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36 Introduction
37 With an estimated 10 million deaths in 2020, cancer is a leading cause of death in
38 populations worldwide [1]. Colorectal cancer (CRC) is the third most diagnosed cancer
39 after breast and lung cancer. Additionally, it is the second most deadly, accounting for
40 9.8% of cancer mortalities [1]. Developed countries show a significantly higher incidence of
41 CRC, with cases 3-4 times more common than in developing nations [2]. However,
42 incidences are rising in developing countries as socioeconomic conditions improve and
43 populations adopt a more western lifestyle [3]. Colorectal cancer is difficult to predict, with
44 only approximately 20% of cases linked with familial risk [4].
45
46 One factor implicated in the development of CRC is the composition of patients' microbiota
47 [5]. Advances in sequencing methods and analytical techniques have revolutionised our
48 understanding of the human microbiome. Composed of 10 13-1014 microbial cells, estimates
49 suggest that cells in and on humans outnumber somatic cells by a factor of 10 [6]. The
50 human genome project was completed in April 2003 and found that human genomes
51 contain ~23,000 protein-coding genes [7]. Comparatively, the human microbiome project
52 suggests that the number of unique genes in just the human gut microbiome may exceed
53 9,000,000 [8, 9].
54
55 The increasing global burden of CRC calls for novel strategies to prevent, detect, and treat
56 CRC. Increasing evidence suggests microbes play a vital role in the initiation,
57 development, and metastasis of CRC [10]. Throughout this review, we examine; factors
58 influencing the microbiota colonisation, tumour suppressive and oncogenic characteristics
59 of bacteria and their associations with CRC and how interactions between bacteria and the
60 immune system can impact cancer detection and eradication. Additionally, we will discuss
61 how current research may benefit future technologies and the limitations of current
62 methods used in labs.
63
64
65 Discussion
66 Causes of Dysbiosis
67 Dysbiosis is a reduction in the diversity of microbial cells within specific microbiomes. With
68 reference to human microbiota, this can result in the loss of beneficial bacteria, alongside
69 a rise in pathobionts, non-harmful bacteria which, under certain conditions, become
70 pathogenic. A significant influencer of dysbiosis is the increasing adoption of a ‘western
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 71 diet,’ high in fats and simple sugars [11]. Both epidemiological and pre-clinical animal
72 studies reinforce hypotheses that diet is a significant risk factor in developing colorectal
73 adenocarcinoma (CAC) [12, 13]. Following a switch to a high-fat diet, David et al. observed
74 changes in the beta-diversity of participants’ gut microbiota within two days [14].
75 An essential indicator of dysbiosis is the Firmicutes/Bacteroidetes ratio within the gut [15].
76 Firmicutes and Bacteroidetes represent two of the most important phyla within the gut
77 microbiota. Imbalances in the Firmicutes/Bacteroidetes ratio indicate dysbiosis and may
78 prevent normal intestinal homeostasis from being maintained [15].
79
80 Obesity, another risk factor in colorectal cancer (CRC), has also been associated with
81 dysbiosis; animal studies demonstrate that obese individuals exhibit higher Firmicutes
82 abundances at a cost to Bacteroidetes [16]. Simultaneously, the colonisation of the gut
83 microbiota may also influence the development of obesity. A gnotobiotic mice study used
84 two groups colonised with either a ‘lean’ or ‘obese’ microbiota. Those colonised with the
85 ‘obese’ microbiota exhibited a greater total body fat when compared with the other group
86 [17].
87
88 Lifestyle choices, including smoking and the consumption of alcohol, also impact the
89 human microbiome [18] [19]. Inhalation of cigarette smoke can increase the gut pH
90 allowing certain bacteria to thrive whilst others decline [20]. A study assessing the
91 microbiome of alcoholics using colonic biopsy samples found that a subset of alcoholics
92 exhibited gut dysbiosis categorised by a lower median abundance of Bacteroidetes and
93 higher of Proteobacteria [21].
94
95 Antibiotic use is associated with decreased microbiome diversity, with patients often
96 prescribed antibiotics unnecessarily or prescribed broad-spectrum antibiotics where a
97 more specific antibiotic would be appropriate [22]. Due to their broad-spectrum effects,
98 many antibiotics can kill or inhibit commensal microbes, thus causing dysbiosis [23].
