Tigecycline mitigates CAC by regulating gut microbiota.
The 16S rRNA sequencing results showed an alteration on bacterial composition in AOM/DSS treated mice. Tigecycline 25mg/kg and 5-FU significantly increased alpha diversity, calculated by different indexes (observed species, Shannon, ACE, and Simpson), in comparison with untreated CAC group (Figure 7A). Besides, the beta diversity determined by weighted unifrac analysis revealed a clear separation in the Principal Coordinates Analysis (PCoA) between healthy and CAC group, indicating that the homeostasis of gut microbiota was dramatically disrupted by the AOM/DSS stimulus (Figure 7B). Remarkably, the 5-FU mice and the lowest dose of tigecycline showed more resemblance to the healthy group (Figure 7B). Consistently, the Venn diagram (prevalence 75%) showed that there were only 4 common OTUs among healthy and CAC groups, and that the highest number of shared OTUs was presented among healthy group and tigecycline group (25 mg/kg, 48 shared OTUs) (Figure 7C).
The gut microbiota taxa and their abundance were also analyzed. As shown in Figure 7D , the relative taxonomic composition at the phylum level mainly contained Bacillota , Bacteroidota , Pseudomonadotaand Verrucomicrobiota. The relative abundance of Bacillotaand Actinomycetota was diminished in the AOM/DSS-induced group in comparison to the control group, while Bacteroidota ,Pseudomonadota , and Verrucomicrobiota abundance was increased (Figure 7D). Notably, the administration of tigecycline mitigated the variation of gut microbiota composition caused by AOM/DSS stimulation (Figure 7D). Moreover, theBacillota /Bacteroidota (named F/B) ratio in the CAC group was significantly lower than in the control mice, and the administration of the lowest dose of tigecycline was able to significantly increase the ratio compared with CAC mice (Figure 7E). At genus level, the heatmap showed that some bacteria abundance was increased in the CAC group when compared to healthy mice (Akkermansia , Turicibacter ,Lachnospiraceae , Desulfovibrio and Enterorhabdus ) (Figure 7F). All these genera abundance was restored by the treatment with tigecycline (Figure 7F). Conversely, different bacterial genera were reduced in untreated mice compared with the healthy group (Coriobacteriaceae , Lactobacillus , and Dubosiella ) (Figure 7F), and even lower in treated CAC mice (Figure 7F).
Volcano plots provide us information about the amount of OTUs that are downregulated and upregulated with respect to the control CAC group. Specifically, in healthy and CAC-FU the higher number of OTUs were upregulated, 93 and 130 OTUs, respectively (Figure 8A). When the Venn diagram was determined with the upregulated OTUs the results showed that none of them were shared between the four groups. Conversely, 1 of them corresponding to Parabacteroides genus was observed among the three treated groups (Figure 8B). Moreover, 6 OTUs were up-regulated and shared among mice treated with the two doses of tigecycline (Figure 8B):Enterobacteriaceae family, Akkermansia andBacteroides genera, as well as, Parabacteroidesdistasonis and uncultured Bacteroidales bacteriumbacterial species (Figure 8B). On the other hand, in tigecycline-treated mice, the higher number of OTUs were found downregulated: 87 and 130 OTUs, respectively (Figure 8B). 3 of them were significantly downregulated and shared in the three groups of treated mice:Dubosiella genus and Dubosiella newyorkinensis andClostridium sp. ASF502 bacterial species (Figure 8B). When tigecycline treated groups were compared only two bacterial genera (Faecalibaculum and A2 from Lachnospiraceae) were decreased and shared (Figure 8B).
Focusing on OTUs shared from treated and healthy mice,Ruminococcus 1 and Erysipelatoclostridium genera,Lachnospiraceae bacterium COE1 and Clostridium sp. Clone-47 species were found down-regulated in healthy, CAC-T50 and CAC-FU groups (Figure 8B). Additionally, the genusRuminococcaceae UCG-013 was decreased in healthy and tigecycline 50 mg/kg treated mice (Figure 8B).
Finally, the heat map constructed with data from control CAC mice and tigecycline treated mice showed a positive correlation between the relative abundance of Akkermansia and P. distasonis and parameters that indicates a good prognosis of the disease such as CD8+ cells levels, fewer number of tumors and levels of inflammation and proliferation markers (Figure 8C).
DISCUSSION
CRC incidence and mortality rates are still high, so the development of novel therapeutic strategies is necessary. In this study, we demonstrated the effectiveness of tigecycline in the treatment of CRCin vitro and in a murine model of CAC. Moreover, our findings provide the first evidence that tigecycline exerts an anti-proliferative and pro-apoptotic effect, as well as immunomodulatory properties and the ability to modulate gut dysbiosis in CRC.
