Most frequently mutated genes in 19 ULMSs studied by exome-sequencing.

<p>Five tumors did not have mutations in any of the six genes listed.</p

Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

<div><p>Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of <i>RSK4</i>, <i>SPARC</i>, and <i>HOXA9</i> were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of <i>RSK4</i>, <i>SPARC</i>, <i>PROM1</i>, <i>HOXA10</i>, <i>HOXA9</i>, <i>WT1-AS</i>, <i>SFRP2</i>, <i>SFRP5</i>, <i>OPCML</i>, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.</p></div

Alignment of human and mouse <i>PIK3CA</i> transcriptional regulatory regions.

<p>Alignment of human and mouse <i>PIK3CA</i> transcriptional regulatory regions.</p

p110α is overexpressed in non-proliferating tumor cells <i>in vivo.</i>

<p>A. Immunohistochemical staining of p110α using high primary antibody concentration (1:50) reveals low expression of p110α diffusely in the cytoplasm of tumor cells in Ki67-positive (Ki67+) regions. B and C. High magnification of non-proliferating (B) and proliferating (C) regions from A.</p

NF-κB binds to the <i>PIK3CA</i> promoter and activates its expression.

<p>A. Illustration of the predicted NF-κB binding site in human (top) or murine (bottom) <i>PIK3CA</i> promoter region. B. Illustration of the constructs comprising luciferase linked to the human <i>PIK3CA</i> promoter with (Luc-2340/316) or without the NF-κB binding site (Luc-378/316). C. Summary of luciferase activities of Luc-2340/316 and Luc-378/316 co-transfected with pCMV- IκBα or pCMV- IκBαM. D. Gelshift assay using ovarian cancer cell nuclear extract after TNF-α stimulation. <u>Lane 1</u>, NF-κB binding site of wild-type human <i>PIK3CA</i> promoter (wt hu<i>PIK3CA</i> NF-κB probe) alone; <u>lanes 2 and 3</u>, wt hu<i>PIK3CA</i> NF-κB probe+nuclear extract; <u>lane 4</u>, control NF-κB probe+nuclear extract, <u>lanes 5 and 6</u>, mutated hu<i>PIK3CA</i> NF-κB probe+nuclear extract; <u>lane 7</u>, mutated control NF-κB probe+nuclear extract; <u>lanes 8 and 9</u>, scramble hu<i>PIK3CA</i> NF-κB probe+nuclear extract. E. Gel shift assay using recombinant NF-κB/p50 protein. <u>Lane 1</u>, wt hu<i>PIK3CA</i> NF-κB probe alone; <u>lanes 2 and 3</u>, wt hu<i>PIK3CA</i> NF-κB probe+p50; <u>lane 4</u>, mutated hu<i>PIK3CA</i> NF-κB probe alone; <u>lanes 5 and 6</u>, mutated hu<i>PIK3CA</i> NF-κB probe+p50; <u>lane 7</u>, control NF-κB probe alone; <u>lanes 8 and 9</u>, control NF-κB probe+p50.</p

5′ upstream regulatory sequence of the human <i>PIK3CA</i> gene.

<p>5′ upstream regulatory sequence of the human <i>PIK3CA</i> gene.</p

<i>PIK3CA</i> is upregulated in non-proliferating tumor cells <i>in vivo.</i>

<p>A to C. Double immunostaining of p110α (green, FITC) and Ki67 (red, Texas Red) in human ovarian cancer B. High magnification of a region from A with low expression of p110α. p110α is expressed at low levels and localizes to the cytoplasm of Ki67-positive tumor cells. C. High magnification of a region from A with high expression of p110α. p110α is expressed at high levels and localizes to the plasma membrane in Ki67-negative tumor cells. D and E. Immunohistochemical localization of Ki67 allows for clear identification of areas of proliferating and areas of non-proliferating tumor cells <i>in vivo</i> in 2008 ovarian xenograft tumors. E. High magnification of area from D showing the boundary between a proliferating and a non-proliferating region. F to H. Strong expression and cell membrane localization of p110α is only found in Ki67-negative areas. F. Section adjacent to D, stained with antibody against human p110α (1:250 dilution). G. High magnification of area from F showing the boundary between proliferating and non-proliferating region. H. High magnification of area from G showing membrane localization of p110α in the non-proliferating region. I. Immunostaining of human cytokeratin identifies tumor cells in proliferating and non-proliferating areas in the 2008 xenograft model. The line traces the boundary between the two areas, as defined by Ki67 staining in adjacent section (see J). Both proliferating and non-proliferating regions are positive for human cytokeratin (FITC, green), indicating tumor cells. Cell nuclei were counterstained with DAPI. J to L. Double p110α and Ki67 immunostaining maps <i>PIK3CA</i> activation in proliferating or non-proliferating areas in 2008 xenograft tumors. J. Ki67 (red, Texas Red) and p110α (FITC, green) exhibit reciprocal expression. K and L. High magnification of proliferating region (I) and non-proliferating region (H) from J.</p

Identification and characterization of the human <i>PIK3CA</i> promoters.

<p>A. Illustration of the structure of human <i>PIK3CA</i> gene and its 5′ upstream regulatory region. B. A region highly rich in GC is found in the <i>PIK3CA</i> 5′TRR. C. Illustration of the primers used for mapping RT-PCR of the <i>PIK3CA</i> transcriptional start site (SST). D. Results of mapping RT-PCR. There is no band between the forward primer F1 located upstream of the SST and the reverse primer R located on exon 1 of <i>PIK3CA</i>. The right size bands could be detected between primer F2 or F3 (both located downstream of SST) and reverse primer R. E. A small splicing variant is found in the 5′UTR of human <i>PIK3CA</i> gene, which can also be detected by mapping RT-PCR (primers F2 and R). F. Summarized results of the transcriptional activity of <i>PIK3CA</i> TRR fragments.</p