1. Introduction
Colorectal cancer (CRC) is the fourth most common and most deadly cancer in the world [
1]. Detecting CRC at a localized stage increases the 5-year survival rate by up to 90%. Therefore, the search for non-invasive and consistent biomarkers such as measurable circulating proteins is of critical importance. The insulin-like growth factor-1 (IGF-1) signaling pathway is involved in the regulation of cell proliferation, survival, differentiation, and apoptosis [
2]. The main constituents of the IGF-1 signaling pathway are the circulating growth factor IGF-1, cell surface receptor IGF receptor 1 (IGF-1R), and IGF binding proteins 1–6 (IGFBP 1–6). IGF-1R is a receptor tyrosine kinase, and its activation stimulates coordinated cell growth by activating downstream mitogenic proteins via the Ras pathway and survival proteins such as AKT [
3,
4]. This ultimately leads to the activation of cell cycle activators such as Cyclin-D and cyclin-dependent kinase 4/6 (CDK4/6) and the inhibition of cell cycle suppressors such as KIP proteins and phosphatase and tensin homologue [
5].
IGFBPs bind to and inhibit the activity of IGF-1. Thus, IGF-1 exerts growth-stimulatory effects, whereas IGFBPs exert growth-inhibitory effects. In healthy adults, IGF-1 is bound to one of the IGFBPs, primarily IGFBP-3, and to a lesser extent, IGFBP-2, in circulation. [
2,
6]. Studies have demonstrated that low IGFBP-3 and high IGF-1 and IGFBP-2 levels are associated with high cancer risk, poor prognosis, and tumor metastasis in several cancers, including CRC, breast cancer, and prostate cancer; however, these findings remain controversial and can be population specific [
7,
8,
9].
Many studies reported that high levels of circulating IGF-1 are associated with CRC; however, other studies showed contradictory results. For instance, a study on approximately 400,000 individuals from the UK biobank showed a positive correlation between circulating IGF-1 and CRC, while another from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort reported no association [
10,
11].
In addition to inhibiting IGF-1, IGFBP-2 and IGFBP-3 have been both shown to have IGF-1-indepenent effects [
12]. IGFBP-2 is involved in pro-oncogenic functions such as increasing cell migration, invasion, and angiogenesis through nuclear and intercellular mechanisms that lead to transcriptional activation of vascular endothelial growth factor (VEGF) [
13,
14,
15,
16]. Increased circulating levels of IGFBP-2 have been detected in both the serum and tumor tissues of most cancers including CRC, and this has been shown to be associated with worse prognosis [
17,
18,
19,
20].
On the other hand, IGFBP-3, the most abundant IGFBP in circulation, is involved in anti-tumor functions such as inhibition of cell proliferation and induction of apoptosis and cell cycle arrest [
21,
22,
23]. IGFBP-3 is upregulated at the transcriptional level by p53, and this activation is necessary for p53-induced apoptosis, and hence, the prevention of abnormal cell growth [
24]. p53, possibly the most important human tumor-suppressor gene, is activated in response to DNA damage to induce DNA repair, cell cycle arrest, or apoptosis and is mutated in 50–60% of all human cancers [
25,
26,
27]. This indicates that IGFBP-3 works in a tumor-suppression mode; therefore, it would be logical for it to be downregulated in cancer. Studies show major discrepancies in this regard, while the majority of studies show lower circulating levels of IGFBP-3 in several cancers including hepatocellular carcinoma [
28], esophageal cancer [
7], and CRC [
29]. However, other studies show contradictory results where no association between IGFBP-3 and CRC is found [
9].
Single nucleotide polymorphisms (SNPs) are the most common genetic variations among individuals. Numerous SNPs have been shown to be associated with clinical characteristics such as cancer risk [
30]. Therefore, SNPs can be used as biomarkers for risk assessment and cancer screening; however, many SNPs show a population-specific association [
31]. Thus, it is necessary to investigate if an SNP can be used as a universal or population-specific biomarker. Single SNPs in IGF-1 have been associated with increased circulating levels of IGF-1 and/or cancer risk [
32,
33]. The most significant SNP that exhibits an association with increased levels of circulating IGF-1 and is also associated with an increased risk of CRC is IGF-1 rs35767 (A > G) [
32]. Although this risk has been reported in several populations, a few studies showed that this is not the case in all populations. For instance, a multiethnic study in the American population showed that rs35767 was associated with a lower risk for CRC in Latinos and a null association for other racial/ethnic groups in the study [
34]. Another polymorphism of interest is IGF-1 rs6214 (C > T), which was observed to be associated with an increased risk for CRC in the Austrian population but not in the Iranian population [
35,
36]. Taken together, these results necessitate the investigation of the effect of IGF-1 SNPs in different populations to establish them as CRC risk indicators.
In this study, we aimed to determine the circulating levels of IGF-1, IGFBP-2, and IGFBP-3 in patients with CRC and to investigate their association with different clinical aspects of CRC, including risk, tumor grade, and tumor stage. We also assessed the genotypic and allelic frequencies of SNPs rs35767 and rs6214, their association with plasma IGF-1 levels, and the risk for CRC.
