceridemia, excessive abdominal obesity, and resultant elevation of serum adipocytokines, such as IL-6, leptin, and Pai-1 compared with values for lean C57BL/6J mice. Such a consequent abnormality is suggested to be involved in the promotion of colorectal carcinogenesis, and is partly considered as a factor for high cancer susceptibility.
The KK-Ay mice were established by cross-mating KK mice, a type 2 diabetes mellitus model, with Ay mice,[5, 6] which carry the Agouti yellow (Ay) gene and feature severe hyperphagia, hyperinsulinemia, and dyslipidemia. Non-diabetic or non-obese mice could be set as control, e.g., young Ay mice or C57BL/6J-Ham-+/+ (+/+) mice. In addition to this, the body weight of ICR mice is almost the same as KK-Ay mice at a young age, and they are also susceptible to induction of colorectal carcinogenesis by AOM treatment. The strains and features that could be the most susceptible to induction of colorectal carcinogenesis by AOM treatment have not been clarified. Thus, we aimed to investigate and find the features and molecules involved in obesity-associated cancer by comparing mouse strains KK-Ay, KK, Ay, +/+, C57BL, and ICR. In the present study, we showed colorectal ACF development was strongly affected by diabetic conditions, and additional features of inflammation derived from agouti gene overexpression may lead to further promotion of cancer development.
- Top of page
- Materials and Methods
- Disclosure Statement
In the present study, obese diabetic KK-Ay and KK mice were found to be highly susceptible to AOM-induced ACF compared to those of Ay, +/+, C57BL/6J, and ICR mice. The KK-Ay and KK mice, but not other mice, showed hyperinsulinemia, hypertriglyceridemia, and hyperglycemia. Other obese and diabetic mice, induced by monosodium glutamate, are also susceptible to AOM-induced colon ACF and β-catenin-accumulated crypts. Regarding the development of ACF, it has been shown that hyperinsulinemia plays an important role. Long-term treatment with long-acting insulin on C57BL/KsJ-db/db mice, which lack a leptin receptor, revealed that insulin increases colonic epithelial cell proliferation, and formation of ACF.[10-12] Moreover, the anti-insulin resistant medicine pioglitazone prevented ACF development in KK-Ay mice through improvement of serum insulin levels, and increase of p27 and p53 mRNA levels in the colorectal mucosa. Hence, it seems likely that hyperinsulinemia in the KK-Ay and KK mice enhanced the development of AOM-induced ACF in the present study. In addition, hyperglycemia and hypertriglyceridemia may also contribute to the development of ACF.[14-16]
To clarify the strain difference regarding susceptibility to AOM-induced colorectal cancer, we treated mice with AOM six times to develop cancerous lesions. Precancerous lesions, both in rodents and humans, have several genetic abnormalities, such as K-ras and APC gene mutations.[17-19] Accumulation of β-catenin protein in the nucleus and cytoplasm are regarded as putative precursors to colorectal adenomas.
In this study, obese diabetic KK-Ay and KK mice developed colorectal carcinomas within very short periods after AOM injection, but not in Ay or ICR mice. Although diabetic conditions were similar between KK-Ay and KK mice (Fig. 2), tumor incidence, multiplicity, and sizes were greater in the KK-Ay mice compared to those of KK mice. Thus, in the next experiments, we attempted to identify the phenotypic differences in the induced lesions in the two strains, KK-Ay and KK. We first carried out immunohistostaining for β-catenin. β-catenin mutations seem to be involved in generating dysplastic lesions. β-Catenin mutations stabilize β-catenin protein in the cytoplasm and activate β-catenin/Tcf signaling to induce target genes, such as cyclin D1, COX-2, and Pai-1.[21-23] Accumulation of β-catenin protein in the nucleus of cancer cells was observed in both KK-Ay and KK mice, along with similar induction of downstream targets of β-catenin, cyclin D1, COX-2, and Pai-1. K-ras mutations contribute to hyperplastic changes. Mutated K-ras activates the MAPK and phosphoinositide-3 kinase/Akt pathways, and leads to overexpression of cyclin D1 and COX-2.
As COX-2 and Pai-1 are also good parameters for showing inflammatory status, we further evaluated other inflammation-associated genes. Among them, molecules that are associated with macrophages, CSF1, F4/80, IL-1β, MCP1, and OPN,[24, 25] were shown to be higher in cancerous parts of KK-Ay mice than in those of KK mice. Indeed, the number of macrophages was observed to be abundant in the tumor tissue of KK-Ay mice compared to that in KK mice. These results suggest that the activation of macrophages may cause promotion of tumor formation in KK-Ay mice. Colony-stimulating factor 1 is a major chemoattractant of macrophages released from tumor cells and recruits macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis. As one of the factors, tumor-associated macrophage-derived IL-1β stimulates Wnt signaling and growth of colon cancer cells and protects colon cancer cells from apoptosis through stabilization of Snail. Indeed, it has been reported that the number of intratumoral tumor-associated macrophages correlates with tumor progression in colorectal cancer. As far as we know, the impact of Ay overexpression on molecules associated with macrophage activation has not been elucidated. However, this issue should be clarified in the near future.
In conclusion, the present studies indicated that KK-Ay mice are the most susceptible to AOM-induced carcinogenesis compared to strains KK, Ay, +/+, C57BL/6J, and ICR. It has become clear that factors such as dyslipidemia and insulin resistance observed in type 2 diabetes could be involved in the susceptibility to colorectal carcinogenesis. Our data indicated that inflammation evoked by macrophages may play important roles in the stages of promotion of colorectal cancer development.