Abstract
Oral squamous cell carcinoma (OSCC) can remain undiagnosed until in an advanced, and sometimes lethal, state. It is often preceded by a potentially malignant lesion, which may manifest as a white patch 'leukoplakia' of the oral mucosa. Previous research has implicated an association between the presence of Candida albicans and the progression of leukoplakias to OSCC. Alcohol may contribute to oral cancer via its conversion to acetaldehyde, a known carcinogen, which is also a product of C. albicans metabolism. The reversible conversion of ethanol to acetaldehyde is catalysed by enzymes known as alcohol dehydrogenases (ADHs) and in C. albicans, it is not known which ADH is responsible for acetaldehyde production.
Aims of this study: To investigate expression of the CaADH genes in vitro and to identify the C. albicans genes responsible for acetaldehyde production. It is also the aim of this study to detect the expression of the CaADH genes in archival formalin-fixed paraffin-embedded (FFPE) samples from leukoplakia biopsies.
Study hypotheses: (i) that production of acetaldehyde by C. albicans via expression of ADH genes can be detected in lesions diagnosed as CHC; (ii) that the presence of C. albicans in CHC lesions is associated with expression of CaADH1 mRNA; (iii) that acetaldehyde production may provide a mechanism for the previously reported putative link between oral infection/colonisation with C. albicans and oral cancer.
Methods: The levels of expression for three C. albicans genes, CaADH1, CaADH2 and CaADH3, were measured under various growth conditions using Northern blot analysis and qRT-PCR. The three C. albicans genes were also cloned and expressed in the model yeast Saccharomyces cerevisiae. CaAdhp polypeptide expression was confirmed by Western blot analysis. Ethanol utilisation was assayed in cell extracts of recombinant S. cerevisiae strains expressing CaADH1 or CaADH2. Disruption of the endogenous ScADH2 gene, which is responsible for production of acetaldehyde in S. cerevisiae, was carried out in the recombinant S. cerevisiae strain that expressed CaADH1 to reduce background ScAdhp activity. The presence of Candida was investigated in archival FFPE samples from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC) and non-CHC dysplastic leukoplakia. Candida was detected in FFPE samples by histology and immunocytochemistry and C. albicans ADH1 and ADH2 mRNAs were detected by RT-PCR and qRT-PCR.
Results: Northern blot analysis showed that CaADH1 mRNA was expressed under a variety of in vitro growth conditions while CaADH2 was only detected during stationary phase in a rich medium. No expression of CaADH3 was detected. Each gene was cloned in S. cerevisiae but CaADH3 again was not expressed. Cell extracts from the Adh1p-expressing S. cerevisiae recombinant, but not the Adh2p-expressing recombinant, or an empty vector control strain, possessed ethanol utilising Adh activity. Expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene had been deleted conferred an NAD-dependant ethanol utilising and hence acetaldehyde producing Adh activity. FFPE samples were analysed by immunocytochemistry for C. albicans and by RT-PCR for C. albicans gene expression. C. albicans was detected by both methods in FFPE samples diagnosed as CHC, but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed the presence of high levels of C. albicans ADH1 mRNA expression, compared to a house-keeping gene, in CHC biopsies but CaADH2 expression was variable.
Conclusions: C. albicans Adh1p was shown to be the major Adh isozyme involved in the production of acetaldehyde by this human commensal yeast. Immunohistopathological evidence was obtained from FFPE samples from patients with previously diagnosed CHC indicating that C. albicans was the predominant species in the lesions. The presence of C. albicans in CHC lesions was associated with a high expression of CaADH1 mRNA. The results of this study provide the first experimental support for the hypothesis of a putative link between acetaldehyde production by oral infection/colonisation with C. albicans and oral cancer despite the lack of malignant transformation in the clinical cases of chronic hyperplastic candidosis studied.