Citations

Abstract Background: The transcription factor Krüppel-like factor 5 (KLF5) is essential for normal homeostasis, stemness, and tumorigenesis. Typically, KLF5 functions as a transcriptional activator, including in esophageal epithelia where KLF5 promotes keratinocyte proliferation and migration by activating EGFR and ILK, respectively, and prevents transformation by activating Notch1. Yet, the function of KLF5 is context dependent, and mutation of the tumor suppressor p53 switches KLF5 from a transcriptional activator to a repressor of p21Waf1/Cip1 and from pro-proliferative to anti-proliferative. Interestingly, KLF5 also inhibits p53 itself, though the mechanism of this inhibition is unknown. We hypothesized that the functional switch of KLF5 from transcriptional activator to repressor involves the recruitment of a co-repressor. Methods: Primary human esophageal keratinocytes immortalized with hTERT were stably infected with inducible lentivirus containing shRNA against KLF5 or Sin3A. p53 mRNA and protein levels were assessed by real-time quantitative PCR and Western blotting, respectively, and p53 protein stability was evaluated by pulse-chase experiments. We employed ChIP across a 4 kb region on the p53 gene, from 2 kb upstream to 2 kb downstream of the transcription start site, to identify the region of KLF5 binding on p53. Promoter activity was assessed with a series of luciferase reporters containing varying lengths of p53 and its upstream regulatory region. Protein-protein interaction was examined by co-IP. Results: KLF5 knockdown significantly increased p53 both at the mRNA and protein levels. In unstressed cells p53 protein undergoes rapid degradation such that p53 levels are typically low. Yet, with KLF5 knockdown, despite increased p53 protein levels, we saw no changes in p53 protein stability. By ChIP, we identified a region of KLF5 binding to p53 between -600 to -356 bp upstream of the transcription start site. Several other KLF family members can interact with the co-repressor Sin3A, and we found that Sin3A also complexed with KLF5 in human primary esophageal keratinocytes. Knockdown of Sin3A abolished the inhibitory effect of KLF5 on p53, resulting in elevated p53 expression, growth inhibition, and increased apoptosis. Conclusion: KLF5 transcriptionally represses p53 via the co-repressor Sin3A. Thus, KLF5 promotes cell proliferation in normal keratinocytes by both activating pro-growth signals and inhibiting the brake machinery of the cell cycle. Citation Format: Yizeng Yang, Taicheng Zhou, Zhen Zong, Gwen Lomberk, Raul Urrutia, Jonathan Katz. Regulation of p53 function by KLF5. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2015-2087

Abstract Background/purpose: Despite recent advances in the development of androgen deprivation therapies (ADT), prostate cancer (PCa) remains the second leading cause of cancer death in American men. Thus, there is a critical need to improve treatment outcomes for men with aggressive PCa. The purpose of this study was to identify and functionally characterize kinases that can serve as both prognostic biomarkers and therapeutic targets in PCa. Methods: To identify kinases associated with metastatic progression of PCa, we utilized high-density oligonucleotide arrays to interrogate the expression of all kinases in 545 prostatectomy samples, obtained from high-risk patients with long-term clinical follow-up (>10 years). We ranked all kinases by their fold change in expression between PCas that subsequently metastasized versus those that did not. We then characterized the top ranked kinase in preclinical PCa models with in vitro mechanistic experiments and in vivo therapeutic studies. Results: We nominated DNA-dependent protein kinase (DNAPK) as the top kinase associated with metastatic PCa progression. Interrogation of pathways associated with DNAPK knockdown in PC3, DU145, VCaP and C4-2B, identified canonical Wnt signaling as the top DNAPK-activated pathway. In PCa cells (PC3, LNCaP C.S. {LNCaP grown under charcoal-stripped serum condition}, C4-2B and LNCaP-AR) DNAPK inhibition significantly abrogates Wnt signaling and abolishes cell proliferation, migration and invasion. Interestingly, we found that DNAPK directly modulates the Wnt signaling independent of the androgen receptor (AR), highlighting the therapeutic potential of DNAPK inhibition in cancers resistant to ADT. Indeed, in VCaP xenograft models, pharmacologic inhibition of DNAPK with NU7441 demonstrated 3.4 fold (p<0.01) reduction of tumor growth at non-toxic doses. Using an independent prostate cancer tissue cohort, we validate DNAPK expression as a biomarker for metastatic disease progression (p = 6.4 e−06, hazard ratio = 2). Conclusion: In summary, we nominate DNAPK as a potential biomarker of disease progression and as a novel therapeutic target in aggressive prostate cancer. Our data suggests that DNAPK mediates PCa progression by upregulating Wnt signaling, and demonstrates that DNAPK inhibition results in significant tumor responses in PCa xenografts. Citation Format: Vishal Kothari, Jonathan F. Goodwin, Shuang Zhao, Elai Davicioni, Jeffrey R. Karnes, Robert B. Den, Rohit Mehra, Karen E. Knudsen, Felix Y. Feng. Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2015-2853