99 Furthermore, studies suggest that antibiotic-induced gut dysbiosis promotes tumorigenesis
100 in mice [24]; additionally, data from a health insurance organisation indicates an
101 association between regular antibiotic use and CRC development [25]. [maybe move to a
102 different section?]
103
104 Tumour suppressive effects of commensal microbes

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105 Protective symbionts colonise the GI tract and produce various beneficial metabolites. The
106 primary metabolites produced via saccharolytic fermentation of fibre and resistant starch
107 are the short-chain fatty acids (SCFAs) [26]. Diet is a known factor in both dysbiosis and
108 CRC development, with the consumption of more than 20g of fibre per day associated with
109 a 25% reduction in CRC risk [27]. The bulk of dietary fibre is non-degradable by human
110 enzymes [28]. Thus, they are mainly degraded by Firmicutes and Bacteroidetes bacteria
111 [29] into acetate, propionate, and butyrate, which comprise circa 80% of all SCFAs formed
112 by gut bacteria [26]. Firmicutes produce the majority of butyrate in the gut, whilst
113 Bacteroidetes produce mainly acetate and propionate [30].
114
115 SCFAs bind to G protein-coupled receptors (GPCRs) and are beneficial to gut health in
116 several ways. These include maintaining intestinal barrier integrity, mucus production,
117 reducing inflammation, regulating immune function, and limiting CRC risk [31]. Literature
118 suggests that of all SCFAs, butyrate demonstrates the most significant tumour
119 suppressant characteristics [32]. Using in vitro techniques, [33] showed treatments of HT-
120 29 adenocarcinoma cells with four separate SCFAs (acetate, propionate, butyrate and
121 valerate). Found that cell growth and differentiation were significantly decreased by
122 butyrate and propionate whilst acetate had no significant effect.
123 Furthermore, gnotobiotic mice colonised with Butyrivibrio fibrisolvens [34] demonstrated
124 that those fed a fibre diet exhibited significantly higher colonic butyrate levels than the
125 other groups. These mice were then treated with the procarcinogens azoxymethane and
126 dextran sodium sulphate to increase tumour incidence. When examined five months later,
127 the experiments group had significantly fewer tumours (mean 1) than the control group
128 (mean 3-4) [34]. These results suggest that high fibre diets combined with butyrate-
129 producing bacteria protect against CRC development in mice. However, to support this
130 relevance in human CRC, the mechanisms involved must be comparable.
131
132 Butyrate’s primary anti-carcinogenic effects are associated with its role in epigenetic
133 regulation. Butyrate inhibits histone deacetylase (HDAC) [35], with one study showing it to
134 induce histone hyperacetylation by 115.4% compared with untreated controls [33]. Histone
135 acetylation causes chromatin to unravel, allowing genes to be transcribed [36]. When
136 prevented, this can cause the silencing of tumour suppressor genes, which has been
137 implicated with the aetiology of multiple cancers, including CRC [37]. Evidence suggests
138 that when HDAC inhibitors are active, benefits include suppressing tumour growth,
139 inducing cell cycle arrest, and apoptosis of cancerous cells [37].
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140 Furthermore, recent studies have shown HDAC inhibitors to suppress angiogenesis, the
141 formation of new blood vessels, in tumours [38]. Therefore, reducing the supply of
142 nutrients and oxygens and limiting tumour growth. This occurs when HDAC1, which
143 SCFAs inhibit, is over-expressed. Resulting in the suppression of two tumour suppression
144 genes, HIF-1α and VEGF, allowing for the stimulation of angiogenesis [39]. Class 1 HDAC
145 inhibitors such as butyrate have been observed to operate under the same mechanisms in
146 humans and mice [40]. Thus, reinforcing the relevance of gnotobiotic mouse models to
147 human CRC development.
148
149 {talk about colonisation preventing other harmful bacteria]
150
151
152 Bacteria associated with colorectal cancer
153 Several bacterial species are associated with the pathogenesis of CRC. Bacteria
154 inhabiting the gut, which are predominantly obligate anaerobes, are most associated with
155 CRC [41]. These include enterotoxigenic Bacteroides fragilis (ETBF), Enterococcus
156 faecalis, Streptococcus bovis, Escherichia coli and Fusobacterium spp. [10].
157
158 Bacteroides fragilis becomes enterotoxigenic (ETBF) when the Bacteroides fragilis toxin
159 gene (BFT) is expressed [17].
