Reproducibility in Cancer Biology: The who, where and how of fusobacteria and colon cancer
The association between the bacterium Fusobacterium nucleatum and human colon cancer is more complicated than it first appeared.
- Johns Hopkins University School of Medicine, United States
Trillions of microbes live on or in the body of a typical human, and this relationship is mostly harmonious. Our colons harbor the highest density of bacteria, with a mucus barrier protecting our gut from them. For 50 years, scientists have researched who amongst these bacteria might cause colon cancer, where these bacteria might act in the colon, and how they might cause colon cancer.
In 2012 researchers at the BC Cancer Agency, Simon Fraser University and the University of Guelph reported that they had used sequence-based technologies to show that Fusobacterium nucleatum, a species not previously linked to cancer, might be associated with colon cancer (Castellarin et al., 2012). They found that the levels of F. nucleatum in colorectal carcinoma were significantly higher than the levels in adjacent normal tissue. An independent group also reported a similar finding at the same time (Kostic et al., 2012). This was a surprise as F. nucleatum is usually found in the mouth.
In 2016, as part of the Reproducibility Project: Cancer Biology, Repass et al. published a Registered Report which explained in detail how they would seek to replicate the experiment (figure 2 in Castellarin et al.) in which quantitative polymerase chain reaction (qPCR) was used to detect F. nucleatum in colon tissues taken from colon cancer patients (Repass et al., 2016). The results of this experiment have now been published as a Replication Study (Repass and Reproducibility Project: Cancer Biology, 2018). In short, F. nucleatum was detected in just 25% of colorectal carcinomas in the Replication Study, and the difference in the level of this species in colorectal carcinomas and adjacent tissues (there was 10% more F. nucleatum in the carcinomas) was not significant.
So why did the Replication Study not find what was reported in the original study? One possible explanation is that Castellarin et al. studied 99 colon tissue pairs (tumor and tumor-adjacent tissues) from colon cancer patients. The Replication Study, on the other hand, included samples from 40 patients with cancer (both tumor and adjacent tissue), along with 40 non-diseased control tissue samples from age, sex and ethnicity matched individuals. A power calculation had suggested that a sample size of 40 would be big enough to see the effect reported in the original paper, assuming that the two populations were clinically similar. However, Castellarin et al. did not fully report the clinical metadata for their study (such as age, gender, ethnicity and risk factors for disease), so differences between the populations might explain why the original results were not replicated. This possibility is also supported by the fact that the relative abundance of F. nucleatum in tumors in the original experiment is much higher than in the Replication Study.
Technical issues might also have contributed to the lack of replication: in particular, in the Replication Study, qPCR suggested that F. nucleatum DNA was present in 26 samples, albeit at low levels, but qPCR amplicon sequencing detected the presence of specific F. nucleatum gene products in just 16 of these samples (10 in carcinomas; 6 in adjacent tissues). Moreover, all the non-specific amplicons (that is, the 10 that were not due to F. nucleatum) were detected very close to the detection limit of this technique.
Given that numerous studies have already shown that there is an association between F. nucleatum and the microbiota of human colon cancer (see Repass et al., 2016 and Gholizadeh et al., 2017 for reviews), the Replication Study is not a reason to change our view of this association. Rather, it provides a critical opportunity to reflect on our growing, yet incomplete, knowledge regarding fusobacteria and colon cancer. First, and most obvious, there is a need for prospective human studies in well-defined populations – using both microbiology and bioinformatics approaches – to carefully probe how risk factor and other clinical features might alter colon health in the presence of fusobacteria. For example, while F. nucleatum has certainly been the species most consistently identified in association with colon cancer to date, many studies have raised the possibility that other species of fusobacteria, or bacteria that co-aggregate in the presence F. nucleatum, could contribute to the pathogenesis of colon cancer (Castellarin et al., 2012; Kostic et al., 2012; Drewes et al., 2017; Bullman et al., 2017).
Further, while it is clear that fusobacteria are highly genetically diverse (Castellarin et al., 2012; Manson McGuire et al., 2014), we do not as yet understand whether the strains associated with colon cancer exhibit unique genetic features or are related to the fusobacteria that are common in the mouth.
