Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease and is the third most lethal cancer in the U.S. While peripancreatic inflammation is widely appreciated as paramount in the context of disease progression, the role of microbial dysbiosis remains under-recognized. Recent studies have revealed that a distinct pancreatic cancer microbiome drives tumor growth by promoting tolerogenic reprogramming of the immune system (Science 2017;357: 1156-1160; Cancer Discov 2018;8:403-416). However, the mycobiome (the fungal counterpart of the bacterial microbiome) has not been implicated in cancer.
In a recent study published in Nature on Oct. 2, we reported that certain fungi move from the gut into the pancreas and encourage pancreatic cancer growth (2019; doi: 10.1038/s41586-019-1608-2.). This is the first study demonstrating a strong evidence that perturbations in the mycobiome can trigger pancreatic oncogenesis, thereby revealing a previously unappreciated role for fungi in PDAC.
Abundant Fungal Microbiome
To test whether PDAC is associated with increased fungal colonization, we performed DNA sequencing from (stool and tissue samples of) mouse models of the disease and human patients with pancreatic cancer. The result demonstrated a ~3,000-fold increase in fungi in tumor tissue compared with normal pancreatic tissue.
To determine the source of the fungi residing in PDAC tissue, we used fluorescent protein-labelled fungi. Upon introducing these tagged fungi into the guts of mice, we were able to track their migration into the pancreas via the sphincter of Oddi in as early as 30 minutes.
Distinct Tumor Mycobiome
We then investigated the link between pancreatic cancer growth and fungi using mice that are engineered to express a KRAS mutation in their pancreatic progenitor cells. These mice develop a slowly progressive PDAC that recapitulates the inflammation and dense desmoplastic reaction seen in human patients with PDAC.
While early in life, no difference could be detected in the fecal mycobiome in mutant mice compared to wild-type mice that don't carry the KRAS mutation, considerable differences were observed at 30 weeks. This late time point is typically characterized by severe dysplasia and high-grade pancreatic intraepithelial neoplasia (PanIN) lesions. The largest population increase in both patient and mice tissues was seen in the genus Malassezia—a yeast naturally found on the skin and body surfaces and has been implicated in common diseases such as atopic dermatitis or dandruff. Recently, studies have linked inflammation caused by overgrowth of Malassezia to inflammatory bowel disease (Cell Host Microbe 2019;25:377-388 e376).
Antifungals Halt Tumor Growth
Next, we tested the effect of fungal ablation on PDAC growth. Our results demonstrated that ablation of the mycobiome using the wide-spectrum antifungal drug amphotericin B halted tumor progression in mice. Fungal ablation also strengthened the anti-cancer effect of a standard chemotherapy, gemcitabine, by 15-25 percent, improving the ability of the chemotherapy to shrink tumors.
To test whether fungal dysbiosis can drive tumor progression, we selectively repopulated mice with Malassezia globosa, Candida sp., S. cerevisiae, or Aspergillus sp. Of these fungal genera, only Malassezia was able to accelerate the growth of PDAC by (20% bigger average tumor weight).
Activation of the Complement Cascade
We next sought to elucidate how Malassezia promotes PDAC growth and tested the association between PDAC and the molecule mannose-binding lectin (MBL). MBL is an acute-phase protein produced in the liver. As a part of an evolutionarily conserved part of the innate immune system, MBL binds to carbohydrate molecules on the surfaces of bacterial or fungal microorganisms and activates the lectin pathway of the complement cascade. The complement cascade is made up of a large number of plasma proteins that react with one another to opsonize pathogens and induce an inflammatory response to help fight infection.
Importantly, we found that deletion of MBL or complement component 3 (C3) in mice resulted in delayed progression of PDAC, which is in line with recent studies that had identified the complement cascade as a potent oncogenic stimulus. Furthermore, Malassezia-associated tumor progression was lost in mice lacking MBL or C3. We therefore hypothesized that fungal pathogens drive PDAC progression through MBL, which in turn activates the C3 complement cascade. Accordingly, administration of recombinant C3a, which is generated as a result of complement activation, was sufficient to accelerate the growth of tumors in mice. Finally, we interrogated The Cancer Genome Atlas to evaluate the role of MBL or C3 as possible prognostic biomarkers in humans. Both MBL and C3 were associated with poor survival in PDAC.
Interpretation, Next Steps
Our results show that in PDAC, Malassezia promotes tumor growth by augmenting pancreatic inflammation through activation of the complement cascade. Our findings therefore reveal a previously unknown mechanism of tumor progression. Since fungi and bacteria coexist in the gut, future studies are needed to further elucidate these microbial networks and how alterations in one community affect the other.
Another unanswered question is whether fungal signatures can be used as biomarkers of disease progression or outcome. However, these findings provide important context and new hope around the therapeutic potential for drugs altering microbial communities by directly targeting specific populations. We also look forward to further exploring the role of Malassezia in other cancers.
BERK AYKUT, MD, is a Postdoctoral Fellow, and GEORGE MILLER, MD, is Director of the S. Arthur Localio Laboratory in the Department of Surgery at NYU School of Medicine.
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