Accumulating evidence indicates that the gut microbiome plays a key role in antitumor immunity and the effectiveness of immune checkpoint inhibitor treatments, but the underlying mechanisms aren’t well understood. Studies by an international team of 40 researchers have now shed new light on the relationship, and identified a causal link between the gut microbiome and the immune system’s ability to fight cancer.

Led by researchers at Sanford Burnham Prebys Medical Discovery Institute, the studies identified a mix of 11 bacterial strains that activated the immune system and slowed melanoma growth in mice. The investigations in mouse models indicated a role for the unfolded protein response (UPR) cellular signalling pathway in the ability to hold back cancer growth, while an analysis of human data also identified reduced UPR in melanoma patients who responded to immune checkpoint therapy.

“Our study establishes a formal link between the microbiome and antitumor immunity and points to the role of the UPR in this process, answering a long-sought question for the field,” commented Ze’ev Ronai, PhD., a professor at Sanford Burnham Prebys’ NCI-designated Cancer Center, who is senior author of the team’s published paper in Nature Communications, “These results also identify a collection of bacterial strains that could turn on antitumor immunity and biomarkers that could be used to stratify people with melanoma for treatment with select checkpoint inhibitors.” The researchers report on their findings in a paper titled, “Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5–/– mice.”

The effectiveness of immune checkpoint inhibitors against different types of cancer is variable, and while such treatments have improved patient survival rates, a significant proportion of patients are resistant to such treatments, the authors explained. “As such a more detailed understanding of the molecular mechanisms underlying selectivity of the tumor response and control of immune checkpoint components is urgently needed.”

Interestingly studies have indicated that changes to the gut microbiome can impact on antitumor immunity in both mice and humans, but the mechanisms underlying these observations aren’t well understood. “Notably, the identification of host genes/pathways that contribute to the regulation of the host microbiota and antitumor immune is expected to be essential for the development of advanced therapeutic strategies, the team wrote.

Ze’ev Ronai, PhD, senior author of the study and a professor at Sanford Burnham Prebys’ NCI-designated Cancer Center. [Sanford Burnham Prebys]
Work at Ronai’s lab has been focused on investigating how cancer responds to stress and becomes resistant to treatment, and their work involves a genetically engineered mouse that lacks the gen for RING finger protein 5 (RNF5), a ubiquitin ligase that helps to remove inappropriately folded or damaged proteins. These Rnf5–/ mice don’t show any outward signs of the effects of gene knockout “We call them the ‘boring mice’ because they don’t have a notable phenotype,” Ronai commented.

RNF5 has also been implicated in the mechanisms that control T-cell priming and immune checkpoint efficiency, so the team investigated whether there was a link between RNF5 knockout on immune checkpoint control and tumor growth.

Initial results demonstrated that melanoma tumors grew more slowly and developed into smaller tumors in Rnf5–/ mice than in normal wild-type (WT) mice, and the knockout animals survived for longer. Further investigation showed that the immune system was involved in the antitumor response in the Rnf5–/ mice, with “marked differences in key immune regulatory networks associated with T, DC [dendritic cell], natural killer (NK), and macrophage function.” Mapping immune components involved also indicated a key role for Toll-like receptors (TLRs). Tests in IECs from the RNF5-deficient animals stimulated DCs and T cells, while there was enhanced activity of DCs in the gut of the knockout animals. “… these data establish that RNF5 plays an important role in control of intestinal DC recruitment and, consistent with previous reports,” the recruitment of DCs underlies T-cell activation and antitumor immunity,” Ronai and colleagues wrote.

The team also found that reduced UPR was a common feature in immune and intestinal epithelial cells, and was sufficient for immune cell activation, with studies indicating that altered UPR in Rnf5–/mice was enough to induce changes in both the gut microbiome and immune response. Interestingly, an analysis of human melanoma samples identified reduced UPR in patients that responded to immunotherapy, and pointed to the UPR components sXBP1, ATF4 and BiP as potential markers of responsiveness.

Notably, melanoma inhibition in the knockout animals required an intact immune system and gut microbiome. Antitumor immunity and tumor inhibition was abolished in Rnf5–/ mice that were either treated with antibiotics, or housed with wildtype littermates, which indicated the importance of the gut microbiome.

Further microbiome and computational analyses led to the identification of 11 bacterial strains that were enriched in the guts of the RNF5-deficient mice. Transfer of these microbial strains to otherwise normal, germ-free mice that lacked an intestinal microbiome induced antitumor immune responses and slowed tumor growth. “… these findings establish the critical role of the select microbiota commensals in the activation of antitumor immunity, resulting in tumor growth inhibition,” the team noted. “

“Our data demonstrate that altered UPR signalling coincides with alteration of gut microbiota and activation of antitumor immunity,” the authors stated. They suggest that cooperation and probable cross-talk between these two mechanisms enables efficient tumor growth inhibition, as observed in the Rnf5–/ mice. “Consequently, defining the crosstalk between the UPR, gut microbiota, and immune checkpoint activity has significant potential expected to advance the development of novel therapeutic strategies,” they concluded.

The researchers aim to identify which bacterial metabolites are responsible for the observed tumor growth inhibition. It may then be possible to test whether these metabolites can directly enhance tumor immunity, or enable the development of prebiotics that could be administered by melanoma patients. “We believe this research applies to another fundamental question pertaining to the balance between antitumor immunity and autoimmunity,” said Ronai. “Because mice that lack RNF5 are also prone to developing gut inflammation – a side effect seen for certain immune checkpoint therapies – we can exploit this powerful model to study how we may tilt the balance between autoimmunity and antitumor immunity, which could help more people benefit from these remarkable therapies.”


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