Gene expression changes during HPV-mediated carcinogenesis: a comparison between an in vitro cell model and cervical cancer

Abstract

We used oligonucleotide microarrays to investigate gene expression changes associated with multi-step human papillomavirus type 16 (HPV16)-mediated carcinogenesis in vitro. Gene expression profiles in 4 early passage HPV16-immortalized human keratinocyte (HKc) lines derived from different donors were compared with their corresponding 4 late-passage, differentiation-resistant cell lines, and to 4 pools of normal HKc, each composed of 3 individual HKc strains, on Agilent 22 k human oligonucleotide microarrays. The resulting data were analyzed using a modified T-test coded in R to obtain lists of differentially expressed genes. Gene expression changes identified in this model system were then compared with gene expression changes described in published studies of cervical intraepithelial neoplasia (CIN) and cervical cancer. Common genes in these lists were further studied by cluster analysis. Genes whose expression changed in the same direction as in CIN or cervical cancer (concordant) at late stages of HPV16-mediated transformation in vitro formed one major cluster, while those that changed in the opposite direction (discordant) formed a second major cluster. Further annotation found that many discordant expression changes involved gene products with an extracellular localization. Two novel genes were selected for further study: overexpression of SIX1 and GDF15, observed during in vitro progression in our model system, was confirmed in tissue arrays of cervical cancer. These microarray-based studies show that our in vitro model system reflects many cellular and molecular alterations characteristic of cervical cancer, and identified SIX1 and GDF15 as 2 novel potential biomarkers of cervical cancer progression.

Figure 1

(a) composition of 232 gene expression changes detected in 4 out of 4 individuals in our in vitro model system. Early: HKc/HPV16 compared with normal HKc; Late: HKc/DR compared with HKc/HPV16; DR_N, changes between HKc/DR and normal HKc, which were derived mathematically from the 2 comparisons listed above. (b) Data mining strategy: summary of all comparisons (explanation in the text). (c) Composition of common gene set between the 2 out of 4 list and the cervical cancer list. Concordant, gene expression changes that occur in the same direction (up or down) in the 2 lists. Discordant, gene expression changes in the opposite direction in the 2 lists; IM specific, gene expression changes in HKc/HPV16 compared to normal HKc, which return to normal in HKc/DR. (d) composition of common gene set between the 2 out of 4 list and other HPV-transformed cell lines list. Gene expression changes were categorized in the same way as in c.

Figure 2

Unsupervised two-way cluster analyses of early and late gene expression changes that overlap with changes detected in cervical cancer or CIN. Cluster to the left of the heat map, gene expression ratios; cluster above the heat map, comparison among cell lines. Colors in the heat maps represent the log ratio of Cy5 over Cy3, with the color-ratio correspondence shown in the colored bar below the heat map on the right. To the left of the heat maps, a colored column shows concordance of expression changes compared to cervical cancer or CIN: blue represents concordant changes, yellow represents discordant changes. Early: cluster of the common gene set between the early stage changes (including changes that occurred in both early and late stages) detected in 2 out of 4 HKc/HPV16 versus normal HKc comparisons, and the cervical cancer/CIN gene list. Late: cluster on the common gene set between late-stage changes detected in 2 out of 4 HKc/DR versus HKc/HPV16 cell line pairs, and the cervical cancer/CIN gene list.

Figure 3

Cellular localization maps of gene products differentially expressed in HKc/DR versus HKc/HPV16. Plasma membranes are represented by a brown band separating the extracellular space from the intracellular space, and nuclei are depicted as light blue ellipses. Genes differentially expressed at both early and late stages are represented by light pink symbols, while late stage-specific changes are symbolized by dark pink. (a) Concordant changes; (b) discordant changes.

Figure 4

Representative immunohistochemical staining for SIX1 and GDF15 in cervical cancer and adjacent normal cervical epithelial tissue, from tissue arrays. (a) SIX1 staining (brown) in a normal cervical specimen (adjacent to cancer tissue). The pie chart presents the summary of staining intensities in all 32 adjacent normal tissue cores. (b) Representative SIX1 staining in a cervical cancer specimen, and summary of the staining intensities in all 40 cervical cancer tissue cores (pie chart). (c) Representative GDF15 staining (brown) in a normal cervical tissue core, and summary of GDF15 staining intensities in all 36 adjacent normal tissue cores (pie chart). (d) Representative GDF15 staining in a cervical cancer tissue core, and summary of GDF15 staining intensities in all 40 cancer tissue cores in the tissue array (pie chart).