Mycobiome: an underexplored kingdom in cancer

SUMMARY

The human microbiome, including bacteria, fungi, archaea, and viruses, is intimately linked to both health and disease. The relationship between bacteria and disease has received much attention and intensive investigation, while that of the fungal microbiome, also known as mycobiome, has lagged far behind bacteria. There is growing evidence showing mycobiome dysbiosis in cancer patients, and certain cancer-specific fungi may contribute to cancer progression by interacting with both host and bacteria. It was also demonstrated that the role of fungi-derived products in cancer should also not be underestimated. Therefore, investigating how fungal pathogenesis contributes to the onset and spread of cancer would yield crucial information for cancer diagnosis, prevention, and anti-cancer therapy.

INTRODUCTION

Globally, cancer is a significant socioeconomic issue, accounting for approximately one-sixth of global deaths and one-quarter of non-infectious deaths (1). An estimated one in five people will have cancer at some point in their lives, while about 1 in 9 males and 1 in 12 females will die from it (1). It is projected that there will be 35 million new cancer cases diagnosed in 2050, according to the demographic estimate. According to clinical statistics, about 13% of the approximately 2.2 million cancer cases that occurred in 2018 were linked to infections (2). Nevertheless, regarding the pathogenic mechanism of cancer, little is known.

The microbiome, which includes bacteria, fungi, viruses, and archaea, is intimately linked to human health (3, 4). Unlike the bacterial microbiome, the role of fungi in diverse diseases has long been neglected. The reasons may include several aspects. First off, it is important to note that there are 10¹¹ bacterial cells/g of feces, which is significantly higher compared to only 10⁵–10⁶ fungal cells. This remarkable contrast suggests that the bacterial population in feces vastly outnumbers the fungal population (5, 6). In other words, fungal genes are approximately less than 1% of the gut microbiome (7). In the TCGA primary tumors, the relative abundance of bacteria is as much as 24 times higher than that of fungi, according to Narunsky-Haziza et al. (8). This further supports the idea that fungi are hundreds of orders of magnitude less common than bacteria (7, 9). Secondly, when extracting DNA, the fungal DNA is more sensitive than bacterial DNA and is more influenced by the existing DNA preparation methods (10). Finally, the existing reference database of fungal genomes is incomplete (11), which also hinders the efforts to study the role of fungi in disease through culture-independent methods.

As a significant part of the microbiome, fungi are found on the surfaces of all human tissues and organ barriers (12). Fungal disorders can lead to diseases including fungal infections and even cancer. Opportunistic pathogenic fungi are prevalent in the environment where we live. An estimated 6.55 million individuals get potential fatal fungal infections annually, and 3.75 million people die from fungal diseases, which are thought to affect over 1 billion people annually (13, 14). The number of new cases is still on the rise (15) due to a number of reasons such as environmental changes and increased susceptible populations. In high-risk groups, the mortality rate after systemic fungal infections can be as high as 50% (16–18). Moreover, cancer patients with immune deficiency are also high-risk populations for fungal infections (19).

Recently, an increasing body of research indicates that fungi are a common presence within a variety of tumor types and may have an impact on the onset and spread of cancer (8, 20–28). The presence of cancer-specific intratumor fungi is associated with tumorigenesis in different body sites (8, 28, 29). The presence of Candida DNA in association with tumors holds promise as a potential biomarker for assessing the survival rates of cancer patients (28). Therefore, it is reasonable to assume that fungi are closely associated with cancer progression and prognosis. Therefore, this review summarizes the possible roles of fungi in cancer, with the aim of offering novel insights for the prevention and treatment of cancer, particularly those strategies targeting fungi.

MYCOBIOME AND CANCER

Different degrees of fungal dysbiosis are detected in a variety of cancer types (Fig. 1), with specific dysbiotic fungal species shown in Table 1. Imbalances in mycobiome may contribute to the advancement of cancer through a complex array of mechanisms, paving the way for groundbreaking therapeutic approaches and enhanced survival outcomes for patients. Herein, we explore the complex relationship between fungi and cancer, highlighting key findings and examining the potential mechanisms that may be involved.

Fig 1.

Schematic overview of cancer types with dysbiotic mycobiome. The composition of the fungal mycobiome has been found to be altered in various body sites associated with tumorigenesis.

TABLE 1.

