Microbial Metabolomics: A Search for the Missing Link to Cancer

The microbes that inhabit the epithelial surfaces of our body, known collectively as the microbiota, vastly outnumber our own cells and genetic material. They have piqued scientific interest since their discovery in the mid-880s.

Thanks to technological advances, the past several decades have witnessed a blossoming appreciation of the varied composition of these microbial communities, their complex and dynamic relationship with the host, and the way they affect health and disease.

Investigators have focused on the gut microbiota, which make up the bulk of an individual’s bacterial ecosystem. It has become increasingly apparent that a complex interplay between the bacteria, the intestinal epithelium, and the immune system can be beneficial. Consequently, changes in the types of bacteria that make up the microbiota can disrupt this mutualistic relationship and have long been associated with the development of various disease states, including cancer.

Despite clear evidence of a link between the microbiota and cancer, it remains unclear whether this is causal or consequential. Understanding how the microbiome functionally influences disease is the critical missing link, and the application of metabolomics could be one of the keys to deciphering it.

Studies have revealed metabolic “chatter” between the host and their microbiota, with both producing a wide variety of metabolites that have a breadth of influence, including potentially driving carcinogenesis.

The Ecological Niche Within

Although the science is in its infancy, research that hones our understanding of microbial metabolomic profiles could allow earlier diagnosis of disease and the development of new prognostic biomarkers for cancer types that are desperately lacking in these areas. New therapeutic strategies aimed at manipulating the microbiota also offer a unique and potentially promising avenue of research.The first recorded observations of bacteria in the intestines of healthy individuals were made more than a century ago.¹ Since then there has been a growing appreciation of the extent of this intestinal ecosystem and its impact on human health. Ultimately, at the turn of the millennium, advances in genome sequencing and research initiatives, such as the National Institutes of Health’s Human Microbiome Project, propelled the microbiota into an exciting new era at the forefront of contemporary medicine.²

The microbial cells in our body outnumber our own cells by as many as 10 to 1 and have staggering genetic diversity in their microbiome. More than 1000 species fall into 2 major phyla: bacteroidetes and firmicutes. They line all epithelial barriers, including the skin, mouth, and vagina, but the majority are found in the gastrointestinal (GI) tract.^3-5

These bacteria begin to colonize humans before birth through the amniotic fluid, placenta, and umbilical cord blood. Transmission from the mother continues upon passage through the birth canal, and the microbiota mature and develop through the influence of breast milk or formula and the introduction of solid foods.⁶

The microbiota are often described as having a “commensal” relationship with the host, meaning that they cause neither harm nor benefit. However, as our understanding has evolved, it has become clear that they form vital components of the intestinal epithelium and contribute to the health of their human host by performing important physiological functions, including harvesting inaccessible nutrients from food, generating energy, and regulating the immune response.

Collectively, they form a dynamic ecosystem of bacterial communities throughout the body, the composition of which varies depending on many factors, such as the surrounding environmental conditions, drug usage, and—garnering the most mainstream attention—diet.⁷

Dysbiosis in Cancer

Our ability to determine the composition of the microbiota and how this changes under different conditions is directly attributable to the development of faster and cheaper technologies for the sequencing of the 16S ribosomal RNA gene. Encoding a subunit of the prokaryotic ribosome, this gene is universally expressed and highly conserved across bacterial species, except for a hypervariable region that is species specific.^8,9Some changes in the microbiota create an imbalance in their mutualistic relationship, known as dysbiosis, which can have negative consequences for the host. A growing body of evidence indicates a link between dysbiosis and the development of disease, including various types of cancer.⁷

The association between gut bacteria and cancer predates our understanding of the microbiota. Certain infectious bacteria, such as Heliobacter pylori, which cause one of the most prevalent infections in the world, can predispose patients to the development of gastric cancer. Both the presence of H pylori and attempts to treat it can lead to dysbiosis of the GI microbiota.^10-14

Since the majority of microbes are found within the GI tract, the largest number of studies to sequence the microbiome have been performed in GI malignancies, in particular colorectal cancers (CRCs). Differences in the composition of the microbiota can be observed in patients with CRC compared with healthy individuals and between the cancerous and adjacent healthy tissues.

