Introduction
Colorectal cancer (CRC) is very common worldwide, which lists third in morbidity and second in mortality overall worldwide [
1], and the trend is still uprising in many countries, such as Russia, China, and Brazil. Twenty percent emergence of CRC could be referred to genetic background, such as hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome), hamartomatous polyposis syndrome, and familial adenomatous polyposis (FAP), with characteristic of family history. The largest fraction of CRC cases is linked to environmental and nonhereditary events. Among them, chronic inflammation is a significant risk factor for CRC development [
2], and some cytokines such as IL-6 and TNF-α play very important role in this process. The activations of Wnt/β-catenin pathway and IL-6/STAT3 pathway have been proved key procedures in CRC carcinogenesis. The environment pathogenic factors of CRC are commonly about personal lifestyle, and the relevant research is useful for disease prevention. High fat diet, insufficient intake of dietary fiber, high consumption of red or processed meat, body fatness, alcohol drinks, gut microbiota disorder [
3], and ulcerative colitis [
4] have been proved to have the relationship with CRC. People with these characteristics usually have obesity problem and many metabolism abnormalities, and also together with sleep apnea problems. Sleep apnea is indeed prevalent among colorectal cancer patients [
5].
Obstructive sleep apnea syndrome (OSAS), also very prevalent in modern society, is a kind of sleep breathing disorder, with repeated partial or complete upper airway collapse. OSAS is characterized by intermittent hypoxia (IH), microarousal, and sleep fragmentation during sleep [
6], affecting at least 2–4% of the adult population [
7]. Many OSAS patients share the common risk factors with CRC patients: obesity body shape, often caused by unhealthy lifestyle. OSAS could induce multi-system disorders, and has been considered an independent risk factor for cardiovascular disease, cerebrovascular disease, and metabolic disease [
8‐
16]. The patients with OSAS usually have elevated cancer burden [
17‐
19]. In patients with lung cancer for example, OSAS is very prevalent [
20,
21]. Some studies also have shown that OSAS could increase the risk of developing colorectal cancer [
22,
23]. A cohort study suggested that OSAS even may promote the CRC development independently beyond the obesity [
24]. These studies have demonstrated the association between OSAS and CRC, but the mechanism has not been investigated.
Intermittent hypoxia (IH) is key characteristic of OSAS, and it could induce multiple organ impairment. Continuous hypoxia condition could promote colon cancer cell proliferation and invasion [
25‐
27], but whether IH could induce the carcinogenesis of CRC is still not clear and lack of related research. Beside the direct hypoxia effect, IH could activate the hypoxia-induced factor (HIF) and NF-κB, promoting the release of inflammatory factor [
28], such as IL-6 and TNF-α. Elevated levels of IL-6 [
29] and TNF-α [
30] have been observed in OSAS patients, which may exert effect on intestinal epithelial cells and induce the inflammation and carcinogenesis in the intestine. Hypoxia and HIFs could also influence both Wnt/β-catenin and IL-6/STAT3 pathways. But whether these hypotheses work in CRC carcinogenesis needs further investigation.
The human large intestine contains the most microorganisms in the body, which play an important role in absorption, metabolism, and storage of ingested nutrients, with potentially profound effects on host physiology. Intestinal microbiota could also influence the inflammation level through interaction with immunocyte and producing short-chain fatty acid (SCFA) by fermentation. Microbiota dysbiosis has been proved to be associated with CRC [
31]. Some specific bacteria, such as Bacteroides fragilis and Fusobacterium nucleatum, could impose effect on E-cadherin and activate Wnt/β-catenin signaling by toxin or adhesin, promoting colorectal carcinogenesis [
32,
33]. These two kinds of pathogenic bacteria are both anaerobic bacteria, which may show more adaptation for the hypoxia microenvironment in OSAS patients compared with common intestinal bacteria. Some research has shown that OSAS could induce the intestinal microbiota dysbiosis [
34], and microbiota dysbiosis correlation with OSAS could induce the inflammation [
35,
36]. Then, we could assume that the intestinal microbiota dysbiosis in OSAS patients may contain more pathogens or induce the inflammation in the intestine, which could induce the CRC carcinogenesis.