A list of the fungal species implicated in various cancer types

Cancer type	Fungal dysbiosis	Ref.
Lung cancer	Malasseziomycetes, Saccharomycetes, Diothideomycetes, Sordariomycetes, Aspergillus, and Malassezia differ in patients and controls.	(8)
	Blastomyces spp. are abundant in lung tumors.	(28)
	Malassezia was more abundant in the patients, whereas Candida was more abundant in the healthy subjects.	(30)
	Enrichment of Alternaria arborescens was found in non-small cell lung cancer cancerous tissue.	(31)
Oral cancer	Candida spp. was detected in patients, with Candida albicans being the most common.	(32–34)
	It is noteworthy that three types of fungi that are particularly associated with OSCCa are Rhodotorula, Geotrichum, and Pneumocystis.	(35)
	Enrichment of C. albicans, Candida etchelsii, and a Hannaella luteola-like species existed in OSCC patients.	(36)
	In patients with OSCC, there is an observed increase in the presence of Prevotella intermedia, Porphyromonas endodontalis, Acremonium exuviarum, and Aspergillus fumigatus, while there is a concurrent decrease in the levels of Streptococcus salivarius subsp. salivarius, Scapharca broughtonii, Mortierella echinula, and Morchella septimelata.	(37)
Colorectal cancer	In CRC, the fungal genera Malassezia, Moniliophthora, Rhodotorula, Acremonium, Thielaviopsis, and Pisolithus were particularly abundant.	(20)
	Significant increases in Microbotryomycetes, Sordariomycetes, Microascaceae, Sordariales, Lasiosphaeriaceae, and Microascales and decreases in the abundance of Pleosporaceae and Alternaria were detected in patients.	(38)
	Colon cancer has the highest Ascomycota-to-Basidiomycota ratio (A:B ratio) compared to other types of cancer.	(8)
	In colon cancer, the burden of Candida tropicalis is significantly increased.	(26)
	Card9−/− mice had higher Saccharomycetes representation and lower occurrences of various Ascomycota members.	(27)
	C. albicans was significantly overrepresented among patients.	(39)
	Compared to the controls, the abundance of S. cerevisiae was 2.68 times lower in CRA patients and 3.94 times lower in CRC patients.	(40)
Head and neck squamous cell carcinoma	The tumor tissues had a much lower level of Glomeromycota than non-tumor tissues.	(41)
	The levels of C. albicans and Rothia mucilaginosa were different in the oral wash of HNSCC patients, whereas Schizophyllum commune was reduced.	(42)
	C. albicans was responsible for at least 96% of the fungal sequencing in the majority of HNSCC patients.	(43)
Cervical cancer	All tested ovarian cancer samples had detectable levels of Cladosporium, Pneumocystis, Acremonium, Cladophialophora, Malassezia, and Microsporidia Pleistophora.	(44)
Breast cancer	Malassezia spp. are abundant in breast tumors.	(28)
	A very strong average hybridization signal for Arthroderma was found in about 95% of ER patients.	(45)
	The healthy controls did not exhibit the fungal signatures of Ajellomyces, Alternaria, Cunninghamella, Epidermophyton, Filobasidiella, Rhizomucor, and Trichophyton that were found in one or more forms of breast cancer.	(46)
Gastric cancer	Candida DNA is enriched in tumors.	(28)
	Solicoccozyma was detected in greater quantity in the tumor group, but Pezizomycetes, Sordariales, Chaetomiaceae, and Rozellomycota were not as prevalent.	(47)
	The higher percentage of opportunistic fungi like Cutaneotrichosporon and Malassezia, as well as the higher ratio of Basidiomycota to Ascomycota, were observed in the patient group compared to the control group.	(48)
	Saliva and tongue coating samples from the GC showed reduced Saccharomyces cerevisiae and increased Malassezia globosa.	(49)
Melanoma	Among multiple cancer types, melanoma had the lowest A:B ratio due to the abundance of Malassezia.	(8)
	Patients with melanoma showed a noticeably greater fungal richness.	(50)
Ovarian cancer	In every ovarian cancer sample that was tested, indications of Pneumocystis, Acremonium, Cladophialophora, Malassezia, and Microsporidia Pleistophora were also found in large amounts. More than 95% of the ovarian cancer samples that were tested had signatures of Rhizomucor, Rhodotorula, Alternaria, and Geotrichum.	(44)
Prostate cancer	The most common fungal families found in prostate cancer include Microsporidia (12), yeasts (15%), zygomycetes (15%), and dermatophytes (31%).	(51)
	The three families that were most significantly different between PCa patients and controls were the Filobasidiales, Pyronemataceae, and Cryptococcus ater spp.	(52)
Bladder cancer	Increased abundance of Tremellales, Hypocreales, and Dothideales in bladder cancer patients compared to controls.	(53)
Liver cancer	Compared to individuals with liver cirrhosis, those with HCC had a significantly lower diversity of the gut mycobiome and a higher abundance of C. albicans.	(54)
Esophageal squamous cell carcinoma	C. albicans was found in patients.	(55, 56)
Pancreatic cancer	PDA tumor tissues exhibit a high enrichment of Malassezia spp.	(57)

^a CRC, colorectal cancer; OSCC, oral squamous cell carcinoma.

Lung cancer

Lung cancer is one of the major causes of death globally and has the highest incidence and mortality rate across all cancer types, according to the most recent cancer statistics (58). Lung cancer risks encompass both genetic factors and environmental influences, such as smoking and exposure to air pollution (59, 60). Numerous recent studies have found the presence of fungi in the tissues of lung cancer patients (8, 28, 31, 61). Higher fungal diversity was observed in patients with non-small cell lung cancer (31). About 2.6% of lung cancer patients will develop invasive aspergillosis (62). Others have suggested that the apparent progression of lung cancer may be due to an invasive aspergillosis infection (63). Recently, Liu et al. found the enrichment of Aspergillus sydowii in the tumor tissues of lung adenocarcinoma (LUAD) patients, isolated a live fungal strain of A. sydowii through culturomics, and demonstrated its tumor-promoting mechanism by using three lung cancer pre-clinical murine models (61). In particular, by promoting interleukin (IL)-1β signaling via the β-glucan/dectin-1/CARD9 pathway (61, 64), A. sydowii improves the recruitment and activation of myeloid-derived suppressor cells (MDSCs, regulators of the immune system) (65). This, in turn, inhibits cytotoxic T-lymphocyte cells and increases the accumulation of PD-1⁺ CD8⁺ T cells, creating an immunosuppressive environment that accelerates the growth of lung tumors (Fig. 2A). Additionally, immunosuppression and a poor prognosis for individuals with lung cancer were linked to the enrichment of A. sydowii (61). In addition, intestinal fungi can serve as non-invasive biomarkers for LUAD (66) and are also associated with recovery of lung function and motor capacity in lung cancer patients undergoing surgical treatment (67). Thus, more attention should be focused on the mechanistic insights into the functional importance of fungi in lung cancer.

Fig 2.

Illustration of possible mechanisms of fungi in the development of lung (A) and pancreatic (B) cancers. (A) Aspergillus sydowii promotes IL-1β secretion-mediated recruitment and activation of MDSCs via the β-glucan/dectin-1/CARD9 pathway. This leads to inhibition of CTL cell activity and aggregation of Tregs and PD-1⁺ CD8⁺ T cells, ultimately leading to lung cancer progression. (B) Intratumor fungi such as Alternaria and Malassezia can promote secretion of IL-33 by cancer cells, which in turn recruits and activates T_H2 and ILC2 cells. This ultimately promotes the secretion of pro-tumorigenic cytokines, including IL-4, IL-5, and IL-13. Moreover, it ultimately promotes tumor progression. ↓, decrease; ↑, increase; CARD9, caspase-recruitment domain 9; CTL, cytotoxic T lymphocyte; IL, interleukin; ILC, innate lymphoid cell; MDSC, myeloid-derived suppressor cell; PDAC, pancreatic ductal adenocarcinoma; Treg, T-regulatory cell; TAM, tumor-associated macrophage.