160 [42] demonstrated an increased prevalence of ETBF in stool samples from CRC patients
161 (38%) compared with control subjects (12%).
162 Additionally, adenocarcinoma tissue from 55 CRC patients exhibited a more significant
163 enrichment [define?] of ETBF compared to healthy paired samples taken from adjacent
164 mucosal tissue [43]. This study used real-time PCR to create a quantitative profile for each
165 sample. Simultaneously, the prevalence of Fusobacterium spp. was assessed using the
166 same methods and patients. Fusobacterium was observed in 82% and 81% of paired
167 tumour and normal samples. However, quantitative results showed a significant increase
168 in the volume of Fusobacterium in tumour samples [43].
169
170 Escherichia coli (E. coli) is a commensal microbe found in 90% of adult gut microbiomes
171 [44] and is one of the first microbes to colonise the gut of newly born infants [45]. Certain
172 E. coli bacteria contain a 54kb genomic island (GI), polyketide synthetase (pks), coding for
173 the synthesis of a known genotoxin colibactin [46]. Findings suggest that pks+ E.coli may

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174 be a cofactor in CAC development. A recent study found 16.7% of CRC patients to be
175 positive for pks+ E.coli compared to only 4.35% of healthy controls [46]
176
177 Streptococcus bovis (S. bovis) is divided into biotypes I and II, with the former
178 demonstrating a more significant association with CRC [47]. [bit more about this]
179
180 Enterococcus faecalis (E. faecalis) is the most prevalent strand of Enterococci to colonise
181 the gastrointestinal tract [(105–107 colony-forming units (CFU)/g] [48]. Compared to other
182 bacteria, fewer studies have investigated the relationship between E. faecalis and CRC.
183 However, studies using DNA extracted from the faeces of CRC patients have shown E.
184 faecalis populations to be significantly higher than those in healthy volunteers [49].
185
186
187 Oncogenic mechanisms of associated bacteria
188 In vitro studies using intestinal epithelial cells have demonstrated that cells infected with
189 pks+ E.coli exhibit an enhanced tumour growth [50]. Colibactins directly induce DNA
190 crosslinks within cells [51]. These are covalent bonds that form between nucleotides on
191 either the same or opposite strands of DNA [52]. Interstrand crosslinks (ICLs) are a source
192 of genomic instability and, if repaired incorrectly or unrepaired, can be involved in CRC
193 pathogenesis [53]
194
195 A recent study provides evidence of an association between E. coli and ETBF and their
196 subsequent role in CRC pathogenesis [54]. It is hypothesised that ETBF causes mucus
197 degradation within the colon, allowing for increased pks+ E.coli colonisation, ultimately
198 facilitating the delivery of colibactin to colonic epithelial cells.
199 BFT is the only known virulence factor of ETBF and has the potential to initiate and
200 promote cancer [55]. This occurs via a multistep inflammatory cascade within colonic
201 epithelial cells (CECs) [56]].
202
203 [Discuss potential interaction with ETBF bft gene/toxin]
204 [Discuss colibactin link to IBD and resulting in increased CRC risk]
205
206 [e coli produce their own ClbS hydrolase, which protects them from mature colibactin [51]]
207

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208 Fusobacterium nucleatum is a putatively pro-carcinogenic bacterium; however, further
209 studies are required to provide a definitive role for F. nucleatum in CRC development.
210 Many studies find F. nucleatum to be enriched in CAC tissues. However, recent studies
211 examining prediagnostic antibody responses to F. nucleatum have found no significant
212 association with CRC risk [57] [58] [suggesting a less direct link?] [move to Immuno
213 section?]
214
215 Although epidemiological studies have demonstrated a strong association between E.
216 faecalis and CRC [49], fewer etiological studies have provided significant results. One
217 study demonstrated that HT-29 epithelial cells showed increased cell damage when co-
218 incubated with wild type E. faecalis. This is hypothesised to be due to the production of
219 extracellular superoxide (O2-) by E. faecalis which in turn leads to a rise in hydrogen
220 peroxide (H2O2), a bona fide genotoxin [59]. [if space here right about how DNA damage
221 was prevented by catalase but not manganese superoxide dismutase]. Under the same
222 conditions, a mutant strain of E. faecalis with reduced (O2-) production demonstrated a
223 significantly lessened level of DNA damage to HT-29 cells [59]. This implies that the
224 production of O2- has a pro-carcinogenic effect. [critique study here?]
