Loss of HLA class I expression in prostate cancer and restoration using adenoviral and adeno-associated viral vectors

Abstract

In the present study, we analyzed HLA-I expression in 42 cryopreserved prostate cancer samples, as well as the molecular mechanisms implicated in the loss of its expression. Immunohistochemical analysis demonstrated that 88% of the studied tumours have at least one type of HLA alteration. 50% of the samples showed complete loss of HLA-I expression either with heterogeneous or totally negative pattern, with a strong positive correlation with tumour relapse, perineural invasion and high D`Amico risk. The rest of the studied tumours demonstrated locus and allelic losses in 26% and 12% respectively. Molecular analysis showed loss of heterozygosity (LOH) at chromosome 6 (where HLA genes are located) in 32% of the studied tumours, while only one tumour showed LOH in chromosome 15. mRNA expression analysis of microdissected samples revealed that HLA-I negative tumours have significantly reduced expression of Beta-2-microglobulin (β2m), antigen processing machinery (APM) components TAP2 and tapasin, and of the transcriptional regulator NLRC5, and an apparent downregulation of LMP2, LMP7 and the Interferon Regulatory Factor (IRF1), as compared to a control group of prostate benign hyperplasia (BH). These data point to a coordinated downregulation of HLA and APM expression as the main mechanism responsible for HLA loss, although large proportion of the studied tumors also showed structural losses in chromosome 6. Moreover, we analyzed twelve previously unreported cell lines derived from prostate tissue of patients with prostate cancer. FACS analysis revealed different types of HLA-I aberrations, ranging from locus and/or allelic downregulation to a total absence of HLA-I expression caused by a 480 pb deletion in one copy of β2m gene and LOH in chromosome 15 harboring another copy. This is the first report of such β2m defect in human prostate cancer. Use of cancer cell lines facilitates the study of HLA-I alterations, as well as allow the manipulation of different factors implied in its expression, modulating or even recovering HLA-I expression. In the second part of the thesis, we analyzed the transduction efficacy of different serotypes of adeno-associated vectors (AAV) and an adenovirus carrying the green fluorescent protein (GFP) gene in several cancer cell lines of different histological origin. The obtained results indicate that adenoviral vector gives the highest trasgene expression, followed by AAV serotype 2 (AAV2). In addition, we constructed two new recombinant vectors, adenoviral and AAV2, both carrying HLA-A2 gene, both of which demonstrated high efficacy in an in vitro A2 gene transfer into human tumor cells of different histotype. Using these vectors we were able to: 1) recover endogenous A2 allele in a cell line which had lost A2 expression due to a haplotype loss; 2) upregulate HLA-A2 expression in cells with low baseline A2 level; and 3) introduce an additional A2 allele into tumour cells that do not have it in their genotype. Moreover, adenovirus mediated co-transfection of HLA-A2 and β2m demonstrated that the de novo expressed proteins associate to form HLA-I/β2m complex on the cell surface, which gives an option of correcting simultaneously multiple defect causing HLA-I loss The high incidence of HLA-I loss observed in prostate cancer, caused by both regulatory and structural defects, are associated with disease progression and may pose a real threat to patient health by increasing cancer progression and resistance to T-cell based immunotherapy. We could speculate that structural genetic aberrations are likely to be continuously accumulating during cancer progression generating tumor escape variants with lower immunogenicity. Our findings suggest that an effective immunotherapy migh benefit from a complementary treatment using gene therapy to bypass the selection and outgrowth of HLA-I-negative tumor cells. The recovery of the endogenous missing HLA-I specificity may be beneficial in the restoration of the natural HLA-I expression responsible for antigen-presentation leading to increased tumour rejection by CTLs. Our results indicate that viral vectors coding for HLA molecules represent a useful tool in upregulation of tumor HLA class I expression which can be used to increase rejection of tumors harboring structural genetic defects responsible for HLA class I loss. Tumor HLA-I upregulation may enable cancer biologists to better understand how to specifically target CTL-mediated immune responses and further improve therapeutic efficacy of cancer immunotherapy.