Breast cancer

Breast cancer is the most common cancer in females, affecting up to one in five of all cancer types in women and representing a high mortality rate (1). Existing evidence has shown microbiome dysbiosis in breast cancer patients. The bacteria in the tumor can promote the metastasis of breast cancer (68), and fungi were also observed in breast tumor tissues (8, 28). Using genome-wide transcriptome amplification and pan-pathogen microarray strategies, the researchers detected shared and specific viruses, bacteria, fungi, and parasites in each type of breast cancer (46). Interestingly, the fungal signatures are relatively unique (45, 46). Ajellomyces, Alternaria, Cunninghamella, Epidermophyton, Filobasidiella, Rhizomucor, and Trichophyton were not found in healthy controls, despite being identified in one or more kinds of breast cancer. Ahmadi et al. investigated the connection between fungi and breast cancer using four groups of mice including normal, tumor, candidiasis (candidiasis only), and tumor/candidiasis groups (69). The study revealed that the interferon gamma (IFN-γ):IL-4 ratio was significantly lower in both the tumor/candidiasis and candidiasis groups when compared to the uninfected control group. Levels of IL-10, transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α) were increased in the candidiasis group. Furthermore, in the tumor/candidiasis group, Candida infection boosted tumor growth and the quantity of Tregs in the tumor microenvironment (TME). These findings imply that fungi, such as Candida albicans, are associated with an increased risk of developing breast cancer and promote the growth of tumors, which merits more research.

Pancreatic cancer

Despite its relatively low incidence, pancreatic cancer has a high mortality rate, with only approximately 8.5% of patients surviving 5 years post-diagnosis. Pancreatic cancer was ranked 12th in incidence and 6th in mortality among all cancer types (1).

Numerous studies have demonstrated the fungal ecological disturbances in pancreatic cancer patients (57, 70, 71). The pancreatic duct is directly connected to the small intestine, providing a pathway for microorganisms to enter into the pancreas from the intestine (72). Alam et al. identified a potential mechanism for Malassezia spp. in the progression of the fungal mycobiome that drives IL-33 secretion and type 2 immunity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) (57). Specifically, tumor tissue-resident fungi promote the secretion of IL-33 by cancer cells, which in turn recruits T_H2 and ILC2 cells in the TME, accelerating the progression of pancreatic cancer (57) (Fig. 2B). The depletion of IL-33 or the administration of anti-fungal therapy leads to a decrease in T_H2 and ILC2 infiltration and enhances survival rates. This highlights the potential clinical utility in PDAC prevention by targeting fungi.

Colorectal cancer

Colorectal cancer (CRC), which includes bowel, colon, and rectal cancers, has the second-highest death rate and the third-highest incidence rate worldwide (1). The incidence of CRC is steadily rising in both high-income countries and among young people, with the incidence rising from 1% to 4% per year (73–75). A notable feature of CRC is its intimate relationship with the gut microbiota, which becomes dysbiotic throughout the development of cancer and actively participates in carcinogenesis, in addition to the significant impact of diet and lifestyle on the disease’s development (76, 77).

In general, it is believed that microbiota affects the distant or proximal tumor tissues indirectly through the immune system and metabolites, especially in CRC, which is physically in close proximity to the gut microbiota. It has been proven that dysregulation of mycobiome in the human gut, such as an increased ratio of Basidiomycota to Ascomycota, is observed in CRC patients (20). Meanwhile, the study revealed that Saccharomyces cerevisiae, which has anti-inflammatory properties (78), was found to be depleted in the gut of CRC patients. This suggests a potentially beneficial role for S. cerevisiae in the gut, which may contribute to the prevention and treatment of CRC (20). In addition, there exist abundant Candida spp. in CRC patients compared to healthy controls (26, 79). C. albicans may encourage the growth of CRC, indicating the presence of C. albicans-driven crosstalk between macrophages and innate lymphocytes in the intestinal environment (25). Glycolysis and the release of inflammatory IL-7 from macrophages in the lamina propria were activated by the increased burden of C. albicans caused by the depletion of the c-type lectin dectin-3 (Fig. 3A). IL-7 secretion stimulates the production of IL-22 by RORγt⁺ (group 3) innate lymphoid cells, which subsequently results in increased p-STAT3 levels in epithelial cells, promoting cancer development (25). In addition, C. albicans was linked not only to the prediction of advanced CRC and metastatic CRC but also with the influence of cell adhesion (28). Malignant cells may employ cell adhesion molecules to facilitate tumor growth (80). Furthermore, low fungal load was associated with higher CARD9 expression (26). Pro-inflammatory cytokines that are very specific to anti-fungal immune responses are released when CARD9 activates the NF-kB and mitogen-activated protein kinase (MAPK) pathways (81). It was found that fungal dysregulation increased the accumulation of MDSCs in tumor tissues, and the impaired bactericidal activity of macrophages resulted in increased fungal load dominated by Candida tropicalis (26). In tumor-bearing CARD9^−/− mice, there was a marked upregulation of S100A9, IL-6, IL-1β, and Cxcl1 expression, with their interplay potentially facilitating the aggregation and activation of MDSCs. Furthermore, in vitro studies revealed that C. tropicalis can trigger MDSC differentiation and augment their immunosuppressive functions (Fig. 3B). In addition, they found that C. tropicalis promoted CRC development through dectin-3-induced NLRP3 inflammatory vesicle activation in MDSCs (82) (Fig. 3D), in addition to promoting chemoresistance to oxaliplatin in CRC by producing lactic acid and inhibiting MLH1 expression (83). Malik et al. found that CARD9^−/− mice treated with AOM-DSS showed a higher tumor burden. The activation of the inflammasome in the colon is primarily triggered by intestinal commensal fungi, which can enhance the inflammasome activation and IL-18 maturation via the SYK-CARD9 axis. IL-18 was reported to aid in the restoration of the epithelial barrier and stimulate the production of interferon-γ by CD8⁺ T cells, thereby mitigating colitis and associated epithelial hyperplasia in the azoxymethane-dextran sodium sulfate mice model. Together, the intestinal commensal fungi-SYK-CARD9-IL-18 axis can provide protection against colon cancer (27) (Fig. 3C). These findings have expanded our understanding of the fungi-CRC relationship, although most experiments were conducted on mice models. Moreover, more realistic, detailed, and conclusive mechanistic studies are necessary to deepen our understanding of the pathological process and to aid in the advancement of clinical trials and applications.

