No abstract

Results of this study suggest that polymorphisms of the HER2 gene may be important susceptibility biomarkers for breast cancer risk, particularly among younger women.

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.

BackgroundHuman papillomavirus (HPV) initiates cervical cancer, and continuous expression of HPV oncogenes E6 and E7 is thought to be necessary to maintain malignant growth. Current therapies target proliferating cells, rather than specific pathways, and most experimental therapies specifically target E6/E7. We investigated the presence and expression of HPV in cervical cancer, to correlate HPV oncogene expression with clinical and molecular features of these tumors that may be relevant to new targeted therapies.ResultsWhile virtually all cervical cancers contained HPV DNA, and most expressed E6/E7 (HPV-active), a subset (8%) of HPV DNA-positive cervical cancers did not express HPV transcripts (HPV-inactive). HPV-inactive tumors occurred in older women (median 54 vs. 45 years, p = 0.02) and were associated with poorer survival (median 715 vs 3046 days, p = 0.0003). Gene expression profiles of HPV-active and -inactive tumors were distinct. HPV-active tumors expressed E2F target genes and increased AKT/MTOR signaling. HPV-inactive tumors had increased WNT/β-catenin and Sonic Hedgehog signaling. Substantial genome-wide differences in DNA methylation were observed. HPV-inactive tumors had a global decrease in DNA methylation; however, many promoter-associated CpGs were hypermethylated. Many inflammatory response genes showed promoter methylation and decreased expression. The somatic mutation landscapes were significantly different. HPV-active tumors carried few somatic mutations in driver genes, whereas HPV-inactive tumors were enriched for non-synonymous somatic mutations (p-value < 0.0000001) specifically targeting TP53, ARID, WNT, and PI3K pathways.Materials and MethodsThe Cancer Genome Atlas (TCGA) cervical cancer data were analyzed.ConclusionsMany of the gene expression changes and somatic mutations found in HPV-inactive tumors alter pathways for which targeted therapeutics are available. Treatment strategies focused on WNT, PI3K, or TP53 mutations may be effective against HPV-inactive tumors and could improve survival for these cervical cancer patients.

Immortalization of human keratinocytes (HKc) by human papillomavirus type 16 (HPV16) is reproducible at a high frequency, is due directly to the presence of the viral sequences in the cells, and occurs independently from the genetic characteristics of the host cells. Ten human keratinocyte strains, each derived from a different individual, were transfected with pMHPV16d and selected with G418. Eight became established lines. Two strains, which failed to grow shortly after successful G418 selection, were negative for HPV16 DNA. No lines were established following transfection of the same HKc strains with vector sequences only. The immortalized lines maintained a constant number of copies of the viral genome integrated into the cellular DNA. Each line showed a unique integration pattern of HPV16 sequences into the cellular genome, but expressed similar patterns of viral messages. Sublines able to grow in the absence of growth factors (epidermal growth factor and bovine pituitary extract), and others which became resistant to differentiation stimuli (serum and calcium) were obtained by selection in growth factor-free medium and serum-supplemented medium, respectively. The establishment of continuous cell lines is a direct consequence of the presence of viral sequences; however, because none of these lines formed tumors in nude mice, additional events must be necessary for progression of malignancy. HPV16-immortalized human keratinocyte lines can be used to investigate and identify the viral factors involved with the modification of growth and differentiation control by HPV16.