One of the important risk factors in colorectal cancer is obesity [3] [4]. β-Catenin is an E-cadherin binding protein that mediates cell-cell adhesion [20] and plays a role in the canonical WNT signaling pathway that controls the coordinated expansion and differentiation of the intestinal crypt stem cells [21]. Degradation of β-Catenin by phosphorylation followed by alteration of destruction complex (APC, GSK-3β and AXIN) results in inactivation if WNT pathway [22]. In our study, we found that was associated with an increased adjusted OR of 3.40 (95%CI=1.42-8.15, P=0.006 for positive nuclear staining) compared to non-CRC samples (Normal or advanced adenoma).
The gatekeeper gene APC is a negative regulator of β-Catenin and is mutated in approximately 80% of sporadic and hereditary colon cancers [23]. There are several mutations that can cause an accumulation of β-Catenin in tumor cells such as mutations of the APC gene, point mutations in GSK-3β or mutations in β-Catenin gene itself [23, 24, 25].
Our positive nuclear staining in CRC (41%) and its association with the positive status for nuclear CTNNB1 intensity compared to non-CRC samples are in contrast to a study by Brabletz et al. [26], which showed that β-Catenin is localized in the cytoplasm and membrane of the tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus. They also mentioned that there was positive nuclear staining at the invasive front as β-Catenin is involved in tumor progression. Such is not the case in our study, indeed even when considering nuclear staining in our specimens, there was no statistically significant differences between stage III/IV cases’ staining versus stages I/II CRC cases levels of staining. The fact that β-Catenin is expressed early in the African American specimens analyzed here might partially explain the aggressive nature of CRC in this population. In addition, we showed that there is uniform membranous staining in normal and increasing cytoplasmic and nuclear staining in advanced adenomas and CRCs. This confirms that the decrease in membranous staining begins with dysplastic changes leading to a progressive disappearance at the membrane level in CRCs.
As we mentioned above, a major risk factor for CRC is obesity, which continues to expand as a pandemic worldwide [27].The American Cancer Society Cancer Prevention Study II, states that there is an increased incidence of CRC, esophageal adenocarcinoma and other cancers with obesity [28]. In our study, we showed that 78% of advanced adenoma patients and 69 % of CRC patients were overweight with BMI > 25. In comparison to advanced adenoma, the percentage was lower in cancer; perhaps due to the late stage of cancer and weight loss in the interim (Tab1e 1). There are several mechanisms by which obesity is believed to promote CRC, this includes increase in leptin levels that cause an increase in growth and proliferation of colon cancer cells [29], altered adipokine levels, altered gut microbiome apart from increased steroid hormones and growth factors [30]. Insulin is however the established biochemical link and the main pathway involved is PIK3/AKT/mTOR pathway. Elevated IGF-1 and insulin act through the insulin receptors and phosphotidylinositide-3 kinase [31].
In addition to the above findings, we also found that overweight and obese patients (BMI>25) did not show a significantly increased expression (p=0.3) of nuclear CTNNB1 (17% positive in normal weight vs. 27% positive in overweight/obese). Morikawa et al. found that in obese patients, nuclear CTNNB1 positivity was associated with significantly better cancer-specific survival suggesting that WNT signaling acts as a switch and when it is on, adipogenesis is repressed. Kennell et al. demonstrated that activated Frz1 (frizzled) promotes β-Catenin stability, inhibits apoptosis, and adipogenesis. Ross, et al. also showed that Wnt signaling acts as a molecular switch that controls adipogenesis. Upregulation of Wnt signaling maintains preadipocytes in an undifferentiated state and when Wnt signaling is prevented they differentiate into adipocytes [17, 32, 33].
Although in our study there was no association between nuclear intensity and CRC between normal and overweight patients (P for interaction = 0.6), there is accumulating evidence to show that the state of chronic inflammation incited by obesity might play a role in promoting colorectal carcinogenesis [8,34]. Of the many markers, TNF-α is important [35, 36], as it activates WNT signaling through the induction of GSK-3β phosphorylation, resulting in increased nuclear localization of β-Catenin [37]. In addition to TNF-α, other humoral agents associated with obesity might also be contributing to the activation of WNT signaling like IL-1β and adiponectin, which is decreased in the obese state and is not an inflammatory cytokine that can modulate GSK3β/β-Catenin signaling pathway [38]. Although multiple mechanisms may be operating in parallel and contributing to the pro-tumorigenic milieu, Wnt is a pivotal tumorigenic pathway [39] , aberrations of which is important in the evolution of most sporadic CRC. In summary, there is positive nuclear staining in CRCs (41%), which was associated with the positive status for nuclear CTNNB1 intensity (adjusted OR: 3.40, 95%CI=1.42%-8.15%, P=0.006 for positive nuclear staining) compared to non-CRC samples (Normal or advanced adenoma). This shows that advanced adenomas and CRCs were associated with activation of β-Catenin in physically fit, overweight and obese patients (Figure 3).