225
226 S. bovis
227
228
229
230
231
232
233 Immune response
234 One salient characteristic of all cancers is their ability to evade the immune system [60].
235 Thus, the relationship between the microbiome and the immune system is relevant to CRC
236 development. F. nucleatum, as mentioned earlier, has been hypothesised to inhibit the
237 function of natural killer (NK) cells and various T cells [61]. This was observed across
238 multiple NK cell lines from different donors. Thereby evincing that F. nucleatum interacts
239 with an inhibitory receptor which is highly conserved throughout different individuals. More
240 specifically, the fap2 protein of F. nucleatum interacted with TIGIT receptors of NK and T
241 cells [61]. Ultimately, this interaction reduces NK cell cytotoxicity, preventing the effective
242 killing of tumorous cells. Furthermore, F. nucleatum cells have shown an affinity to
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243 intestinal epithelial cells, with FadA and Fap2 proteins promoting adhesion to these cells
244 [62].
245
246 Although less well studied, another mechanism by which F. nucleatum facilitates CRC is
247 via the increased M2 polarisation of macrophages [63]. When comparing two groups of
248 mice, the density of M2 macrophages and the incidence and volume of intestinal tumours
249 were significantly higher in those infected with F. nucleatum than in the control group [63].
250
251 Sodium butyrate metabolite… bacteria ratio…[64]
252 Application of research
253 Advances in high-throughput sequencing technology have allowed accurate sequencing of
254 metagenomic data at a lower cost than ever. With further research, there is a potential to
255 use metagenomic microbiome data from stool samples as a non-invasive biomarker for
256 CRC. One study used literature from PubMed was used to compile a database of CRC
257 associated microbes from metagenomic studies, which were classified at three different
258 levels based on statistical significance [65]. Databases such as these can identify potential
259 CRC biomarkers in patients. However, due to conflicting information from different studies,
260 further work is required to produce replicable results. A recent study found significant
261 differences in metagenomic microbiota data from patients with early-stage CAC than CRC
262 patients [66]. This suggests that data from faecal samples could be used to diagnose and
263 treat CRC earlier. This would be revolutionary as the survival rate for stage I cancer is
264 92%, whilst late-stage metastatic cancer yields only a 12% survival rate [2]. Additionally,
265 only an estimated 39% of cases are diagnosed at stage I [67].
266
267 Immunotherapy is a biological cancer therapy that has succeeded in treating several
268 different cancers, particularly melanoma [68]. More recently, immunotherapy has shown
269 efficacy in patients with metastatic CRC; however, treatment results vary massively
270 amongst patients [68]. One factor influencing immunotherapy success is the composition
271 of patients’ gut microbiomes [69].
272 Cancer patients will have often undergone numerous highly toxic treatments such as
273 chemotherapy which dramatically alter the composition of their microbiota. Thus,
274 Applications of histone modification
275
276 Finally, information about the gut microbiome will be influential in preventing CRC…
277
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278 Limitations of Methods
279 Metagenomic data from the human microbiome has provided us with a massively
280 improved understanding of the interactions between microbes and somatic cells.
281 Nevertheless, it remains challenging to elucidate the exact relationships of microbes
282 concerning the development of CRC. This is partly due to conflicting literature; however, it
283 is primarily due to difficulty linking epidemiological studies to etiological ones [check makes
284 sense]… CRC etiological studies tend to either be based on animal, usually mice, models
285 or in vitro experiments using cultured human adenocarcinoma cells.
286 Although the lab mouse (Mus musculus) is an invaluable in vivo model in biomedical
287 research, there are limitations to its use in CRC research.
288 When diagnosed, circa one-third of humans have metastasised CRC. Comparatively,
289 carcinogen-induced mouse models rarely show the development of metastases [70]. It is
290 also difficult to directly compare the microbiomes of humans and mice, as research into
291 human microbiomes has been far more extensive. Over 1500 species have been isolated
292 from the human GI tract compared to just circa 100 species from mice [71]. Furthermore,
293 the genetics of microbes found in either species differed dramatically, with only 4% of
294 genes demonstrating an identity >95% and coverage >90% when aligned [71].