Abstract Genome-wide association studies (GWAS) have identified several susceptibility regions that confer risk of two or more cancers. A region at 19p13 is associated with both breast and ovarian cancer risk in the general population as well as in BRCA1/BRCA2 mutation carriers. Using the iCOGs custom genotyping array, we analyzed 438 SNPs in this region in 7,435 ER-negative breast cancer cases and 42,599 controls from BCAC, and 15,347 ovarian cancer cases and 30,845 controls from OCAC and 15,252 BRCA1 mutation carriers. We identified two significant peaks of association. Peak 1, consisting of 13 highly correlated SNPs (r2>0.95), spans a 19.4kb region harboring the BABAM1, ANKLE1 and ABHD8 genes. Peak 1 SNPs are associated with serous ovarian cancer (top hit rs4808075, P = 9.2×10-20), ER-negative breast cancer (rs61494113, P = 1.1×10-13) and breast cancer in BRCA1 mutation carriers (rs61494113, P = 7.7×10-16). Peak 2 spans over 100kb and contains 7 SNPs associated with breast cancer in BRCA1 mutation carriers only (rs4464206, P = 8.9×10-8). Multiple assays were used to establish the putative functional roles of risk SNPs and candidate susceptibility genes in this region. Expression quantitative trait locus (eQTL) analyses identified significant genotype-gene expression correlations between genotype at the Peak 1 SNP rs480816 and ABHD8 expression in ovarian tumors (P = 3.0×10-5) and normal breast tissues (P = 2.8×10-3). The Peak 2 SNP, rs7246243, was significantly associated with BABAM1 expression (P = 5×10-3) in normal breast tissue only. Overexpression of BABAM1 in normal ovarian and breast cells did not modulate neoplastic phenotypes, whereas overexpression of ABHD8 induced a significant reduction in migration in both cell types in vitro. Differentially expressed genes in ABHD8 models, identified by RNAseq, were associated with cellular movement (P<1.82×10-44). Chromosome conformation capture (3C) identified an interaction between the ABHD8 promoter and a region containing 4 risk SNPs (rs4808075, rs10419397, rs56069439 and rs4808076) in breast and ovarian normal and cancer cells but only rs56069439 coincides with epigenetic enhancer marks in breast and ovarian cells. CRISPR/Cas9 genome editing was used to delete a 57bp region containing rs56069439 and expression of BABAM1, ANKLE1 and ABHD8 were measured. Taken together, these data suggest a common functional mechanism underlying risk of breast and ovarian cancer at this locus, mediated by rs56069439 variants differentially regulating expression of ABHD8, a gene involved in cell motility. Citation Format: Kate Lawrenson, Siddhartha Kar, Karoline Kuckenbaeker, Stacey Edwards, Qiyuan Li, Jonathan Tyrer, Jonathan Beesley, Kyriaki Michailidou, Susan Ramus, Alison Dunning, Fergus Couch, Alvaro Monteiro, Jacques Simard, Matthew Freedman, Douglas Easton, Georgia Chenevix-Trench, Paul Pharoah, Antonis Antoniou, Simon Gayther, OCAC, BCAC and CIMBA. Common functional mechanisms underlying pleiotropy at the 19p13.1 breast and ovarian cancer cusceptibility locus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2783. doi:10.1158/1538-7445.AM2015-2783

Abstract Emerging evidence demonstrates that the DNA repair kinase DNA-PK exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PK functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PK inhibits tumor metastases. Clinical assessment revealed that DNA-PK is significantly elevated in advanced disease, and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PK in advanced tumors is highly activated, independently of DNA damage indicators. Combined, these findings put forth new paradigms for DNA-PK function, identify DNA-PK as a potent driver of tumor progression and metastases, and nominate DNA-PK as a therapeutic target for advanced malignancies. Citation Format: Jonathan F. Goodwin, Vishal Kothari, Justin M. Drake, Shuang Zhao, Emanuela Dylgjeri, Jeffry L. Dean, Matthew J. Schiewer, Christopher McNair, Michael S. Magee, Robert B. Den, Ziqi Zhu, Nicholas A. Graham, Ajay A. Vashisht, James A. Wohlschlegel, Thomas G. Graeber, R Jeffrey Karnes, Mandeep Takhar, Elai Davicioni, Scott A. Tomlins, Nima Sharifi, Owen N. Witte, Felix Y. Feng, Karen E. Knudsen. DNA-PK-mediated transcriptional regulation drives tumor progression and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1860. doi:10.1158/1538-7445.AM2015-1860