Therefore, we hypothesized that IH and intestinal microbiota dysbiosis in OSAS patients may have the ability to activate one or more CRC carcinogenesis pathways, and inflammation may be involved in this process. To validate our hypotheses, we exposed Immorto-Min colonic epithelial (IMCE) cells to IH and sterile fecal supernatant from OSAS to establish precancerous cell model, mimicking CRC premalignancy in OSAS patients, and the expressions of genes and inflammation cytokines associated with colorectal cancer, such as β-catenin, STAT3, HIF-1α, IL-6, TNF-α, c-myc, and cyclinD1, were analyzed to evaluate the effect of IH and microbiota of OSAS on CRC development.
Discussion
MCE cell line is phenotypically normal, but it has proven to be susceptible to transformation, so it is a very suitable model system available for studying malignant progression in colon cancer [
56]. In our study, we exposed IMCE cells to IH for 4, 8, and 12 h in the first part. It was observed that the expression of HIF-1α, cyclinD1, and STAT3 was upregulated in the IH group, and p-STAT3 moving into the nucleus was also elevated in the IH group, and both of them getting to the most after 12 h IH; the mRNA of β-catenin was elevated, but the β-catenin protein moving into the nucleus showed no significant change under IH. IL-6 and TNF-α also showed no significant change under IH. Then, we combined the IH exposure and fecal fluid incubation together to treat IMCE cells. Data showed that HIF-1α, STAT3, and p-STAT3 in the nucleus were elevated in the OSAS group compared with the healthy control group. Expressions of IL-6 and TNF-α were elevated in the OSAS group for mRNA level at 4 h and 8 h IH exposure, though without significant change for protein level. The expression of β-catenin in mRNA level and β-catenin protein moving into the nucleus also showed no significant changes between the two groups. The expressions of cyclinD1 and c-myc were upregulated in the OSAS group significantly at 4 h IH exposure, but downregulated in the OSAS group at 12 h IH exposure.
Cancer cells proliferate quickly and consume much oxygen, so malignant tumors usually contain hypoxic regions, which is the characteristic of tumor microenvironment. Tumor cells develop corresponding mechanisms for hypoxia environment adaptation. HIFs are activated in hypoxia conditions, regulating the expression of many genes that code for proteins involved in angiogenesis [
57], glucose metabolism, and cell proliferation [
58], which play important role for hypoxia tolerance. HIF-1α is the most important one among HIFs. Overexpression of HIF-1α in tumor cells is closely connected with increased resistance to radio- and chemotherapies, increased risk of metastasis, more aggressive phenotype, and strengthened immune suppression [
59]. HIFs are also involved in COX2/mPGES-1/PGE2, WNT, and STAT3 signaling pathways correlating with CRC carcinogenesis [
25,
60,
61], and implicated in CRC development and metastasis [
62‐
67]. But the conclusions above were acquired under continuous hypoxia condition.
Chronic IH is the key pathogenesis of OSAS damage. Different from continuous hypoxia, IH is in cycling reoxygenation, and reactive oxygen species could be generated in this progress, which is more similar with ischemia-reperfusion process, so OSAS could be regarded as an oxidative stress disorder [
68]. The shift frequency of hypoxia and reoxygenation in OSAS patients could be very high, up to more than 5–100 times per hour [
69], and blood oxygen saturation may be changed violently, even down to 20%. Multiple organs in OSAS patients would show damage in function and tissue, such as the cardiovascular, brain, liver, pancreas, and kidney. OSAS could aggravate the hypoxia in tissues. But seldom research focused on whether OSAS would promote CRC carcinogenesis. In HCT 116 cells, HIF-1α could be activated in a hypoxia dose-dependent manner under IH condition [
70], but whether the pathway correlating with CRC carcinogenesis could be activated under chronic IH condition is still not clear. Our data showed that IH could promote p-STAT3 elevated in the nucleus, and mRNA expression of cyclinD1 also increased at the same time as its downstream target gene, suggesting that STAT3 may be activated under IH condition.
