Patricia Piña-Sánchez

Genome wide expression analysis in HPV16 Cervical Cancer: identification of altered metabolic pathways

BackgroundCervical carcinoma (CC) is a leading cause of death among women worldwide. Human papilloma virus (HPV) is a major etiological factor in CC and HPV 16 is the more frequent viral type present. Our aim was to characterize metabolic pathways altered in HPV 16 tumor samples by means of transcriptome wide analysis and bioinformatics tools for visualizing expression data in the context of KEGG biological pathways.ResultsWe found 2,067 genes significantly up or down-modulated (at least 2-fold) in tumor clinical samples compared to normal tissues, representing ~3.7% of analyzed genes. Cervical carcinoma was associated with an important up-regulation of Wnt signaling pathway, which was validated by in situ hybridization in clinical samples. Other up-regulated pathways were those of calcium signaling and MAPK signaling, as well as cell cycle-related genes. There was down-regulation of focal adhesion, TGF-β signaling, among other metabolic pathways.ConclusionThis analysis of HPV 16 tumors transcriptome could be useful for the identification of genes and molecular pathways involved in the pathogenesis of cervical carcinoma. Understanding the possible role of these proteins in the pathogenesis of CC deserves further studies.

Characterization of the global profile of genes expressed in cervical epithelium by Serial Analysis of Gene Expression (SAGE)

BackgroundSerial Analysis of Gene Expression (SAGE) is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE), useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV), where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC).ResultsWe report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation.ConclusionThis first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.

Human papillomavirus genotypes among females in Mexico: a study from the Mexican institute for social security.

BACKGROUND The aetiological relationship between human papillomavirus (HPV) infection and cervical cancer (CC) is widely accepted. Our goal was to determine the prevalence of HPV types in Mexican women attending at the Mexican Institute for Social Security from different areas of Mexico. MATERIALS AND METHODS DNAs from 2,956 cervical samples were subjected to HPV genotyping: 1,020 samples with normal cytology, 931 with low-grade squamous intraepithelial lesions (LGSIL), 481 with high grade HGSIL and 524 CC. RESULTS Overall HPV prevalence was 67.1%. A total of 40 HPV types were found; HPV16 was detected in 39.4% of the HPV-positive samples followed by HPV18 at 7.5%, HPV31 at 7.1%, HPV59 at 4.9%, and HPV58 at 3.2%. HPV16 presented the highest prevalence both in women with altered or normal cytology and HPV 18 presented a minor prevalence as reported worldwide. The prevalence ratio (PR) was calculated for the HPV types. The analysis of PR showed that HPV16 presents the highest association with CC, HPV 31, -33, -45, -52 and -58 also demonstrating a high association. CONCLUSIONS The most prevalent HPV types in cervical cancer samples were -16, -18, -31, but it is important to note that we obtained a minor prevalence of HPV18 as reported worldwide, and that HPV58 and -52 also were genotypes with an important prevalence in CC samples. Determination of HPV genotypes is very important in order to evaluate the impact of vaccine introduction and future cervical cancer prevention strategies.

Differentially expressed genes between high-risk human papillomavirus types in human cervical cancer cells.

Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. Human papillomavirus (HPV) infection is the most important etiologic factor for CC. Of the oncogenic types, HPV16 and HPV18 are found in 60–70% of invasive CCs worldwide. HPV18 appears to be associated with a more aggressive form of cervical neoplasia than HPV16 infection. At present, there are no studies on differentially expressed cellular genes between transformed cells harboring HPV16 and HPV18 sequences. Based on previous complementaryDNA microarray data from our group, 13 genes were found to be differentially overexpressed between HPV16- and HPV18-transformed cells. These genes were as follows: E6BP, UBE4A, C20orf14, ATF7, ABCC8, SLC6A12, WASF3, SUV39H1, SPAG8, CCNC, E2FFE, BIRC5, and DEDD. Differential expression of six selected genes was confirmed by real-time reverse transcription–polymerase chain reaction (RT-PCR). All real-time RT-PCRs confirmed differential expression between HPV18 and HPV− samples. The present work identifies genes from signaling pathways triggered by HPV transformation that could be differentially deregulated between HPV16+ and HPV18+ samples.