295
296 In vitro studies using adenocarcinoma cell lines provide insight into direct reactions of
297 cancerous cells to treatments in the laboratory. This cost-effective method provides highly
298 reproducible results as labs across the globe can study the same cells [73]. However,
299 genetic and phenotypic drift can occur within these cell lines as mutations form, and those
300 adapted best to the lab environment are selected for. This means that cells may become
301 increasingly different from their parental origin over multiple generations [73]. Additionally,
302 cell lines are primarily 2D and therefore not necessarily representative of cancerous
303 growths in vivo. One solution is the growth of multicellular tumour spheroids (MCTS);
304 these allow for more relevant observations of tumour growth and proliferation, immunes
305 interactions and angiogenesis [73]. Furthermore, these cells' protein and gene expression
306 profiles are more similar to those in in vivo trials than in 2D cultures [73]. However, the
307 downside to this technique is that it is currently relatively expensive, and results are more
308 challenging to reproduce than in 2D cell lines.
309
310
311 Conclusion

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312 With rapid advances in technology, our understanding of the human microbiota has grown
313 enormously. Current epidemiological research indicates an association between
314 microbiota dysbiosis and CRC risk, with microbes exhibiting antagonistic roles in CRC
315 development. Although most mechanisms are yet to be well documented, numerous
316 studies indicate that microbiota, particularly in the gut, play a significant role in CRC
317 pathogenesis. Microbes are hypothesised to influence CRC in three major ways. Firstly, by
318 exhibiting tumour suppressing qualities, either directly or via the reduction in potency of
319 CRC cofactors such as IBD. Secondly, via pro-carcinogenic routes, bacteria can induce
320 genomic instability resulting in oncogenesis and can accelerate tumour development by
321 enhancing processes such as angiogenesis. Lastly, the interaction between the
322 microbiome and the immune system can play a significant role in an individual’s ability to
323 detect and kill cancerous cells.
324
325 Although contradictions in the surrounding literature currently exist, with more clinical and
326 pre-clinical trials, the microbiota has the potential to revolutionise the prevention, detection,
327 and treatment of CRC.
328
329 1. Sung, H., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality
330 Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians, 2021. 71(3): p. 209-
331 249.
332 2. Rawla, P., T. Sunkara, and A. Barsouk, Epidemiology of colorectal cancer: incidence, mortality,
333 survival, and risk factors. Gastroenterology Review, 2019. 14(2): p. 89-103.
334 3. Arnold, M., et al., Global patterns and trends in colorectal cancer incidence and mortality. Gut,
335 2017. 66(4): p. 683-691.
336 4. Lynch, H.T. and A. De La Chapelle, Hereditary Colorectal Cancer. New England Journal of Medicine,
337 2003. 348(10): p. 919-932.
338 5. Huybrechts, I., et al., The Human Microbiome in Relation to Cancer Risk: A Systematic Review of
339 Epidemiologic Studies. Cancer Epidemiology Biomarkers & Prevention, 2020. 29(10): p. 1856-1868.
340 6. Ursell, L.K., et al., Defining the human microbiome. Nutrition Reviews, 2012. 70: p. S38-S44.
341 7. Collins Francis, S., M. Morgan, and A. Patrinos, The Human Genome Project: Lessons from Large-
342 Scale Biology. Science, 2003. 300(5617): p. 286-290.
343 8. Turnbaugh, P.J., et al., The Human Microbiome Project. Nature, 2007. 449(7164): p. 804-810.
344 9. Yang, X., et al., More than 9,000,000 Unique Genes in Human Gut Bacterial Community: Estimating
345 Gene Numbers Inside a Human Body. PLoS ONE, 2009. 4(6): p. e6074.
346 10. Cheng, Y., Z. Ling, and L. Li, The Intestinal Microbiota and Colorectal Cancer. Frontiers in
347 Immunology, 2020. 11.
348 11. Martinez, K.B., V. Leone, and E.B. Chang, Western diets, gut dysbiosis, and metabolic diseases: Are
349 they linked? Gut microbes, 2017. 8(2): p. 130-142.
350 12. Veettil, S.K., et al., Role of Diet in Colorectal Cancer Incidence. JAMA Network Open, 2021. 4(2): p.
351 e2037341.