Fig 3.

A depiction of various potential mechanisms through which fungi may facilitate the progression of colorectal cancer. (A) A higher load of Candida albicans causes macrophages to undergo glycolysis and secrete IL-7, which contributes to the development of CRC via generating IL-22 through ILC3. Fungal load and IL-22 frequency in tumor tissues have a positive correlation, indicating that this regulatory axis is closely linked to human illness. (B) The general composition of the gut microbiota changes, and the quantity of fungus, particularly Candida tropicalis, increases as a result of CARD9^−/− macrophages’ impaired fungicidal action. Intestinal MDSCs are more prevalent in CARD9^−/− mice, but CTL is less prevalent. (C) Intestinal fungal sensing activates the inflammasome through the CARD9-SYK signaling pathway and produces IL-18, which promotes the repair of the epithelial barrier and the T-cell response, thus inhibiting the development of inflammatory bowel disease and colon cancer. CARD9 or SYK deficiency alters the intestinal microecology, leading to impaired activation of the inflammasome and increasing the susceptibility to inflammatory bowel disease and colon cancer. (D) Candida tropicalis promotes transcription of NLRP, pro-caspase-1, and IL-1β genes by activating the JAK-STAT1 signaling pathway, and mtROS mediates C. tropicalis-induced activation of the NLRP3 inflammasome. Overall, Candida tropicalis induces NLRP3 inflammasome activation in MDSC to promote CRC progression. CARD9, caspase-recruitment domain 9; CTL, cytotoxic T lymphocyte; IL, interleukin; ILC, innate lymphoid cell; MDSC, myeloid-derived suppressor cell; mtROS, mitochondrial reactive oxygen species.

In addition, fungi can also interact with bacteria through physical or biochemical mechanisms (84, 85). For instance, bacteria and fungus together can form biofilms that worsen intestinal inflammation (86, 87). Hager et al. found that the relationship between bacteria and fungi was Candida specific and inhibited the emergence of germination when the probiotic filtrate was co-incubated with C. albicans (88). Thus, the role of this interaction in CRC progression cannot be ignored, which may provide new ideas for future anti-fungal and anti-cancer therapy (89).

Moreover, it was discovered that patulin (PAT), a toxin secreted by fungi such as Aspergillus spp. which often contaminate fruits, is able to change intestinal epithelial cells (90). Long-term low-dose PAT exposure increases cell proliferation, migration, and invasion, while in vivo evidence suggests that PAT-exposed Wistar rats develop colonic aberrant crypt foci, a marker for early CRC (90).

In conclusion, an increasing body of research has identified associations between fungi and CRC, confirming the complex mechanisms by which fungi promote cancer and their interactions with other microorganisms. Our comprehension of the complex interplay among fungi, gut microbiota, and CRC development will be significantly enhanced by research in this field.

Gastric cancer

Gastric cancer (GC), which accounted for nearly 1 million new cases in 2020, was ranked as the sixth largest malignant tumor in the world (1). The high-risk populations of GC include those who smoke, drink alcohol, have a history of stomach disorders, or have GC in the family tree of a first-degree relative (91).

The abundance of Pezizomycetes, Sordariales, Chaetomiaceae, and Rozellomycota was lower in tumor tissues compared to normal tissues (47). Moreover, Solicoccozyma is differentially enriched in tumor tissues and may be used as a fungal biomarker to differentiate between stage I and stage II and III with an area under the receiver-operating curve (AUROC) of 0.7061 (47). Additionally, using functional prediction, the expression of Solicoccozyma in tumor tissue was favorably correlated with the metabolic pathway connected to amino acids and carbohydrates (47). C. albicans, Fusicolla acetilerea, Arcopilus aureus, and Fusicolla aquaeductuum were enriched in GC, while Candida glabrata, Aspergillus montevidensis, Saitozyma podzolica, and Penicillium arenicola were considerably decreased (92). They found that Malassezia globosa was shown to be more abundant in samples of saliva and tongue coating obtained from patients with GC, while Saccharomyces cerevisiae was decreased. Moreover, Malassezia globosa can be used as a functional marker to detect GC (49). Furthermore, patients with GC have also been found to have Malassezia disorders (47, 93), and Malassezia is believed to be a gastric fungus associated with programmed death ligand 1 (PD-L1) expression and with overall survival in patients with GC (93), which are worth looking into for learning more about how this fungus and PD-L1 work.

In addition, it was found that Candida had a favorable correlation with Lactobacillus and negatively correlated with Helicobacter pylori. The upregulation of pro-inflammatory cytokines, especially IL-6, may contribute to the progression of cancer (94). Moreover, inflammation could promote the colonization of Candida, which in turn could maintain a pro-inflammatory environment by increasing its own inflammatory response (28). Consequently, the overall survival rate for cancer patients may be raised by preventing and treating Candida infections. While these studies indicate a potential correlation between specific fungi and GC, with diagnostic implications, there is currently insufficient evidence to establish causality. Therefore, ongoing attention and further research by scientists are necessary in the future.

Liver cancer

Liver cancer ranks sixth in incidence among all cancer types and third in all cancer deaths (1). According to the recent statistics, opportunistic fungal species including Malassezia and Candida are more prevalent in the gut microbiota of patients with hepatocellular carcinoma (HCC) than in healthy subjects and cirrhosis patients (95). Fungal diversity was also reduced in HCC and cirrhosis patients in contrast to healthy subjects. In addition, the abundance of C. albicans in patients with early HCC is lower than that in patients with advanced stages of HCC. Aflatoxins, which are produced by Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus, are produced in the liver by cytochrome P450 epoxidation to form genotoxins (e.g., aflatoxin B1 exo-8,9 epoxides). After being introduced into the DNA, these extremely electrophilic epoxides quickly alkylate N7-guanine residues (96). This can then lead to mistakes in DNA-adduct replication or repair, leading to liver cancer.