NF-kappaΒ-inducing kinase regulates stem cell phenotype in breast cancer

Breast cancer stem cells (BCSCs) overexpress components of the Nuclear factor-kappa B (NF-κB) signaling cascade and consequently display high NF-κB activity levels. Breast cancer cell lines with high proportion of CSCs exhibit high NF-κB-inducing kinase (NIK) expression. The role of NIK in the phenotype of cancer stem cell regulation is poorly understood. Expression of NIK was analyzed by quantitative RT-PCR in BCSCs. NIK levels were manipulated through transfection of specific shRNAs or an expression vector. The effect of NIK in the cancer stem cell properties was assessed by mammosphere formation, mice xenografts and stem markers expression. BCSCs expressed higher levels of NIK and its inhibition through small hairpin (shRNA), reduced the expression of CSC markers and impaired clonogenicity and tumorigenesis. Genome-wide expression analyses suggested that NIK acts on ERK1/2 pathway to exert its activity. In addition, forced expression of NIK increased the BCSC population and enhanced breast cancer cell tumorigenicity. The in vivo relevance of these results is further supported by a tissue microarray of breast cancer samples in which we observed correlated expression of Aldehyde dehydrogenase (ALDH) and NIK protein. Our results support the essential involvement of NIK in BCSC phenotypic regulation via ERK1/2 and NF-κB.

Mesenchymal stromal cells derived from cervical cancer tumors induce TGF-β1 expression and IL-10 expression and secretion in the cervical cancer cells, resulting in protection from cytotoxic T cell activity.

Cervical cancer (CeCa) tumors are characterized by increased expression of TGF-β1 and IL-10, which are correlated with downregulated expression of major histocompatibility complex class I antigens (HLA-I) on cancer cells and a reduced immune response mediated by cytotoxic T lymphocytes (CTLs). Mesenchymal stromal cells (MSCs) are important components in the tumor microenvironment that have been suggested to contribute to cancer progression through the induction of TGF-β1 and IL-10. In this study, we provided evidence that MSCs derived from cervical tumors (CeCa-MSCs) cocultured with CeCa cells induced significant expression of TGF-β1 and secretion of IL-10 by CeCa cells compared to MSCs derived from the normal cervix (NCx-MSCs) and normal bone marrow (BM-MSCs; gold standard). This increase in expression was associated with a significant downregulation of HLA-I molecules and protection of the cells against specific CTL lysis. Interestingly, the addition of the neutralizing antibody anti-TGF-β to the CeCa/CeCa-MSCs coculture strongly inhibited the expression and production of IL-10 by CeCa cells. Anti-TGF-β as well as anti-IL-10 also abolished HLA-I downregulation, and reversed the inhibition of CTL cytotoxicity. These results provide evidence that TGF-β1 and IL-10 could play an important role in the downregulation of HLA-I molecules on CeCa cells induced by tumor MSCs. Our findings suggest a novel mechanism through which MSCs may protect tumor cells from immune recognition by specific CTLs.

Human papillomavirus genotyping by Linear Array and Next-Generation Sequencing in cervical samples from Western Mexico

BackgroundThe Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS.MethodsTwo hundred thirty three cervical samples from women Without cervical lesions (WCL, n = 48), with Cervical intraepithelial neoplasia grade 1 (CIN I, n = 98), or with Cervical cancer (CC, n = 87) were recruited, DNA was extracted, and HPV positivity was determined by PCR amplification using PGMY09/11 primers. All HPV- positive samples were genotyped individually by LA. Additionally, pools of amplicons from the PGMY-PCR products were sequenced using 454 NGS technology. Results obtained by NGS were compared with those of LA for each group of samples.ResultsWe identified 35 HPV genotypes, among which 30 were identified by both technologies; in addition, the HPV genotypes 32, 44, 74, 102 and 114 were detected by NGS. These latter genotypes, to our knowledge, have not been previously reported in Mexican population. Furthermore, we found that LA did not detect, in some diagnosis groups, certain HPV genotypes included in the test, such as 6, 11, 16, 26, 35, 51, 58, 68, 73, and 89, which indicates possible variations at the species level.ConclusionsThere are HPV genotypes in Mexican population that cannot be detected by LA, which is, at present, the most complete commercial genotyping test. More studies are necessary to determine the impact of HPV-44, 74, 102 and 114 on the risk of developing CC. A greater number of samples must be analyzed by NGS for the most accurate determination of Mexican HPV variants.