This study agrees with and supports previous studies that have deemed some antibiotics as promising candidates for cancer therapy (Senet al. , 2018). In fact, different antibiotic drugs, including tigecycline, have been recently reported to impact growth of various types of tumor cells and show positive therapeutic effects in cancer patients (Li et al. , 2015). The antitumor mechanisms of tigecycline remain unidentified, but one of them could be the capacity of tigecycline to regulate cell proliferation rate. In this regard, almost 80% of CRC patients (Korinek et al. , 1997) show mutations in APC gene which lead to alterations on Wnt/β-catenin signaling pathway. Thus, CTNNB1 aberrant activation and nuclear translocation boost the expression of oncogenes involved in cell cycle and proliferation such as AXIN2, MYC and CCND1 (Shang et al., 2017) , increasing cell proliferation and promoting the tumorigenesis process, including migration, invasion, apoptosis evasion and chemoresistance (Tenbaum et al. , 2012). The present study demonstrates that tigecycline suppressed tumorigenesis by downregulating the Wnt/β-catenin signaling pathway, confirming previous studies in cervical squamous cell carcinoma (Li et al. , 2015). Additionally, we also show that tigecycline can enhance the phosphorylation of CTNNB1 in tumor cells, even when the Wnt/β-catenin pathway is overstimulated with Wnt3a ligand. This action increases the degradation of CTNNB1 downregulating the expression of genes such as AXIN2 andMYC .
Other signaling pathways are also altered in the tumoral process, including JAK/STAT3 and PI3K/AKT/mTOR. These pathways are involved in the proliferation, survival, and metastasis of CRC, as well as immune and apoptosis evasion and poor patient outcomes (Luo et al. , 2017; Malinowsky et al. , 2014). In this regard, there is evidence that STAT3 signaling contributes to the stimulation of CCND1 andMYC expression promoting cell-cycle progression and proliferation (Luo et al. , 2017). Besides, STAT3 favors cell survival and invasion through the up-regulation of Bcl2 and Mmp9 (Luet al. , 2017; Xiong et al. , 2008). Accordingly, we found that tigecycline treatment decreased the expression of these markers, which could explain its capacity to inhibit STAT3 activation. Other antibiotics have also displayed antitumor effects through the inhibition of STAT3 in different types of cancer (Lu et al. , 2017).
In addition, it has been demonstrated that individual CRCs contain cells at various stages of differentiation, with cancer stem cells (CSCs) driving tumor growth and progression. Many studies have confirmed that CSC initiate the tumor process and are the main responsible for resistances to treatment and recurrences in CRC (Das et al. , 2020). CSCs display survival mechanisms as apoptosis evasion or the capability to establish quiescent state, thus circumventing conventional antineoplastic treatments. In this regard, tigecycline interferes with the generation of CSCs, reducing the population of LGR5⁺CD44⁺ cells. Our findings have clinical relevance since tigecycline could be considered for conducting future clinical trials on the treatment of CRC, including “pre-malignant” and advanced metastatic process. Supporting this finding, tigecycline has previously shown capacity to eradicate CSC across different tumor types (breast, ductal carcinoma in situ, ovarian, prostate, lung, pancreatic, melanoma, and glioblastoma) (Lamb et al. , 2015), maybe mediated by mitochondrial inhibition.
Additionally, EMT process is crucial for CSC generation and, therefore, for the development and progression of CRC (Dongre et al. , 2019). Tigecycline has shown that interferes with the expression of SNAI1, a marker involved in the EMT process, in this study, and earlier, in melanoma (Hu et al. , 2016).
As it is widely known, evasion of cell death is one of the hallmarks of cancer. Previous studies have evidenced that tetracyclines can induce apoptosis in different cell lines, including tumor cells (Tolomeoet al. , 2001). Our findings corroborate these results and, also, demonstrate the apoptotic activity of tigecycline in vitro andin vivo . Interestingly, molecular mechanisms involved in tigecycline-mediated apoptosis are different from those displayed by 5-FU. The latest activates intrinsic apoptosis through TP53 upregulation, whereas tigecycline exerts its action downstream, at BAX level. Moreover, our findings support that CASP3 and CASP7 are the main apoptosis executioners in 5-FU treatment, whereas CASP7 is involved in the tigecycline-mediated apoptosis. In any case, both treatments lead to PARP1 cleavage and cell death. This action is shared by different anticancer drugs, such as etoposide, being considered as an early marker of chemotherapy-induced apoptosis (Kaufmann et al. , 1993).
We also show that both treatments target ER-mediated apoptosis markers, favoring the induction of cell death through this pathway. 5-FU increased the ER-mediated apoptosis through the activation of the two isoforms of JNK. Tigecycline also acts at this level, however, it only favors the activation of p54 isoform, which, according to Waetzig V et al., 2003, is enough for taxol-mediated apoptosis (Waetzig et al. , 2003). Dissimilar to 5-FU, tigecycline also activates this pathway through its action on ATF6.