Moreover, Candida spp., especially C. albicans, are essential to the etiology and development of nearly all hepatobiliary disorders (6). It was found that C. albicans could affect the development of HCC (54). In contrast to individuals with cirrhosis, they discovered that patients with HCC had a higher quantity of C. albicans and a lower diversity of gut microbiota. Additionally, it was noted that oral gavage of C. albicans enhanced tumor weight and size in a mouse model, as well as the expression of nucleotide oligomerization domain-like receptor family pyrin domain containing 6 (NLRP6). However, colonization by C. albicans did not influence tumor growth in NLRP6-deficient mice. Thus, aberrant colonization by C. albicans could promote NLRP6-dependent HCC progression (Fig. 4A).

Fig 4.

Potential mechanisms of fungi in the development of hepatocellular carcinoma (A), esophageal squamous cell carcinoma (B), and oral cancer (C). (A) Increased intestinal Candida albicans load promotes hepatocellular carcinoma by upregulating NLRP6. (B) The IKK-α^KA/KA mouse model suggests that autoreactive CD4 T cells contribute to esophageal squamous cell carcinoma by promoting inflammation through fungal infection and tissue injury. (C) One mechanism suggests that Candida albicans promotes infiltration of TAMs via the IL-17A/IL-17RA pathway and that attracted TAMs polarize into M2-like macrophages that highly express PD-L1 and GAL-9 to influence tumor progression. Another mechanism is thought to be that Candida can influence cancer progression by producing alcohol dehydrogenase, which converts alcohol to acetaldehyde. ↓, decrease; ↑, increase; EGFR, epidermal growth factor receptor; GAL-9, galectin-9; IL, interleukin; NLRP6, nucleotide oligomerization domain-like receptor family pyrin domain containing 6; PD-L1, programmed death ligand 1; TAM, tumor-associated macrophage.

Cervical cancer

The global women’s health is impacted by cervical cancer, a malignant tumor with a high morbidity and death rate (1). Candida is a symbiotic fungus found in the healthy vagina, and it has a high prevalence in patients with vulvovaginal candidiasis (VVC) (97). Yeast forms of Candida are mostly present in healthy women, while hyphal forms of Candida can be isolated from patients with severe VVC. This result supports the correlation between the morphology of Candida and pathogenicity (98). Candida’s morphological change is significantly influenced by the interaction between fungi and bacteria. For example, the growth of Candida and its transformation from the non-toxic yeast form to the toxic hyphal form are generally inhibited by low pH and bactericidal chemicals released by bacteria (99, 100). According to a different study, one of the key constituents of the vaginal microbiome can enhance the local immune response of vaginal epithelial cells and reduce the pathogenicity of C. albicans. This is achieved by increasing the expression of IL-2, IL-6, and IL-17 while simultaneously decreasing the expression of IL-8 (101). These findings imply that Lactobacillus and Candida interactions may be crucial for preserving the balance of the vaginal microbiota and that disturbance of Candida may result in infection (102).

Esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a major disease affecting human health and quality of life, ranking 11th in incidence and accounting for 4.6% of all cases of death (1). Fungal dysbiosis, ulcers, and esophagitis are common in ESCC patients, suggesting a potential correlation between fungi and ESCC development (55). The relationship between ESCC and fungal infection was investigated by using the kin-dead IKk-α knockin (IKk-α^KA/KA) mice model. The mice developed esophageal squamous cell cancer, exhibiting autoinflammation, a persistent fungal infection, and reduced central tolerance. The damaged thymus microenvironment produces autoreactive T cells, which allow fungal infection and trigger tissue damage and inflammation. Moreover, the researchers detected fungi in human ESCC tissue specimens but not in normal human tissue, and a statistically significant difference was observed between fungal infections and human ESCC. In addition, epidermal growth factor receptor (EGFR) is the target of IKKα, an oncogene that can drive the development of squamous cell carcinoma (103). Inhibiting inflammation or IKKα’s target EGFR activity reduces fungal load. Anti-fungal therapy or auto-reactive CD4 T-cell depletion can promote ESCC treatment (Fig. 4B). The findings suggest that deficiencies in immune and epithelial cells work together to promote chronic fungal infections and the development of ESCC.

Oral cancer

The oral microbiota, which is the second most abundant microbiome in the human body, is present in the oral cavity (104). Oral cancer (OC) is also a serious threat to people’s health and a serious public health problem (105). Most OCs are diagnosed after they have progressed significantly, so they have a high mortality rate (106). Several studies have found that patients with oral Candida infections have an increased risk of OC compared to the control (32, 107). About 74% of OC patients in a case-control study had Candida, with C. albicans emerging as the predominant strain (84%) (33). One possible mechanism suggests that Candida can produce ethanol dehydrogenase, which converts alcohol to acetaldehyde (ACH), impacting cancer development (108, 109) (Fig. 4C). ACH consumed with alcoholic beverages has been identified as a Group I human carcinogen (110). Excessive alcohol intake can increase the production of ACH in saliva (111). Non-drinkers may develop OC as a result of oral metabolism of glucose into ACH by C. albicans (112). Individuals with poor oral health can detect twice as much ACH as those with good oral hygiene (109). There was a strong correlation between the amount of ACH in saliva and the prevalence of Candida (113). Among these, it has been demonstrated that OC is associated with the virulence characteristics of C. albicans and the production of ACH (34). Certain host cellular proteins may interact with alcohol dehydrogenase, resulting in an immunological reaction, pro-inflammatory cytokines, and other mediators that ultimately cause inflammation and DNA damage (109). By generating carcinogenic nitrosamines, invasion of Candida hyphae may possibly be a factor in oral leukoplakia’s malignant development (114, 115). Proto-oncogenes that cause malignant transformation in the host may be activated by nitrosamine complexes (116).