Overexpression of MEOX2 and TWIST1 Is Associated with H3K27me3 Levels and Determines Lung Cancer Chemoresistance and Prognosis

Lung cancer is the leading cause of death from malignant diseases worldwide, with the non-small cell (NSCLC) subtype accounting for the majority of cases. NSCLC is characterized by frequent genomic imbalances and copy number variations (CNVs), but the epigenetic aberrations that are associated with clinical prognosis and therapeutic failure remain not completely identify. In the present study, a total of 55 lung cancer patients were included and we conducted genomic and genetic expression analyses, immunohistochemical protein detection, DNA methylation and chromatin immunoprecipitation assays to obtain genetic and epigenetic profiles associated to prognosis and chemoresponse of NSCLC patients. Finally, siRNA transfection-mediated genetic silencing and cisplatinum cellular cytotoxicity assays in NSCLC cell lines A-427 and INER-37 were assessed to describe chemoresistance mechanisms involved. Our results identified high frequencies of CNVs (66–51% of cases) in the 7p22.3–p21.1 and 7p15.3–p15.2 cytogenetic regions. However, overexpression of genes, such as MEOX2, HDAC9, TWIST1 and AhR, at 7p21.2–p21.1 locus occurred despite the absence of CNVs and little changes in DNA methylation. In contrast, the promoter sequences of MEOX2 and TWIST1 displayed significantly lower/decrease in the repressive histone mark H3K27me3 and increased in the active histone mark H3K4me3 levels. Finally these results correlate with poor survival in NSCLC patients and cellular chemoresistance to oncologic drugs in NSCLC cell lines in a MEOX2 and TWIST1 overexpression dependent-manner. In conclusion, we report for the first time that MEOX2 participates in chemoresistance irrespective of high CNV, but it is significantly dependent upon H3K27me3 enrichment probably associated with aggressiveness and chemotherapy failure in NSCLC patients, however additional clinical studies must be performed to confirm our findings as new probable clinical markers in NSCLC patients.

HOXA9 is Underexpressed in Cervical Cancer Cells and its Restoration Decreases Proliferation, Migration and Expression of Epithelial-to-Mesenchymal Transition Genes.

HOX transcription factors are evolutionarily conserved in many different species and are involved in important cellular processes such as morphogenesis, differentiation, and proliferation. They have also recently been implicated in carcinogenesis, but their precise role in cancer, especially in cervical cancer (CC), remains unclear. In this work, using microarray assays followed by the quantitative polymerase chain reaction (qPCR), we found that the expression of 25 HOX genes was downregulated in CC derived cell lines compared with nontumorigenic keratinocytes. In particular, the expression of HOXA9 was observed as down-modulated in CCderived cell lines. The expression of HOXA9 has not been previously reported in CC, or in normal keratinocytes of the cervix. We found that normal CC from women without cervical lesions express HOXA9; in contrast, CC cell lines and samples of biopsies from women with CC showed significantly diminished HOXA9 expression. Furthermore, we found that methylation at the first exon of HOXA9 could play an important role in modulating the expression of this gene. Exogenous restoration of HOXA9 expression in CC cell lines decreased cell proliferation and migration, and induced an epithelial-like phenotype. Interestingly, the silencing of human papilloma virus (HPV) E6 and E7 oncogenes induced expression of HOXA9. In conclusion, controlling HOXA9 expression appears to be a necessary step during CC development. Further studies are needed to delineate the role of HOXA9 during malignant progression and to afford more insights into the relationship between downmodulation of HOXA9 and viral HPV oncoprotein expression during cercical cancer development.

Increased Expression of HOXB2 and HOXB13 Proteins is Associated with HPV Infection and Cervical Cancer Progression

BACKGROUND Cervical cancer (CeCa) is the second most common cancer in women in developing countries, and human papilloma virus (HPV) is the primary etiological factor. Aberrant expression of HOX transcription factors has been observed in several types of cancer. To date, however, no reports exist on the expression of HOXB2 and HOXB13 proteins during neoplastic progression in CeCa and its correlation with HPV infection. MATERIALS AND METHODS Expression of HOXB2 and HOXB13 proteins was assessed in tissue microarrays from normal cervical epithelium, cervical intraepithelial neoplasias grade 1-3, and CeCa. HPV was detected by PCR and sequencing. Expression of HOX-positive cells was determined in each diagnostic group. RESULTS Percentage of HOXB2- and HOXB13-positive cells gradually increased from means of 10.9% and 16.7%, respectively, in samples from healthy women, to 75.2% and 88.6% in those from CeCa patients. Frequency of HPV infection also increased from 13% in healthy tissue samples to 92.3% in CeCa. Both HOXB2 and HOXB13 proteins were preferentially expressed in HPV+ samples. CONCLUSIONS The present study represents the first report on the expression of both HOXB2 and HOXB13 proteins through cervix tumorigenesis, providing evidence that increased expression of such proteins is a common event during progression to CeCa.