A key feature of CRC is an overabundance of Bacteroides, Prevotella, and, in particular, Fusobacterium species. Other studies have found that certain bacterial species are underrepresented in stool and mucosal samples from patients with CRC, including Bifidobacterium longum, Clostridium clostridioforme, and Ruminococcus bromii. A recent study even found that the composition of the microbiota differs in patients with tumors from different CRCs (eg, distal and proximal tumors).^15-19

Bacterial dysbiosis in the gut and other epithelial surfaces has now been linked to an increasing number of cancer types (Figure).^17,18,20-42 An abundance of the bacteria responsible for poor oral health, Porphyromonas gingivalis and Aggregatibacter actinomycetecomitans, have been linked to an increased risk of pancreatic cancer, while Fusobacterium and its genus Leptotrichia were associated with a reduced risk.²³

Impact on Immunotherapy

P gingivalis and a second oral microbe, Tannerella forsythia, have also been linked to an increased risk of esophageal cancer and Streptococcus and Neisseria with less risk of this cancer type.²⁴ Meanwhile, an abundance of Corynebacterium and Kingella was associated with a reduced risk of head and neck cancer.⁴²Apart from their proposed role in carcinogenesis, the microbiota may also be important in mediating the effects of anticancer therapies. The presence of specific bacterial species has been linked to the efficacy and toxicity of chemotherapeutic drugs and treatment outcomes with immunotherapy.^43,44

A study examining the oral, gut, and fecal microbiota of 112 patients with melanoma who were treated with immune checkpoint inhibitors found that responders had a higher diversity and different composition of gut microbiota compared with those who failed to respond. Responders had an increased abundance of Faecalibacterium, Ruminococcaceae, and Clostridiales, while nonresponders had higher levels of Bacteroidales.⁴⁵

Furthermore, the use of broad-spectrum antibiotics up to a month before treatment with a checkpoint inhibitor has been shown to be associated with shorter progression-free and overall survival in patients with metastatic renal cell carcinoma.⁴⁶

Cause or Consequence?

In separate studies, Bifidobacterium spp, Bacteroides thetaiotaomicron, and nontoxigenic Bacteroides fragilis have been shown to improve the efficacy of immune checkpoint inhibitors in mouse models.^33,34 A phase II trial is currently ongoing that will further assess the link between the microbiota and response to immunotherapy (NCT02853318).Despite a better grasp of the specific microbiota changes that underlie dysbiosis in patients with cancer, there is not yet a definitive consensus on precisely if and how these changes drive carcinogenesis. It is not clear whether dysbiosis is a cause or a consequence of cancer. Do the altered bacteria directly drive the development of the tumor or do certain bacterial species merely thrive in the specific environmental conditions surrounding the tumor?

Indeed, it is entirely possible that both events are true, that there are certain “driver” bacteria that are directly involved in tumorigenesis and other “passenger” bacteria that exploit the new microenvironment established by the tumor.⁴⁷

Evidence suggests that the microbiota likely influence the initiation and progression of cancer by modulating the host processes that contribute to the cancer hallmarks, including inflammation, immune evasion, and DNA damage.⁴⁸

Although our understanding of the composition of the microbiota in health and disease has grown dramatically in the past several decades, there is a gap in our knowledge of their functional role. It will be necessary to bridge this gap in order to determine if changes in the microbiota have any causal impact on the etiology of disease.

Investigators are, therefore, increasingly turning to the use of proteomics, transcriptomics, and metabolomics to functionally annotate the microbiome. The latter has provoked particular interest because the metabolism of the microbiota and host are inextricably linked and the host is directly influenced by microbial metabolites.

The metabolites present within a host can be broadly grouped into those that the microbiota produces from dietary components, those that are produced by the host, and those that are synthesized de novo by the microbiota. Metabolites produced by the host can be biochemically modified by the microbiota.

