In this study, we clearly found that the composition of the gut microbiota differs between right- and left-sided colon cancer patients and after curative colectomy using T-RFLP analysis and 16S rRNA gene amplicon sequencing. These results demonstrated the specific change in gut microbiota in each group, suggesting that gut microbiota are closely associated with the development of colon cancer and physiological conditions after partial colectomy.
T-RFLP analysis showed a reduced ratio of clostridial clusters XIVa in SCC patients and clostridial clusters IX in RCC patients, although these changes were not seen in RCR and SCR patients. Clostridial cluster XIVa includes most butyrate producers that belong to the Firmicutes phylum in the human colon and Clostridial cluster IX propionate-producing bacteria [28–30]. SCFAs such as butyrate or propionate are known to have an important role in preserving gut barrier functions and exerting immunomodulatory and anti-inflammatory properties [29, 31]. The antiproliferative, apoptotic and differentiating properties of the various SCFAs are linked to the degree of induced histone hyperacetylation . Fewer SCFAs in feces are observed in patients with IBD or colorectal cancer compared with normal patients . Our study indicates that different types of main SCFA-producing bacteria are lower in colon cancer patients and that those of clostridial clusters IX are lower in the RCC group and those of clostridial clusters XIVa are lower in the SCC group. However, this kind of change was not observed in patients after colectomy. Therefore, we speculate that low SCFA-producing bacteria may be related to colon carcinogenesis and are high-risk markers of colon cancer.
We further analyzed gut microbiota in patients with RCC and SCC and with curative colectomy by 16S rRNA gene amplicon sequencing with QIIME 2, which is a next-generation microbiome bioinformatics platform. We found that the diversity of the gut microbiota in the RCC group was higher than that in the control group, but that in the RCR group was at the same level as that in the control group. In contrast, diversity in the SCR group was significantly higher than that in the RCR group. We also found that gut microbial genus composition using principal component analysis of the log-transformed relative abundances showed a separation between the control group and RCC group and between the RCR group and SCR group. A previous study suggested that patients with colon cancer have a less diverse microbiome than do healthy individuals . However, other reports found higher richness in the microbiomes of patients with colon cancer than controls, partly by the expansion of species derived from the oral cavity. A study of microbiota after curative colon surgery suggested that the right hemicolectomy group showed a tendency to decrease in terms of richness and diversity at the genus level . These results are consistent with our current data. Diversity of the gut microbiome is defined as the number and relative abundance distribution of distinct types of microbiomes in the gut. Previous studies have suggested that dysbiosis of the gut microbiome is becoming increasingly recognized for its influence on host immunity and may influence the good response to a variety of cancer therapies, such as chemotherapy, radiotherapy, and immunotherapy . From this point of view, it is very interesting that diversity and richness are different in patients with both pre- and postoperative colorectal cancer according to the tumor location, and high diversity in the SCC group may be associated with favorable outcomes in the SCR group.
Analysis of microbiota at the class level revealed that Clostridia and Bacilli belonging to Firmicutes were significantly dominant in the RCC group compared to the control group. The SCC group had a higher abundance of Verrucomicrobiae belonging to Verrucomicrobia. These characteristics were not observed in patients in the postcolectomy group. In particular, Firmicutes was more abundant and Verrucomicrobia was lower in the SCR group than in the RCR group. This tendency was consistent with the findings of a previous long-term study after curative colectomy . These results indicated that there may be specific profiles of the gut bacterial population at the phylum and class taxonomic levels related to colon cancer, and this alteration was different between the locations of cancer. Furthermore, colectomy may also differentially lead to new gut microbiota compositions depending on the removal site.
Next, we performed discriminant analysis by using the LEfSe approach, which was applied to show the key taxa responsible for the difference between several groups and identified several gut microbes mainly at the genus and family levels. Among these bacteria, especially Ruminococcaceae, Streptococcaceae, Clostridiaceae, Gemellaceae, and Desulfovibrio, which are seen in the RCC group, these species have already reported as constituting colon cancer-associated microbiomes [2, 3, 4, 37]. However, the exact mechanism by which these bacteria affect the development and progression of colon cancer has not been fully elucidated. In the SCC group, Porphynomonas, Parvimonas, Peptoniphilus, and Peptococcaceae were identified. These species are gram-positive anaerobic cocci mainly located among oral bacteria and influence mucosal gene expression, which might contribute to the development of colon cancers [3, 4, 38]. In addition, Butyricicoccus, which produces butyrate, was less abundant in the SCC group. Butyrate is an essential metabolite in the human colon and is the preferred energy source because colon epithelial cells have immunomodulatory and anti-
inflammatory properties . Some cross-sectional studies reported that, compared with control individuals, patients with colorectal cancer had a lower abundance of butyrate-producing species and lower fecal levels of butyrate, which is consistent with our current data.