Beside genetic heredity, CRC could be divided into sporadic CRC and colitis-associated cancer (CAC), according to the molecular mechanism by which cancer was triggered and promoted. For sporadic CRC, classical “normal mucosa-adenoma-carcinoma” sequence has been considered to be the core mechanism, and Wnt/β-catenin pathway plays an important role. Once activated, β-catenin would move into the nucleus, regulating its target gene such as cylinD1, c-myc, and MMP-7 which control the cell proliferation [
71]. But for CAC, chronic inflammation is a crucial cancer promoter. Two key genes in the inflammatory process, cyclooxygenase-2 (COX-2) and nuclear factor kappaB (NF-kappaB), provide a mechanistic link between inflammation and cancer, while some cytokines, such as TNF-α and IL-6-induced signaling, have been recently shown to promote tumor growth in experimental models of colitis-associated cancer [
72]. IL-6 could activate STAT3, which regulates the transcription of regulators of cellular proliferation (cyclinD1, proliferating cell nuclear antigen), survival (BCL-xL, surviving), and angiogenesis (VEGF) [
73]. The activation of STAT3 signal pathway has been proved to be key in the CAC carcinogenesis process [
73]. How about the mechanism of CRC carcinogenesis correlated with OSAS patients is still unknown to date. Our data showed that STAT3 pathway may be involved in this process. Because STAT3 mediates signaling from multiple inflammation cytokines, not only IL-6, but also IL-11, IL-21, and IL-22, all of which play roles in CRC development [
74‐
80], so the activated STAT3 suggested that chronic inflammation may be involved in this process.
At present, it has been proved that hypoxia and HIFs could influence both Wnt/β-catenin and IL-6/STAT3 pathways. HIF-1 could activate and maintain the Wnt/β-catenin pathway [
81]. In HCT 116 cells, Wnt/β-catenin pathway and cell proliferation are inhibited after HIF-1α was blocked [
82]; hypoxia could downregulate the APC expression in mRNA and protein level through HIF-1α-dependent mechanism [
83]. HIF and p-STAT3 are upregulated in mice and human colon cancer cells, and HIF could promote colon cancer cell proliferation by activating JAK-STAT3 pathway [
84]. HIF-1α knockdown leads a significant decrease in the expression levels of STAT3 in human colon cells [
85]. But the conclusions above were under continuous hypoxia condition and from cancer cells, how about the effect of chronic IH on colon premalignant cells is still unknown. Our results showed that IH could upregulate the expression of β-catenin mRNA, but we did not observe the increase of β-catenin protein in IMCE cell nucleus, and this indicated that Wnt/β-catenin pathway was not activated indeed in our model. Restricted to experiment conditions, we could not test the effect of longer IH duration on β-catenin. IH could elevate the expression of STAT3 and promote p-STAT3 protein entering to the nucleus, which indicated that STAT3 pathway could be activated by IH. But curiously this STAT3 activation seemed not to be through IL-6 in this process, because we did not see the elevation of IL-6 protein during IH exposure.
Many researches have shown that chronic IH could mimic the OSAS pathogenic process. But only IH itself may not be perfectly suitable for intestine research, because intestinal epithelium is inconstant contact with intestinal microbiota in vivo. It has been proved that the function and disease of intestine are affected by microbiota and their metabolic products. Some research has indicated that intestinal microbiota dysbiosis was induced in OSAS patients [
34,
86] and OSAS animal models [
87], and the similar situation even occurred in oral [
88] and lung [
89]. This change may play crucial role in pathogenesis process of OSAS-related hypertension [
90,
91]. The changed intestinal microbiota by chronic IH could not be reversed even after normoxic recovery [
92]. Those experiments above suggested that intestinal microbiota dysbiosis plays important role in OSAS pathogenic process, so it is not perfectly suitable that using IH only to mimic OSAS for intestine research without microbiota.