Recent advances in analytical chemistry are helping to advance efforts to distinguish types of metabolites. These advances include nuclear magnetic resonance imaging, mass spectroscopy, and the development of computational models. A growing list of microbial metabolites has been identified.

Microbial Metabolic Pathways

Various strategies have been developed to elucidate their functions in health and disease. Using antibiotics (both broad-spectrum and those that are more targeted to specific groups of bacteria), germ-free mice, and dietary modifications, researchers can manipulate the abundance of certain bacteria that produce specific metabolites and examine the consequences to the host. The metabolite/phenotype relationship can be inferred by transplanting microbial communities into germ-free mice.^49,50The best-studied microbial metabolic pathways involve the production of short-chain fatty acids (SCFAs) and secondary bile acids (SBAs). SCFAs are produced when the gut microbiota metabolize dietary fibers that cannot be digested by the host. The most abundant are propionate and acetate, which are produced predominantly by the bacteroidetes, and butyrate, which is produced mainly by the firmicutes.

SCFAs are taken up by numerous cells in the body, where they function as substrates or signaling molecules. The majority of SCFAs, particularly butyrate, are taken up by the absorptive cells of the intestine where, in their substrate capacity, they serve as a primary energy supply. Some SCFAs pass into the liver and the peripheral blood stream.

They also function as both intracellular- and extracellular-signaling molecules, binding to G-protein—coupled receptors on host cells and activating signaling pathways involved in the regulation of lipid and glucose metabolism.⁵¹ They can also impact transcription by inhibiting epigenetic enzymes, such as histone deacetlyases.⁵² Finally, they have important roles in the regulation of the immune response and inflammation.

Under conditions of dysbiosis, the levels of SCFA-producing bacterial species, and thus of SCFAs, have been shown to be markedly reduced. SCFAs are thought to have a variety of anticancer effects, including their stimulation of the antitumor immune response. In addition, because of the altered metabolism of cancer cells, they cannot readily use butyrate as an energy supply; as a result, it accumulates in the cytoplasm and sensitizes the cancerous colonocytes to apoptosis.⁵³

Primary bile acids are produced in the liver and secreted into the gut to aid fat digestion. They can be metabolized by the microbiota to form SBAs, mainly deoxycholic and lithocholic acid. Contrary to SCFAs, SBAs are thought to promote carcinogenesis, due to their role in DNA damage, because they contribute to the production of oxygen radicals and reactive nitrogen species. Elevated levels of SBAs and reduced levels of SCFAs might explain the association between high-fat diets and increased risk of CRC and high-fiber diets and reduced cancer risk.^54,55

Investigative studies have now begun to include metabolomics analyses into study design. The incidence of and mortality rates from CRC are higher in the black population in America than any other ethnic population in the United States. A study revealed dramatically different microbiota composition in American blacks compared with native Africans. The former was dominated by Bacteroides and the latter by Prevotella. African Americans had increased expression of genes encoding for SBAs, in addition to higher levels of SBAs in fecal samples.⁵⁶

A study examined intestinal bacteria and metabolites present in fecal samples from patients with CRC (n = 11) compared with healthy patients (n = 10). Several bacterial genera, including butyrate-producing species, were underrepresented in the samples from patients with CRC. Butyrate concentrations were higher in the stool samples of healthy individuals, while those of CRC patients had higher levels of acetate. There were also higher levels of amino acids in CRC samples and of poly- and monounsaturated fatty acids and ursodeoxycholic acid in healthy patient samples.⁵⁷

More recently, paired tissue samples from colon tumors and adjacent normal and mucosal tissue were examined in 83 patients who underwent partial or total colectomy for CRC. Investigators found that tumors with dMMR, a common defect in the DNA repair pathway, had distinct microbial and metabolic profiles.⁵⁸

In addition to therapeutic applications, investigators are also looking at the microbiome as a potential source of biomarkers for cancer. Identifying bacterial species or their metabolic products that are indicators of cancer risk could allow earlier intervention, which could prove particularly impactful in cancers that are currently difficult to diagnose at a treatable stage, such as pancreatic cancer. They could also be used as prognostic biomarkers or to predict treatment outcomes.^59,60

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