352 13. Benninghoff, A.D., et al., Consumption of the Total Western Diet Promotes Colitis and Inflammation-
353 Associated Colorectal Cancer in Mice. Nutrients, 2020. 12(2): p. 544.
354 14. David, L.A., et al., Diet rapidly and reproducibly alters the human gut microbiome. Nature, 2014.
355 505(7484): p. 559-563.
10
356 15. Stojanov, S., A. Berlec, and B. Štrukelj, The Influence of Probiotics on the Firmicutes/Bacteroidetes
357 Ratio in the Treatment of Obesity and Inflammatory Bowel disease. Microorganisms, 2020. 8(11): p.
358 1715.
359 16. Magne, F., et al., The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese
360 Patients? Nutrients, 2020. 12(5): p. 1474.
361 17. Turnbaugh, P.J., et al., An obesity-associated gut microbiome with increased capacity for energy
362 harvest. Nature, 2006. 444(7122): p. 1027-1031.
363 18. Gui, X., Z. Yang, and M.D. Li, Effect of Cigarette Smoke on Gut Microbiota: State of Knowledge.
364 Frontiers in Physiology, 2021. 12.
365 19. Qamar, N., et al., Meta-analysis of alcohol induced gut dysbiosis and the resulting behavioral
366 impact. Behavioural Brain Research, 2019. 376: p. 112196.
367 20. Tomoda, K., et al., Cigarette smoke decreases organic acids levels and population of bifidobacterium
368 in the caecum of rats. The Journal of toxicological sciences, 2011. 36(3): p. 261-266.
369 21. Mutlu, E.A., et al., Colonic microbiome is altered in alcoholism. American journal of physiology.
370 Gastrointestinal and liver physiology, 2012. 302(9): p. G966-G978.
371 22. Elvers, K.T., et al., Antibiotic-induced changes in the human gut microbiota for the most commonly
372 prescribed antibiotics in primary care in the UK: a systematic review. BMJ Open, 2020. 10(9): p.
373 e035677.
374 23. Zhang, S. and D.-C. Chen, Facing a new challenge: the adverse effects of antibiotics on gut
375 microbiota and host immunity. Chinese medical journal, 2019. 132(10): p. 1135-1138.
376 24. Xu, C., et al., Antibiotics-induced gut microbiota dysbiosis promotes tumor initiation via affecting
377 APC-Th1 development in mice. Biochemical and Biophysical Research Communications, 2017.
378 488(2): p. 418-424.
379 25. Dik, V.K., et al., Frequent Use of Antibiotics Is Associated with Colorectal Cancer Risk: Results of a
380 Nested Case–Control Study. Digestive Diseases and Sciences, 2016. 61(1): p. 255-264.
381 26. Nogal, A., A.M. Valdes, and C. Menni, The role of short-chain fatty acids in the interplay between
382 gut microbiota and diet in cardio-metabolic health. Gut Microbes, 2021. 13(1): p. 1-24.
383 27. Baena, R. and P. Salinas, Diet and colorectal cancer. Maturitas, 2015. 80(3): p. 258-264.
384 28. den Besten, G., et al., The role of short-chain fatty acids in the interplay between diet, gut
385 microbiota, and host energy metabolism. Journal of lipid research, 2013. 54(9): p. 2325-2340.
386 29. Parada Venegas, D., et al., Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune
387 Regulation and Its Relevance for Inflammatory Bowel Diseases. Frontiers in Immunology, 2019. 10.
388 30. Louis, P. and H.J. Flint, Formation of propionate and butyrate by the human colonic microbiota.
389 Environmental Microbiology, 2017. 19(1): p. 29-41.
390 31. Silva, Y.P., A. Bernardi, and R.L. Frozza, The Role of Short-Chain Fatty Acids From Gut Microbiota in
391 Gut-Brain Communication. Frontiers in Endocrinology, 2020. 11.
392 32. Wu, X., et al., Effects of the intestinal microbial metabolite butyrate on the development of
393 colorectal cancer. Journal of Cancer, 2018. 9(14): p. 2510-2517.
394 33. Hinnebusch, B.F., et al., The Effects of Short-Chain Fatty Acids on Human Colon Cancer Cell
395 Phenotype Are Associated with Histone Hyperacetylation. The Journal of Nutrition, 2002. 132(5): p.
396 1012-1017.