Besides, as previously commented, the tumor microenvironment has an essential role in tumor progression and prognosis. Thus, chronic inflammatory stimuli triggers cancer initiation and progression, as occurs in this experimental CAC model, and IBD patients, who have an increased risk of developing CRC (Clarke et al. , 2019). On the other hand, the immune system also plays an important role attacking the tumor cells. In CRC, altered Th1/Th2 response contributes to tumor progression, while enhanced Th1 population is associated with better prognosis (Ling et al. , 2016). Furthermore, CTLs are the preferred immune cells for tumor surveillance and are the most powerful effectors in immune cancer targeting (Raskov et al. , 2021). Accordingly, we showed that the development of CAC was associated with a depletion of T cells, specifically CTLs in the colon of non-treated CAC mice. However, the treatment with tigecycline and 5-FU restored CTL levels. In fact, 5-FU and the lowest dose of tigecycline, significantly increased Tc1 cells, which plays an important role in the antitumor effect of these lymphocytes, being IFNγ producers and considered an indicator of CTL activation (Bhat et al. , 2017).
Finally, it is well accepted that alterations in gut microbiota composition contributes to tumor development and are associated with CRC (Garrett, 2019; Tjalsma et al. , 2012). Although it is not clear whether dysbiotic changes have a causal role or follow environmental changes produced by tumor onset, there is enough evidence supporting the role of certain bacteria in cancer initiation and progression as well as cancer protection (Garrett, 2019; Tjalsma et al. , 2012). Therefore, modulation of this altered microbiota in CRC with drugs such as antibiotics, could be considered a complementary therapeutic approach (Fong et al. , 2020). Correspondingly, our results confirm the existence of a dysbiotic status in the CAC group characterized by a reduced microbial diversity, as seen before (Ibrahim et al. , 2019), while the lower dose of tigecycline restored this microbial diversity. Supporting the clinical importance of the latest, other antibiotics, including tetracyclines, have shown the ability to modulate the dysbiosis in oncology patients (Ghanem et al. , 2021).
Over the last decade, several bacteria species have gained interest for their involvement in colorectal carcinogenesis (Garrett, 2019).Akkermansia muciniphila stands out among these species for its anti-inflammatory and protective functions on gut barrier (Zhai et al. , 2019). The relationship between A. muciniphila and CRC remains controversial. Different studies have shown an enrichment ofAkkermansia in CRC patients (Colombo et al. , 2022), while others have reported that it can mitigate tumorigenesis in CRC with capacity to boost the effect of existing antitumor treatments (Houet al. , 2021). In this study we observed an increase inAkkermansia genus in CAC mice compared to healthy mice, as well as increased levels in mice treated with tigecycline, which may contribute to its beneficial effect. Additionally, P. distasonisis a gram-negative anaerobic bacterium that has been associated with an attenuation of tumorigenesis and inflammation by interfering production of cytokines and proliferation mediators, such as AKT (Koh et al. , 2020). Tigecycline treatment increased levels of P. distasonis , which can be related to the modulation of the inflammatory markers and the promotion of the intestinal barrier integrity, as reported previously in other studies (Koh et al. , 2020). Indeed, the correlation analysis showed a negative association betweenAkkermansia genus and P. distasonis and some macroscopic parameters such as tumor number, size and DAI, as well as molecular markers of proliferation (STAT3, CTNNB1 and Ccnd1 gene expression), survival (BCL2), stemness (SNAI1) or inflammation (Il6, Il17a, Il23a, Tnfa gene expression). Moreover,Akkermansia sp. and P. distasonis abundance are positively correlated with apoptosis (Tunel and CASP7) and cytotoxic activity (CD8+ and Tc1 response) (Figure 8C). Consequently, the connection between both bacteria species can contribute to tumorigenesis attenuation.
Human colonic microbiota can process a wide range of substrates that escape digestion by the host. Among Bacillota , theLachnospiraceae and Ruminococcaceae species hydrolyze starch and other sugars to produce butyrate and other short chain fatty acids (SCFAs). Initially, these SCFA-producing species have been associated with an improvement of the outcomes in different clinical settings including CRC and IBD (Mirzaei et al. , 2021). Despite this, other studies have reported an increase in Lachnospiraceaeand Ruminococcus genera in CRC patients (Yang et al. , 2019). Here, we found down-regulation of Ruminococcus andLachnospiraceae species in healthy and treated groups when compared with control CAC.
On the other hand, bacterial species from theErysipelatoclostridium genus, such as E. ruminantium andE. ramosum, have been reported to be increased in adenoma (Wuet al. , 2021). In this regard, we found a lower abundance ofErysipelatoclostridium in both, healthy and tigecycline treated groups. Indeed, in this study we show a positive correlation between tumor size and markers associated with tumor progression and the abundance of Ruminococcaceae UCG-013, Ruminococcus 1, Lachnospiraceae A2, Lachnospiraceae bacterium COE1 andErysipelatoclostridium (Figure 8C).
In conclusion, this study has demonstrated the capacity of tigecycline to induce a sustained attenuation of tumorigenesis in CRC, evidenced by a reduced tumor number and characterized by reduction of tumor cells proliferation, induction of apoptosis, as well as amelioration of inflammation and modulation of gut dysbiosis. Therefore, our data support the treatment with tigecycline as a novel therapeutic strategy against CRC.