Paper presentation

1. Paperpresentation Rocío Rojo PhD 13th May 2019

2. Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus Date of publication: 26thJuly 2018 Journal: Cell Impact factor (JCR): 31.398 Citations (reportedbyScopus) so far: 8 total, 7 type A Authors:Ping Gao, Ji-Han Xia, CsillaSipeky, Xiao-Ming Dong, Qin Zhang, Yuehong Yang, Peng Zhang, Sara Pereira Cruz, Kai Zhang, Jing Zhu, Hang-Mao Lee, SufyanSuleman, NikolaosGiannareas, Song Liu, The PRACTICAL Consortium, Teuvo L.J. Tammela, AnssiAuvinen, Xiaoyue Wang, Qilai Huang, Liguo Wang, AkiManninen, Markku H. Vaarala, Liang Wang, Johanna Schleutker,Gong-Hong Wei First and correspondingauthors: Affilitations:Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland Scopus reports: Gao Ping, PhD Wei Gong-Hong, PhD Papers published: h-index: Citation overview (2015-2019): 13 7 199 total 157 type A 35 13 1,754 total 1,565 type A

3. Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus In Brief: A non-coding risk allele associated with aggressive prostate cancer creates a transcription factor binding site that in turn promotes oncogenesis by impacting expression of nearby genes. Graphical Abstract:

4. Overview Incidence and mortality of prostate cancer (PCa): Source: https://onlinelibrary.wiley.com/doi/full/10.3322/caac.21492 PCa subtypes: • Indolent non-fatal PCa • Aggressive PCa

5. Overview • So far, Genome-Wide Association Studies (GWAS) have identified a number of Single-Nucleotide Polymorphisms (SNPs) associated with PCa risk. • In 2014, meta-analyses of cohorts with European ancestry identified the SNP “rs11672691” at 19q13 and found that its risk allele is associated with predisposition to aggressive PCa and with worse progression to PCa-specific mortality following diagnosis [2-3]. • This study, confirmed such association in a Finnish cohort (2,738 PCa patients vs 2,427 controls), investigated the molecular mechanisms that are potentially involved and experimentally validated their results. Source: https://www.ncbi.nlm.nih.gov/snp/rs11672691

6. Confirmation of the presence of rs11672691 in the population of interest and assessment of a potential clinical relevance in PCa Finnish cohort (2,738 PCa patients vs 2,427 controls) Clinical stratification of PCa patients (aggressive vs non-aggressive) SNP genotyping (blood DNA + Illumina iSelect SNP genotyping array) Case-control unconditional regression analysis Estimation of per allele odds ratio • Findings: • rs11672691 was present at locus 19q3 in the population studied • The allele G of rs11672691 is associated with predisposition to aggressive PCa and with worse progression to PCa-specific mortaility following diagnosis.

7. Identification of candidate genes by eQTL analysis at the 19q13 aggressive PCa locus The allele G of rs11672691 is associated with overexpression of CEACAM21 in different cohorts of PCa patients R package “Matrix eQTL” to find association between genotype and gene expression Fig 1. The aggressive PCa risk allele G at rs11672691 associates with increased expression of CEACAM21in the Swedish (A), TCGA (B) and Wisconsin (C) cohorts of prostate specimens and PCAT19(D) in the Wisconsin cohort of prostate tissues. Linear model p values assessed by Matrix eQTL.

8. Identification of candidate genes by eQTL analysis at the 19q13 aggressive PCa locus Downregulation of any of the two loci of interest in PCa cell lines negatively affects biological functions involved in aggressiveness Fig 1. PCAT19 (E) or CEACAM21 (F) promotes PCa cell proliferation measured by XTT colorimetric assays (mean ± SD of triplicate experiments), and aggressiveness by migration and invasion assays (mean ± SEM of triplicate experiments) in 22Rv1 cells infected with control or gene-specific shRNAs. 22Rv1: PCacell line; lentivirus-mediatedshRNAagainstthetwo loci of interest

9. Identification of candidate genes by eQTL analysis at the 19q13 aggressive PCa locus Ectopic expression of CEACAM21 in a normal prostate cell line promotes an aggressive phenotype in vitro and enriches transcription of genes associated with the MYC and mTOR signaling pathways: RWPE1: Normal-prostatecell line; ectopicexpressioninductionwasplasmid- orlentivirus vector-based Transcription of PCAT19 and CEACAM21 is increased in different cohorts of PCa patients and it correlates with a worse prognosis: PrimaryPCatissues Metastasis Prostate carcinoma

10. rs11672691 resides in the binding site of a transcriptional complex including HOXA2 rs11672691 is a putative enhancer region as it shows enrichment of marks related to transcriptional activity: Active enhancer Silent / inactive TF Binding HOXA2 directly binds at the rs11672691 enhancer with preference to the aggressive PCa risk-associated G allele: Tumor tissue PCa cell line Normal prostate cell line

11. HOXA2 as an Oncogene with Prognostic Potential in PCa HOXA2 downregulation decreases aggressiveness-related biological activity in PCa cell lines: HOXA2 mRNA is upregulated in primary and metastatic PCa tumors: Higher levels of HOXA2 show predictive values for increased biochemical recurrence and reduced survival:

12. HOXA2 as regulator of rs11672691 eQTL Genes Expression of HOXA2 and the potential target loci are correlated both in vitro and in vivo: PCAT19 exerts and enhancer-like function to promote expression of CEACAM21 in PCa: PCAT19 and CEACAM21 chromatin interaction might be directly attributed to rs11672691 :

13. Direct effect of rs11672691 on its eQTL gene expression and PCa cell proliferation The G risk allele of rs11672691 directly contributes to enhance expression of PCAT19 and CEACAM21 as well as to PCa cell proliferation and severity:

14. Summary Authors identified a regulatory circuit among several novel genes underlying the association of rs11672691 and an aggressive PCa phenotype. They revealed that rs11672691 is an enhancer variant and synergizes with HOXA2 to drive the expression of its target loci. CEACAM21 has not been thoroughly studied, but it plays roles in cell adhesion, invasion and metastasis; which might explain the outcome of this research. Data imply that rs11672691 might affect indirectly on CEACAM21 expression through PCAT19, and this mechanism involves transcription factors such as HOXA2. This group obtained direct evidence of a role for rs11672691 allele G in enhanced chromatin binding of HOXA2, increased PCAT19 and CEACAM21 expression and PCa aggressiveness. Results correlated with the clinical scenarios in a number of cohorts of PCa patients. Altogether, these findings might lead to improved prognosis for PCa patients.

15. “Pros” I consider it is very useful to develop research projects that amalgamate bioinformatics and lab-based research and that hold a clinical aim. I (think I) increased (a little bit) my knowledge on bioinformatics. “Cons” Ethnicity was quite limited in the analyzed population. Potential area of opportunity to use primary tumor cells when studying these kind of phenomena. Data was (sometimes) unnecessarily too dense (e.g. shRNA plots). Methods were not thorough enough to assure experimental reproducibility. There was some missing information regarding description of figures/results.

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