Our data clearly suggest that the tumor microbiota between right- and left-sided colorectal cancer patients shows differential microbial diversity and bacterial taxa at several levels, meaning that the RCC and SCC groups may clearly exhibit different specific microbiome compositions.
Genetically, right-sided tumors are commonly associated with microsatellite instability and are highly immunogenic, presenting with BRAF mutations, whereas left-sided tumors show chromosomal instability with mutations in KRAS, APC, PIK3CA, and p53 [9, 11]. In fact, Fusobacterium nucleatum signatures in proximal colorectal cancer tissue are correlated with shorter patient survival and molecular alterations such as hypermutation with microsatellite instability and BRAF mutations [3, 39]. Clinically, patients with right-sided tumors present a worse prognosis than those with left-sided tumors [4, 40].
Therefore, there is a possibility that the gut microbiome affects the development and progression of colon cancer differently according to the tumor location.
Although there are a few studies regarding the differences in the tumor microbiota between right- and left-sided colon cancer, compositional alterations in the microbiota are not restricted to cancerous tissue and differ between distal and proximal cancers. . Another report of analysis of on- and off-tumor microbiota suggests that the right and left colon show distinctive bacterial populations; however, the presence of a colonic tumor leads to a more consistent microbiota between locations . Therefore, when we investigate the gut microbiota of colon cancers using feces, we should consider that this might be influenced by not only tumor-associated microbiota but also by the surrounding nontumor microbiota.
Because the number of patients enrolled in this study was small, we could not demonstrate clinical characteristics, such as DM, in postcolectomy patients. However, a previous study reported that right hemicolectomy patients rather than left anterior resection patients had higher serum fasting glucose than controls, implying that the proximal colon may play an important role in glucose control . In contrast, a large study demonstrated an increased risk of clinically recorded type 2 diabetes among patients who had undergone total and partial colectomy, with the risk being elevated only among individuals who had the left part of their colon removed . Therefore, recently, the gut microbiota has been shown to play an important role in the development of metabolic diseases, including obesity and metabolic syndrome [16, 25].
In our current study, the RCR and SCR groups showed clear alterations in microbiome composition. At the family level, Gemellaceae, the members of which modulate immunological reactions, was less abundant in the RCR group compared with the other groups. In contrast to the RCR group, in the SCR group, a variety of gut bacteria belonging to the Firmicutes phylum were abundant, but the abundance of Fusobacteriaceae was low. A previous study reported that the ratio of Firmicutes to Bacteroidetes was significantly lower in patients with DM . However, our study showed a high Firmicute-to-Bacteroidetes ratio in the SCR group. Further studies are needed to determine how gut microbiome composition is related to clinical manifestations in patients with colectomy.
Finally, we performed predictive metagenome analysis to clarify the functional enzyme spectrum in each group. We revealed the differential expression of several genomes related to glucose metabolism in each group. A previous meta-analysis of metagenomic studies identified the microbiome function of gluconeogenesis and the putrefaction and fermentation pathways as being associated with colorectal cancer . Furthermore, we revealed that cholesterol transport- and metabolism-related enzymes were specifically upregulated in the RCC group and that cobalamin metabolism-related enzymes were downregulated in the SCC group. These results supported a previous reports in which cholesterol may inhibit the proliferative capacity of certain human colonic adenocarcinomas [42, 43] and plasma vitamin B12 concentrations is the risk of colorectal cancer [44, 45]. Furthermore, some SCFA-related enzymes were identified in both the RCC and the SCC groups. However, these specific characteristics were not observed in the postoperative groups. These results support the idea that pathological microbial dysbiosis is responsible for the gut of patients with colorectal cancer. Future shotgun metagenomic studies of the intestinal mucosa-associated microbiome will be important to further refine the list of colorectal cancer-associated gut microbes.
In conclusion, although there are several limitations in our study, such as small sample sizes or several unclear backgrounds of patients, using T-RFLP analysis, 16S rRNA gene amplicon sequencing and metagenome analysis, we clearly found that the composition of the gut microbiota dramatically differs between right- and sigmoid colon cancer patients and between right hemicolectomy and sigmoidectomy patients. Our findings support the hypothesis of tumor location-specific microbiota. We found that high richness and diversity were associated with the RCC and SCR groups and the different gut microbiota compositions in each group. It is difficult to determine whether gut microbiota could influence the pathophysiological condition of patients or whether
gut condition could alter microbiota. However, in assessing the gut microbiota in patients with colon cancer, we should consider tumor location.
We hope that the results herein can provide useful information for using gut microbes as biomarkers to assess the location and progression of colon cancer or lead to interventional targets to control the development of this disease.