There are also other gaps in our understanding. For example, some experiments that used daily inoculations of F. nucleatum in mouse models of colon cancer suggest that F. nucleatum acts early in colon cancer (Kostic et al., 2013; Yang et al., 2017), whereas germ-free mouse experiments – which are, arguably, a more definitive way to test the role of F. nucleatum on its own in tumor induction – refute this result (Tomkovich et al., 2017). In contrast, at least two studies (Castellarin et al., 2012; Bullman et al., 2017) suggest that F. nucleatum acts late in the tumor process, and research by the present author and co-workers could not discern an association with tumor stage (Drewes et al., 2017).
There is also uncertainty about where F. nucleatum is found: some researchers have reported that it is found with increased frequency in cancers on the right side of the colon (Mima et al., 2016), whereas this effect was not seen in other experiments (Drewes et al., 2017). The question of 'where?' is also complicated by the fact that right colon cancer contains prominent mucus-invasive bacterial biofilms, and about one third of these exhibit blooms of F. nucleatum.
Lastly, we lack a clear picture of how F. nucleatum contributes to the emergence of human colon cancer. Limited data suggest a wide range of putative mechanisms (including Wnt nuclear signaling, immune cell recruitment, checkpoint molecules and specific miRNA induction). While each mechanism is plausible, we lack a cohesive, step-by-step story for F. nucleatum carcinogenesis (Gholizadeh et al., 2017; Kostic et al., 2013; Yang et al., 2017).
While the Replication Study did not replicate the results of Castellarin et al., it provided useful information about the importance of population differences and the need for accurate F. nucleatum detection methods, and highlighted how much we need to learn about the links between F. nucleatum and colon cancer. A better understanding of the who (which Fusobacterium species and/or associates), the where (where in the colon, where in the world) and the how (which disease mechanisms) will help with the development of new prevention approaches, diagnostics and/or therapies for a cancer that is increasing in the young and also across the globe.
Note
Cynthia L Sears was the Reviewing Editor for the Registered Report (Repass et al., 2016) and the Replication Study (Repass and Reproducibility Project: Cancer Biology, 2018).
References
-
Carcinogenesis mechanisms of Fusobacterium nucleatumBiomedicine & Pharmacotherapy 89:918–925.https://doi.org/10.1016/j.biopha.2017.02.102
-
Fusobacterium nucleatum in colorectal carcinoma tissue according to tumor locationClinical and Translational Gastroenterology 7:e200.https://doi.org/10.1038/ctg.2016.53
Article and author information
Author details
Publication history
- Version of Record published: March 13, 2018 (version 1)
Copyright
© 2018, Sears
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,915
- Page views
-
- 235
- Downloads
-
- 16
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Reproducibility in Cancer Biology: The who, where and how of fusobacteria and colon cancer
eLife 7:e28434.
https://doi.org/10.7554/eLife.28434
Further reading
-
- Cancer Biology
- Computational and Systems Biology
Lung squamous cell carcinoma (LUSC) is a type of lung cancer with a dismal prognosis that lacks adequate therapies and actionable targets. This disease is characterized by a sequence of low- and high-grade preinvasive stages with increasing probability of malignant progression. Increasing our knowledge about the biology of these premalignant lesions (PMLs) is necessary to design new methods of early detection and prevention, and to identify the molecular processes that are key for malignant progression. To facilitate this research, we have designed XTABLE (Exploring Transcriptomes of Bronchial Lesions), an open-source application that integrates the most extensive transcriptomic databases of PMLs published so far. With this tool, users can stratify samples using multiple parameters and interrogate PML biology in multiple manners, such as two- and multiple-group comparisons, interrogation of genes of interests, and transcriptional signatures. Using XTABLE, we have carried out a comparative study of the potential role of chromosomal instability scores as biomarkers of PML progression and mapped the onset of the most relevant LUSC pathways to the sequence of LUSC developmental stages. XTABLE will critically facilitate new research for the identification of early detection biomarkers and acquire a better understanding of the LUSC precancerous stages.
-
- Biochemistry and Chemical Biology
- Cancer Biology
Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.