Candida spp., Malassezia spp., Saccharomyces spp., and Aspergillus spp. exhibited a significant prevalence in individuals with oral squamous cell carcinoma (OSCC) compared to those without OSCC (117), and there is in vitro and in vivo evidence that C. albicans is involved in malignant promotion and progression of OC (118). From the standpoint of TME, Zhi-Min Yan’s group identified a possible mechanism by which C. albicans encourages OC (119). Specifically, C. albicans can promote tumor-associated macrophage (TAM) infiltration through the IL-17A/IL-17RA pathway, while the TAMs are polarized into M2-like macrophages that express PD-L1 and galectin-9 at high levels. Conversely, in mice infected with C. albicans, both the neutralization of IL-17A and the depletion of macrophages were observed to reduce the number of TAMs and the size of the tumor (Fig. 4C).

Thus, it was demonstrated that fungi can contribute to the development of OC, and further research in other cancer models and environments is also required.

Skin cancer

The skin, which is the body’s outermost covering, is typically where environmental microbes and the host first come into touch with (120). Cutaneous melanoma is also one of the cancers that affect human health and causes the most skin cancer-related deaths globally (1). Malassezia, which relies on long-chain fatty acids, is the main symbiotic skin fungus (8, 120). Vitali et al. conducted a meta-analysis of intestinal microbiota in patients with metastatic melanoma and examined the makeup of intestinal bacteria and fungus in patients with stage I and stage II melanoma (50). They observed high levels of Prevotella copri and yeast in the cancer group. Another important finding was that the composition of the gut microbiota changed along a gradient from in situ to malignant melanoma in early melanoma. Alterations in the microbiota may be associated with the clinical outcome of advanced melanoma and response to immunotherapy through direct or indirect immunomodulatory effects. Similarly, evidence from Shiao et al. suggests that fungi can modulate the TME through immune regulation, thereby affecting the therapeutic efficacy of tumors (121). However, the causal relationship between fungi and melanoma development awaits further validation.

Certain fungi, primarily Penicillium, can produce clavaflatoxin PAT (122), which mainly contaminates fruits such as ripe apples (123). In mammalian cells, PAT can induce DNA damage, chromosome aberration, and genotoxicity (124, 125). By producing reactive oxygen species (ROS) and damaging DNA, it may aid in the development of cancer (126, 127). The results of Saxena et al. showed that the skin of mice treated with PAT could promote the accumulation of ROS production; further induce the MAPK signaling pathway; activate downstream c-fos, c-jun, and NF-kB protein expression; and finally lead to cell proliferation, indicating that PAT had the potential to promote skin cancer (128). However, the carcinogenic mechanism of this toxin requires further study.

Head and neck squamous cell carcinoma

In tumor samples of head and neck squamous cell carcinoma (HNSCC), Glomeromycota is significantly decreased compared with non-tumor tissues (41).

Candida is the most common oral fungal genus in both HNSCC patients and healthy individuals (42, 43) and is intimately associated with the risk of developing various types of cancer, including HNSCC (32). Compared with the control group, the diversity of oral wash fluid in HNSCC patients was decreased. While Schizophyllum commune was decreased, C. albicans and Rothia mucilaginosa were both differentially abundant (42). Consistently, Vesty et al. showed that C. albicans dominated the fungal community in the saliva of HNSCC patients, accounting for 96% of the total fungal community (43), and there was a positive correlation between C. albicans abundance and IL-1β or IL-8 (43).

Other types of cancer

Prostate cancer

One of the leading causes of morbidity and death among men is prostate cancer (1). Banerjee et al. identified microbial signatures associated with prostate cancer, mostly with fungal genera derived from Ascomycota (51). Similarly, plasma samples from patients with prostate cancer have been found to exhibit fungal dysbiosis, and certain fungal genera are associated with prostate-specific antigen levels and pathological stages (52). However, further comprehensive study is required to elucidate the exact mechanism and ascertain the existence of a causal relationship.

Bladder cancer

Bukavina et al. performed internal transcribed spacer sequencing on stool samples from 52 bladder cancer patients and the healthy control to evaluate the similarities and differences in intestinal mycobiome. By examining antigen presentation, it was found that patients with a “favorable” fungal microbiota composition, characterized by high diversity and low levels of Agaricomycetes and Saccharomycetes, may exhibit a more robust systemic immune response to chemotherapy (53). These findings shed light on the makeup of the gut fungal population in patients with bladder cancer as well as the possible influence of these fungi on the efficacy of treatment.

Leukemia

Leukemia is the most common childhood tumor, accounting for 28% of cases, which is one of the human malignant tumors (1, 129). Patients with acute myeloid leukemia (AML) are particularly vulnerable to invasive Aspergillus infections. Such infections occurring during the first induction chemotherapy can delay subsequent treatment plans and are among the factors that affect the recovery of AML patients (130). Additionally, it has been the most frequent infectious cause of mortality for leukemia patients, particularly those with AML.

Ovarian cancer

The microbiome in ovarian cancer patients exhibited a significant disruption, with 18 distinct fungal signatures identified in the tumor samples that were absent in the control group. Pneumocystis, Acremonium, Cladophialophora, Malassezia, and Microsporidia Pleistophora are notably present in tumor tissues (44).

Although fungal research lags behind that of bacteria, tumor-associated fungal research is now rapidly evolving. Although there are a lot of interesting outcomes right now, more research is required in the future to establish the causal link between fungal traits and the development of cancer. Future research in this area is promising and rewarding, which will provide additional insights into the intricate interplay between fungi and cancer development.

MYCOBIOME AND ANTI-CANCER THERAPY

Given the critical role of fungi in cancer progression, it was urgent and necessary to develop potential therapeutic strategies by targeting fungi for cancer prevention and control (Fig. 5).

Fig 5.

The role of fungi in anti-cancer therapy. New indirect effects of fungi on cancer may be utilized in the diagnosis and treatment of cancer. (A) On the therapeutic side, fungal components, such as β-glucan, can be a target for cancer therapy. (B) Fungi could be supplemented as “precision probiotics” or could be engineered to specifically release cancer-suppressive vectors. (C) Secondary metabolites of fungi have potential for pharmaceutical applications. (D) The disordered intestinal mycobiome could be modulated by fecal transplantation.