397 34. Donohoe, D.R., et al., A Gnotobiotic Mouse Model Demonstrates That Dietary Fiber Protects against
398 Colorectal Tumorigenesis in a Microbiota- and Butyrate-Dependent Manner. Cancer Discovery,
399 2014. 4(12): p. 1387-1397.
400 35. Yuille, S., et al., Human gut bacteria as potent class I histone deacetylase inhibitors in vitro through
401 production of butyric acid and valeric acid. PLOS ONE, 2018. 13(7): p. e0201073.
402 36. Gräff, J. and L.-H. Tsai, Histone acetylation: molecular mnemonics on the chromatin. Nature Reviews
403 Neuroscience, 2013. 14(2): p. 97-111.
404 37. Qin, J., et al., Histone Modifications and their Role in Colorectal Cancer (Review). Pathology
405 oncology research : POR, 2020. 26(4): p. 2023-2033.
406 38. Ellis, L., H. Hammers, and R. Pili, Targeting tumor angiogenesis with histone deacetylase inhibitors.
407 Cancer Letters, 2009. 280(2): p. 145-153.

11
408 39. Kim, M.S., et al., Histone deacetylases induce angiogenesis by negative regulation of tumor
409 suppressor genes. Nature Medicine, 2001. 7(4): p. 437-443.
410 40. Wagner, J.M., et al., Histone deacetylase (HDAC) inhibitors in recent clinical trials for cancer
411 therapy. Clinical Epigenetics, 2010. 1(3-4): p. 117-136.
412 41. Jahani-Sherafat, S., et al., Role of gut microbiota in the pathogenesis of colorectal cancer; a review
413 article. Gastroenterology and hepatology from bed to bench, 2018. 11(2): p. 101-109.
414 42. Ulger Toprak, N., et al., A possible role of Bacteroides fragilis enterotoxin in the aetiology of
415 colorectal cancer. Clinical Microbiology and Infection, 2006. 12(8): p. 782-786.
416 43. Viljoen, K.S., et al., Quantitative Profiling of Colorectal Cancer-Associated Bacteria Reveals
417 Associations between Fusobacterium spp., Enterotoxigenic Bacteroides fragilis (ETBF) and
418 Clinicopathological Features of Colorectal Cancer. PLOS ONE, 2015. 10(3): p. e0119462.
419 44. Tenaillon, O., et al., The population genetics of commensal Escherichia coli. Nat Rev Microbiol,
420 2010. 8(3): p. 207-17.
421 45. Mueller, N.T., et al., The infant microbiome development: mom matters. Trends in Molecular
422 Medicine, 2015. 21(2): p. 109-117.
423 46. Iyadorai, T., et al., Prevalence and association of pks+ Escherichia coli with colorectal cancer in
424 patients at the University Malaya Medical Centre, Malaysia. PLOS ONE, 2020. 15(1): p. e0228217.
425 47. Murray, P.R.B.E.J., Manual of clinical microbiology. 2007, Washington, D.C.: ASM Press.
426 48. De Almeida, C.V., A. Taddei, and A. Amedei, The controversial role of Enterococcus faecalis in
427 colorectal cancer. Therapeutic Advances in Gastroenterology, 2018. 11: p. 175628481878360.
428 49. Balamurugan, R., et al., Real-time polymerase chain reaction quantification of specific butyrate-
429 producing bacteria, Desulfovibrio and Enterococcus faecalis in the feces of patients with colorectal
430 cancer. (1440-1746 (Electronic)).
431 50. Cougnoux, A., et al., Bacterial genotoxin colibactin promotes colon tumour growth by inducing a
432 senescence-associated secretory phenotype. Gut, 2014. 63(12): p. 1932-42.
433 51. Bossuet-Greif, N., et al., The Colibactin Genotoxin Generates DNA Interstrand Crosslinks in Infected
434 Cells. mBio, 2018. 9(2).
435 52. D’Andrea, A.D., 4 - DNA Repair Pathways and Human Cancer, in The Molecular Basis of Cancer
436 (Fourth Edition), J. Mendelsohn, et al., Editors. 2015, W.B. Saunders: Philadelphia. p. 47-66.e2.
437 53. Huang, Y. and L. Li, DNA crosslinking damage and cancer - a tale of friend and foe. Translational
438 cancer research, 2013. 2(3): p. 144-154.