The microbiota dysbiosis in OSAS patients may correlate to sleep disorders and metabolic comorbidities. OSAS patients with Prevotella enterotype would exhibit worse sleep disruption [
93]. Compared with common people, short-chain fatty acid (SCFA)-producing bacteria was decreased in OSAS patients, accompanied by increased pathogens and elevated levels of IL-6 and homocysteine. Stratification analysis revealed that the Ruminococcus enterotype posed the highest risk for patients with OSAS [
34]. Research from real-life patients showed that gut microbiota dysbiosis and decreased SCFA in vivo significantly correlated with CRC [
94,
95], so we could hypothesize that gut microbiota dysbiosis and SCFA decrease may be involved in the process of CRC carcinogenesis in OSAS patients. In mouse model, IH exposure could induce higher abundance of Firmicutes and a smaller abundance of Bacteroidetes and Proteobacteria phyla; at the level of dominant microbiota families and genera, Prevotella, Paraprevotella, Desulfovibrio, and Lachnospiraceae increased, whereas Bacteroides, Odoribacter, Turicibacter, Peptococcaceae, and Erysipelotrichaceae decreased in the feces [
87]. The co-occurrence of Prevotella and Desulfovibrio suggests a mucin-degrading niche [
53], because the sulfate which liberated during Prevotella-mediated mucin degradation could back inhibit this process, but Desulfovibrio could remove the sulfate [
96,
97]. The lack of mucin on the epithelial layer of the intestine could potentially lead to a significant alteration in intestinal permeability [
87]. The enriched bacteria Desulfovibrio reduces sulfate in order to produce hydrogen sulfide (H2S), which has been reported as a possible contributing risk factor of colorectal cancer [
98,
99]. It has been proved that IH can directly impair cellular function and increase epithelium permeability [
100], and translocation of nonpathogenic bacteria via the transcellular routes would increase under hypoxia [
101]. Failure of intestinal epithelium barrier would lead to chronic inflammation [
102]. Plasma IL-6 and TNF-α have been proved to increase in patients with OSA [
30,
103‐
107], and this increase of inflammation cytokines was even not improved by CPAP therapy [
103,
108,
109], suggesting the inflammation seems not coming from IH only, which may be partially attributed to gut microbiota dysbiosis and destruction of intestinal epithelium barrier.
Our study showed that both IH and gut microbiota from OSAS patients could promote p-STAT3 entering to the nucleus, but we observed no change for β-catenin pathway in this process. It has been proved that gut microbiota dysbiosis of OSAS patients could induce inflammation, but we only observed elevation of inflammation cytokines for mRNA level in our model, which suggests that our cell model is far from perfect.
Our study had many limitations. We did not test the cell proliferation after IH exposure in this study, just tested the genes related to proliferation. Restricted to experiment conditions, it was not accurate for temperature modulation in IH exposure chamber, so the cells were not in good growth conditions during IH exposure, and this would lead to cell viability decrease or even cell death after longtime IH exposure. The outcome would be affected more when IH duration was longer, so we had to restrict IH duration to 12 h at most. Cell proliferation test, such as MTT test, may be meaningless in this condition. The amount of fecal sample was not sufficient enough, so we did not test the composition of feces microbiota in this study. Though we found STAT3 activated, but the pathway upstream is still not clear. Our IH parameters were performed according to previous teamwork [
42,
43], and it was the first time used for intestine cells. We did not try any other IH parameters, or the outcome may be different. Conclusions of cell research still need further validations in animal models and clinical researches. So, our findings are very limited, and there is still very long distance from this conclusion to clarify CRC carcinogenesis associated with OSAS.
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