Fungal components

Through the stimulation and/or activation of innate immune cells, β-glucan, one of the fungal cell wall components that represents pathogen-associated molecular patterns, can regulate host immune responses (131). Zhang et al. demonstrated that systemic injection of certain β-glucans can successfully regulate TME, which, in turn, greatly inhibits tumor development and distant metastasis (132). According to the findings, β-glucan may be an immunomodulator that enhances the efficacy of immunotherapy against cancer (133). In addition to systemic administration, oral glucan treatment can regulate the transformation of M2 macrophages into M1-like phenotypes, which significantly reduces tumor load and delays tumor growth (134, 135). Prophylactic treatment with pure β-glucan can considerably increase the longevity of tumor-bearing mice in the tumor mice model and block lung metastasis in a dose-dependent manner. Therapeutic administration of purified β-glucan also significantly inhibited the proliferation of tumor cells (136). Others have also found that combined β-glucan can reduce tumor burden, improve overall survival in experimental animals, and increase the efficacy of immunotherapy (137, 138).

Another clinical study was conducted to evaluate the short-term effects of oral β-glucan on peripheral blood mononuclear cells, involving 23 advanced stages of breast cancer patients (139). Patients with breast cancer had a much lower baseline lymphocyte count than those without the disease (P = 0.04). At day 15 of administration, the mean monocyte count in breast cancer patients was noticeably higher (P = 0.015). On CD14-positive monocytes, CD95 (Apo1/Fas) expression was increased from 48.17% at the start of treatment to 69.23% on day 15 (P = 0.002). Additionally, there was a noteworthy increase in CD45RA expression in CD14-positive monocytes (P = 0.001).

Imprime PGG, which is a soluble β-glucan and an innate immunomodulator for anti-cancer immunotherapy, directly activates innate immune cells and induces a coordinated anti-tumor immune response (140). This anti-cancer effect is contingent upon the formation of an immunological complex involving naturally occurring anti-α-glucan antibodies (141). In summary, the results of the above studies have proven the utility of β-glucan in anti-cancer prevention and treatment.

Driving infection and host immunological responses requires the cytolytic peptide toxin known as candidalysin, which is released by Candida spp. (142–144). Candidalysin and C. albicans both activate EGFR, and in vitro administration of EGFR kinase inhibitors, such as the Food and Drug Administration-approved gefitinib, prevents the release of neutrophil-activating cytokines and MAPK signaling that is triggered by candidalysin and Candida albicans (145). In addition, a team of researchers has also found that C. albicans induces upregulation of PD-L1 (a crucial indicator of tumor immune evasion and tumor development) in OSCC both in vitro and in mice models (146).

It is evident that molecules such as EGFR, PD-L1, β-glucan, and others are potential targets for therapeutic interventions in cancer types where fungal infection serves as a potential oncogenic mechanism and deserves further investigation in the future for their potential applications in cancer therapy.

Probiotics

Probiotics, which can inhibit biofilm formation in C. albicans or C. tropicalis (88) and also mediate anti-cancer effects through the immune system, are a promising anti-cancer biotherapeutic drug (147–149).

Galinari et al. evaluated the activity of the cell wall component α-d-mannan fractions for antioxidant, anti-proliferative, and immunostimulating properties (150). It was shown that Aspergillus oryza heptelidic acid can have anti-tumor effects via the p38-MAPK signaling pathway in an in vitro pancreatic cancer cell and in vivo xenograft model (149).

A probiotic called Saccharomyces boulardii has anti-bacterial and anti-inflammatory properties that can regulate host inflammatory response by down-regulating Erk1/2 MAP kinase activities (151). Apart from its anti-inflammatory properties, S. bouldarii inhibits the EGFR signaling pathway and other receptor tyrosine kinases, the activation of which leads to cell proliferation, invasion, and inhibition of apoptosis (152). The EGFR-Mek-Erk pathway in colon cancer cells is inactivated by S. bouldarii (151), which inhibits the growth and colony formation of colon cancer cells, deactivates Akt, a crucial signaling protein for cell survival, and triggers apoptosis in cancer cells (153). Therefore, the clinical application of S. bouldarii in the prevention and management of intestinal disorders is worthy of further study. In addition, S. cerevisiae, S. bouldarii (S. cerevisiae variant), and Schizophyllum commune have been shown to exert beneficial effects such as antioxidants (40, 154). Consequently, the fungus itself may be utilized as a probiotic to treat and prevent cancer. In addition, fungi-bacterial interactions can also provide insights into anti-fungal treatments. Hager et al.’s team found that the levels of C. tropicalis, Escherichia coli, and Serratia marcescens in patients with Crohn’s disease were elevated, and C. tropicalis formed polymicrobial biofilms with E. coli and S. marcescens (88, 155). Furthermore, they developed a new probiotic combination consisting of S. bouldarii, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium breve that can reduce the tumor burden of CRC through prevention and treatment of polymicrobial biofilms (88). Additionally, Saccharomyces burra can prevent bacterial growth by producing significant quantities of acetic acid, which may be related to its probiotics (156). Fungal biomarkers performed better, and the AUROCs were enhanced when combined with bacterial markers, according to the results of a study that used fecal metagenomic data from 862 samples of nine different cohorts to predict gut microbial response to the immune checkpoint inhibitor (ICI). It identified a fungal profile of ICI response and multi-kingdom microbial markers, which may increase the overall efficacy of ICI therapy (157). Multi-kingdom biomarkers that combined bacterial and fungal biomarkers enhanced the performance of CRC diagnostic models, according to another study of 1,368 samples from eight different geographic cohorts (23). This suggests that multi-kingdom biomarkers can be applied as both a potential therapeutic target and a diagnostic model for CRC.

The depletion of intestinal symbiotic fungi could increase the efficacy of radiotherapy, while loss of symbiotic bacteria leads to the expansion of pathogenic fungi and the depletion of symbiotic fungi (121, 158). This study suggested the importance of targeting microbial communities to enhance the efficacy of anti-cancer treatment. Therefore, the combination of probiotics and radiotherapy may enhance the therapeutic efficacy against cancer.

Fungal metabolites

Fungal secondary metabolites are important in the pharmaceutical and healthcare sectors, which may be useful in pharmaceutical applications (159–161).