439 54. Tomkovich, S. and C. Jobin, Microbial networking in cancer: when two toxins collide. British Journal
440 of Cancer, 2018. 118(11): p. 1407-1409.
441 55. Wu, S., et al., The
442 <i>Bacteroides fragilis</i>
443 Toxin Binds to a Specific Intestinal Epithelial Cell Receptor. Infection and Immunity, 2006. 74(9): p.
444 5382-5390.
445 56. Chung, L., et al., Bacteroides fragilis Toxin Coordinates a Pro-carcinogenic Inflammatory Cascade via
446 Targeting of Colonic Epithelial Cells. Cell Host & Microbe, 2018. 23(2): p. 203-214.e5.
447 57. Butt, J., et al., Antibody Responses to Fusobacterium nucleatum Proteins in Prediagnostic Blood
448 Samples are not Associated with Risk of Developing Colorectal Cancer. Cancer Epidemiology
449 Biomarkers & Prevention, 2019. 28(9): p. 1552-1555.
450 58. Lo, C.-H., et al., Prediagnostic Antibody Responses to Fusobacterium nucleatum Proteins Are Not
451 Associated with Risk of Colorectal Cancer in a Large U.S. Consortium. Cancer Epidemiology
452 Biomarkers & Prevention, 2021. 30(6): p. 1279-1282.
453 59. Huycke, M.M., V. Abrams, and D.R. Moore, Enterococcus faecalis produces extracellular superoxide
454 and hydrogen peroxide that damages colonic epithelial cell DNA. Carcinogenesis, 2002. 23(3): p.
455 529-536.
456 60. Hanahan, D. and A. Weinberg, Robert, Hallmarks of Cancer: The Next Generation. Cell, 2011.
457 144(5): p. 646-674.
458 61. Gur, C., et al., Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory
459 Receptor TIGIT Protects Tumors from Immune Cell Attack. Immunity, 2015. 42(2): p. 344-355.

12
460 62. Sun, C.-H., et al., The role of Fusobacterium nucleatum in colorectal cancer: from carcinogenesis to
461 clinical management. Chronic diseases and translational medicine, 2019. 5(3): p. 178-187.
462 63. Chen, T., et al., Fusobacterium nucleatum promotes M2 polarization of macrophages in the
463 microenvironment of colorectal tumours via a TLR4-dependent mechanism. Cancer Immunol
464 Immunother, 2018. 67(10): p. 1635-1646.
465 64. Ma, X., et al., Sodium butyrate modulates gut microbiota and immune response in colorectal cancer
466 liver metastatic mice. Cell Biology and Toxicology, 2020. 36(5): p. 509-515.
467 65. Zhou, Z., et al., Human Gut Microbiome-Based Knowledgebase as a Biomarker Screening Tool to
468 Improve the Predicted Probability for Colorectal Cancer. Frontiers in Microbiology, 2020. 11.
469 66. Wu, Y., et al., Identification of microbial markers across populations in early detection of colorectal
470 cancer. Nature Communications, 2021. 12(1).
471 67. Moghimi-Dehkordi, B. and A. Safaee, An overview of colorectal cancer survival rates and prognosis
472 in Asia. World journal of gastrointestinal oncology, 2012. 4(4): p. 71-75.
473 68. Ganesh, K., et al., Immunotherapy in colorectal cancer: rationale, challenges and potential. Nature
474 Reviews Gastroenterology & Hepatology, 2019. 16(6): p. 361-375.
475 69. Gopalakrishnan, V., et al., The Influence of the Gut Microbiome on Cancer, Immunity, and Cancer
476 Immunotherapy. Cancer Cell, 2018. 33(4): p. 570-580.
477 70. Tong, Y., W. Yang, and H.P. Koeffler, Mouse models of colorectal cancer. Chinese Journal of Cancer,
478 2011: p. 450-462.
479 71. Hugenholtz, F. and W.M. De Vos, Mouse models for human intestinal microbiota research: a critical
480 evaluation. Cellular and Molecular Life Sciences, 2018. 75(1): p. 149-160.
481 72. Nguyen, T.L.A., et al., How informative is the mouse for human gut microbiota research? Disease
482 Models & Mechanisms, 2015. 8(1): p. 1-16.
483 73. Katt, M.E., et al., In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the
484 Right Platform. Frontiers in Bioengineering and Biotechnology, 2016. 4.
485

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