4. Discussion
Differences in circulating IGF-1 and its inhibitors (IGFBP-2 and IGFBP-3) have been associated with the risk of several cancers, thus proposing these indicators as non-invasive biomarkers for cancer risk and prognosis [
7,
8,
9]. In our study, plasma IGF-1 levels were lower in patients than they were in controls (
Table 2). Although studies that previously investigated IGF-1 levels in CRC have yielded inconsistent results, some demonstrated that IGF-1 levels were higher in patients with cancer, whereas others reported that IGF-1 levels were lower in patients than they were in controls. However, studies that demonstrated that increased IGF-1 levels were associated with an increased risk of CRC were performed prior to the diagnosis of CRC, whereas those that revealed lower levels in patients compared to those in controls were performed in patients with advanced disease, after the beginning of treatment and disease progression. Therefore, it appears that higher levels of IGF-1 were detected in patients before diagnosis up to disease onset; however, after disease onset, IGF-1 has been demonstrated to decrease with disease progression [
41]. This has been observed in a number of cancers, including breast cancer, hepatocellular carcinoma, and CRC, and is in agreement with our findings [
42,
43,
44].
In our study cohort, IGFBP-2 levels were higher in patients with CRC than they were in the controls (
Table 2), and IGFBP-2 levels increased with increasing histological grade of tumor differentiation (
Table 6). As IGFBP-2 is the main binding protein and inhibitor of IGF-1 and therefore inhibits its pro-proliferative action, it would be logical for IGFBP-2 to be downregulated in patients with cancer. Conversely, high IGFBP-2 expression has been reported in the serum and tissues of several cancers [
44]. In addition to its role as an IGF-1 inhibitor, IGFBP-2 is a developmental protein that is highly expressed during fetal development and embryogenesis; however, its expression decreases significantly after birth [
45]. Additionally, IGFBP-2 is upregulated in several cancers and promotes several pathways involved in oncogenic signaling, including the stimulation of proliferation, epithelial–mesenchymal transition, invasion, metastasis, and angiogenesis, all of which are independent of IGF-1 [
46,
47]. Furthermore, IGFBP-2 levels were positively correlated with tumor size and decreased significantly in patients following curative surgery [
48]. Finally, IGFBP-2 levels were low in well-differentiated tumors and normal tissues but high in poorly differentiated tumors [
49]. This suggests that IGFBP-2 circulating levels are positively associated with tumor load and can be used to measure disease progression and response to therapy. Therefore, they can be used as prognostic biomarkers.
In contrast to IGFBP-2, IGFBP-3 expression was lower in patients than it was in controls (
Table 2) and was not associated with tumor grade or stage (
Table 6). Again, this is consistent with previous reports of an IGFBP-2 correlation with an increased malignant status of the tumor but not IGFBP-3. This correlation was reported in cancers of the colon, lung, ovaries, prostate, and central nervous system [
50,
51,
52,
53,
54]. The most well-known function of IGFBP-3 is IGF-1 inhibition. IGFBP-3 also increases IGF-1 function by stabilizing IGF-1 and protecting it from degradation. High circulating concentrations of IGFBP-3 are associated with reduced cancer risk; however, once cancer develops, IGFBP-3 levels in cancer patients drop significantly compared to levels in control groups [
55,
56,
57]. Decreased IGFBP-3 expression correlates with disease progression and exhibits antitumor activities that are IGF-1-independent, including pro-apoptotic and anti-proliferative functions [
58,
59,
60]. Additionally, IGFBP-3 has been demonstrated to induce apoptosis and decreases survival when stimulated by p53 in response to DNA damage in breast cancer and CRC cells [
61]. Taken together, these results lead to the conclusion that IGFBP-3 exhibits tumor suppressor activity; therefore, it would be logical for IGFBP-3 to be reduced in patients with cancer. It remains unclear if this is the cause or result of cancer.
Most CRC cases are sporadic and may be caused by a plethora of lifestyle and environmental factors [
62]. However, approximately 30% are inherited. Nearly 5% are associated with highly penetrant inherited mutations, whereas the remaining 25% are likely to be caused by variations in less penetrant but more common single genes, including SNPs [
63].
Our study revealed no association between IGF-1 SNPs and CRC (
Table 3). All genotypes for both SNPs were represented in the cohort and in Hardy–Weinberg equilibrium. The SNPs rs6214 and rs35767 have been investigated for their association with different cancers, but the results have been inconsistent. While certain studies demonstrated an association, others failed. Interestingly, studies that observed an association reported that it may be race specific [
39,
64].
The rs6214 SNP is located in the 3′-UTR, and this may indicate its importance in the translation, stability, and localization of IGF-1 mRNA. rs35767 is located in the promoter region, and this may indicate that it affects IGF-1 mRNA expression, and therefore, protein levels [
65]. Both SNPs possess the potential to affect circulating IGF-1 levels; however, inconsistent results have been obtained regarding the association between these SNPs and circulating IGF-1 levels by many studies [
35,
39]. One study reported that this association may be race specific, as it was present in Caucasian women but absent in African American women [
66]. In our study, none of the SNPs were associated with CRC or IGF-1 and IGFBP-2 levels (
Table 3 and
Table 4). However, it is interesting to note that rs6214 was associated with IGFBP-3 levels in patients but not in controls, where the C allele was associated with an increase in IGFBP-3 expression (
Table 5). This may indicate that this SNP affects IGFBP-3 expression via IGF-1, as it has been reported that IGF-1 increases IGFBP-2 and -3 expression in a cell-dependent manner [
67,
68]. Although rs6214 is in the UTR of exon 4, it may have regulatory functions or may be in strong linkage disequilibrium with functional variants that influence IGF-1 [
69]. Furthermore, as IGFBP-3 expression is affected by IGF-1, it may also be affected. These findings warrant further investigation.
Our study possesses some limitations. First, the sample size was moderate for this type of study, and this limited its statistical power. Also, a limitation to overcome is increasing the number of patients with tumor grade information to detect a power above that of 70%. Additionally, this was a retrospective clinical study. Finally, the lack of information on CRC risk factors for most patients such as diet, physical activity, and smoking limited our ability to evaluate the association between these factors, IGF-1 levels, and CRC risk.