Recently, there are 61 endophytic fungal strains discovered from the bark of plants like Albizzia julibrissin and Ginkgo biloba (162). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay showed that the growth inhibition of human esophageal cancer cells was greater than 50% at 200 µg/mL (162). He et al. isolated the endophytic fungal secondary metabolites from Ginkgo biloba leaves and discovered that they could dramatically reduce the formation of Hela-implanted tumors in mice, promote the death of Hela cells, and limit the proliferation of Hela cells (163). López-Legarda et al. identified Schizophyllum radiatum and found that its external polysaccharides exhibited anti-tumor activity (164).

Schisandra polysaccharide (SPG) is a substance produced by the fermentation of Schisandra chinensis that can be used to treat cervical cancer (165). It had strong immunomodulatory activities in vivo. After ultrasonic treatment, SPG fractions had stronger immunomodulatory and anti-tumor effects (166).

Lung cancer is strongly inhibited by fungal immunomodulatory proteins, which are broadly distributed in fungi. The 114 amino acid residues that make up the fungal immunomodulatory protein from Nectria haematococca (FIP-nha) can prevent lung cancer cells from growing in vivo by blocking the PI3K/Akt pathway (167). Similarly, colletofragarone A2 isolated from the fungus Colletotrichum spp. (13S020) reduced the level of mutant p53 and inhibited cancer cell growth in vivo (167). Darvishi et al. found that a new yeast L-asparaginase generated from Yarrowia lipolytica inhibits the proliferation and migration of lung (A549) and breast (MCF7) cancer cells, providing enzyme therapies that can be used in anti-cancer treatment (168).

Overall, these results supplement our understanding of fungal secondary metabolites as small molecules for anti-cancer therapy, and whether they can be applied to human anti-cancer therapy warrants additional research.

Fecal microbiota transplantation

Fecal microbiota transplantation (FMT) restores the normal microbiota composition and function, which can effectively inhibit recurrent Clostridium difficile infection (CDI) (169, 170). Nevertheless, it was also discovered that a significant concentration of C. albicans in donor stool is linked to reduced efficacy of FMT. In CDI animal models, C. albicans decreases the efficacy of FMT; however, anti-fungal therapy can increase the efficacy, confirming the connection between intestinal fungal dysbiosis and decreased FMT efficacy in CDI patients (171). Furthermore, clinical response is linked to Candida abundance prior to FMT, whereas disease improvement is indicated by a decrease in Candida abundance following FMT (172). Thus, FMT has been successfully used in the treatment of ulcerative colitis. Additionally, the data imply that FMT might lessen the pro-inflammatory immunological response caused by Candida (172). Serum IFN-γ, IL-4, and TGF-β levels were significantly reduced, and apoptosis was induced by gavage with C. albicans isolated from the feces of healthy older adults (173).

Therefore, FMT may offer benefits in the treatment of CRC (174), which awaits further in-depth investigation and more clinical evidence.

DISCUSSION AND FUTURE PERSPECTIVES

Although lagging far behind the study of bacteria in cancer progression, the investigation of fungi is still a promising field. By inducing inflammation through fungal components, biofilm formation, interactions with bacteria, or the generation of secondary carcinogenic compounds, fungi can have a role in both the onset and development of cancer (175). However, fungi are involved in both the diagnosis and therapy of cancer in addition to the progression of disease (174, 176). The idea of employing various fungal combinations as biomarkers for various malignancies is further made possible by the discovery that fungal ecologies, particularly fungi-bacterial interactions, are distinct in different cancer types (8, 22). Furthermore, in mouse models of melanoma and breast cancer, gut fungus can alter the efficacy of radiation therapy (121). Fungal components and the secreted small molecule metabolites also have anti-cancer properties, providing a promising but remaining premature therapeutic strategy.

Although increasingly acknowledged, the involvement of fungi in tumorigenesis remains a relatively novel field of investigation. This compelling evidence increased our notion that fungi could be linked to particular cancer types, and it is reasonable to assume that tumor types harboring intratumor bacteria might also contain fungi (177). This presents both promising hypotheses and poses significant challenges for research in this field. There are some inconsistencies and even conflicting results in the current findings (8, 28, 178, 179), which may stem from advancements in the pipeline and analytical methods used (178, 180). This underscores scientists’ great enthusiasm and scientific rigor in the field while also emphasizing the importance of employing standardized protocols for identifying and decontaminating low-biomass and highly contaminant-prone data, such as the microbiome in tissues (8, 28, 70, 178–180). When sampling, it is important to include normal tissues whenever ethically permissible and feasible (8). Negative controls should be set for steps that could introduce contamination, such as sampling, DNA extraction, and library preparation (8, 28, 61). Data should be processed using the most comprehensive analytics pipeline currently available, tailored to the type and purpose of the data, while incorporating the latest iterations of existing analytics. Additionally, the reference genome is being updated (181, 182), and the comprehensive version should be selected for analysis. Confirming the presence of intratumor microbiota through staining, culturing, and other experimental methods would provide more rigorous and solid evidence (8, 61, 183). Ultimately, after the data analysis has generated the underlying hypothesis, more extensive and refined experiments should be carried out to test it (61, 68). Finally, as sequencing technology and bioinformatics analysis continue to progress, it is promising to fully understand the direct carcinogenic or anti-cancer effects of fungal species through integrated multi-omics analysis, aiming to achieve the tertiary prevention of cancer.

Biographies

Yan-Yan Sun received her B.A. in Nursing from Shanghai Jiao Tong University School of Medicine and conducted her Master research in Ning-Ning Liu's lab, where she focused on the mechanistic study of fungal infection and novel antifungal agent identification.

Ning-Ning Liu obtained his B.A. from Shanxi Agricultural University and his Ph.D. from Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, in 2012. After graduation, he continued his research in Harvard Medical School/Boston Children’s Hospital as a post-doctoral researcher. In 2017, he joined the School of Public Health in Shanghai Jiao Tong University School of Medicine, where he is still a faculty member. Dr. Liu’s research has been focused on fungal infection and cancer progression, with particular interest in the mechanism of fungus-host interaction and the causal relationship between fungi and cancer.