Abstract Background: Genome-wide association studies (GWAS) have so far reported 12 loci associated with serous epithelial ovarian cancer (EOC) risk. We hypothesized that some of these loci function through nearby transcription factor (TF) genes and that putative target genes of these TFs as identified by coexpression may also be enriched for additional EOC risk associations. Methods: We selected TF genes within 1 Mb of the top signal at the 12 genome-wide significant risk loci. Mutual information, a form of correlation, was used to build networks of genes strongly coexpressed with each selected TF gene in the unified microarray dataset of 489 serous EOC tumors from The Cancer Genome Atlas. Genes represented in this dataset were subsequently ranked using a gene-level test based on results for germline SNPs from a serous EOC GWAS meta-analysis (2,196 cases/4,396 controls). Results: Gene set enrichment analysis identified six networks centered on TF genes (HOXB2, HOXB5, HOXB6, HOXB7 at 17q21.32 and HOXD1, HOXD3 at 2q31) that were significantly enriched for genes from the risk-associated end of the ranked list (P < 0.05 and FDR < 0.05). These results were replicated (P < 0.05) using an independent association study (7,035 cases/21,693 controls). Genes underlying enrichment in the six networks were pooled into a combined network. Conclusion: We identified a HOX-centric network associated with serous EOC risk containing several genes with known or emerging roles in serous EOC development. Impact: Network analysis integrating large, context-specific datasets has the potential to offer mechanistic insights into cancer susceptibility and prioritize genes for experimental characterization. Cancer Epidemiol Biomarkers Prev; 24(10); 1574–84. ©2015 AACR.

ObjectiveThe Lymphatic Malformation Function (LMF) instrument is a preliminary parent‐report assessment designed to measure outcomes in children with cervicofacial lymphatic malformation (LM). This study aimed to assess the measurement properties of the LMF, refine it, test criterion validity, and evaluate the test‐retest reliability.Study DesignCross‐sectional.SettingTwo pediatric tertiary referral centers.SubjectsParents of 60 children from 6 months to 15 years old with cervicofacial LM.MethodsParents were recruited via mail and online. The LMF was administered on paper or online initially and again within 21 days. Response distributions and interitem correlations were examined for item reduction. Exploratory factor analysis was conducted on retained items. Cronbach’s α, Spearman correlation, and intraclass correlation (ICC) coefficients were calculated to test internal consistency, criterion validity (compared to stage), and test‐retest reliability, respectively.ResultsOne item was removed due to a floor effect. The response scale was collapsed from a 5‐point scale to a 3‐point scale due to skewness. Six items were discarded due to redundancy (interitem correlations >0.7); 2 items were discarded due to factor loadings <0.4. Exploratory factor analysis revealed a 2‐factor structure explaining 84% of variance, and the domains Signs and Impacts had good internal consistency (all Cronbach’s α >0.80 and <0.90), significant association with stage (P <. 05), and good overall test‐retest reliability (ICC, 0.82).ConclusionThe LMF has been refined into a 12‐item, 2‐domain instrument measuring LM‐specific signs and impacts with internal consistency, criterion validity, and test‐retest reliability.

Abstract Purpose: Chemotherapy resistance remains a major challenge in the treatment of ovarian cancer. We hypothesize that germline polymorphisms might be associated with clinical outcome. Experimental Design: We analyzed approximately 2.8 million genotyped and imputed SNPs from the iCOGS experiment for progression-free survival (PFS) and overall survival (OS) in 2,901 European epithelial ovarian cancer (EOC) patients who underwent first-line treatment of cytoreductive surgery and chemotherapy regardless of regimen, and in a subset of 1,098 patients treated with ≥4 cycles of paclitaxel and carboplatin at standard doses. We evaluated the top SNPs in 4,434 EOC patients, including patients from The Cancer Genome Atlas. In addition, we conducted pathway analysis of all intragenic SNPs and tested their association with PFS and OS using gene set enrichment analysis. Results: Five SNPs were significantly associated (P ≤ 1.0 × 10−5) with poorer outcomes in at least one of the four analyses, three of which, rs4910232 (11p15.3), rs2549714 (16q23), and rs6674079 (1q22), were located in long noncoding RNAs (lncRNAs) RP11-179A10.1, RP11-314O13.1, and RP11-284F21.8, respectively (P ≤ 7.1 × 10−6). ENCODE ChIP-seq data at 1q22 for normal ovary show evidence of histone modification around RP11-284F21.8, and rs6674079 is perfectly correlated with another SNP within the super-enhancer MEF2D, expression levels of which were reportedly associated with prognosis in another solid tumor. YAP1- and WWTR1 (TAZ)-stimulated gene expression and high-density lipoprotein (HDL)-mediated lipid transport pathways were associated with PFS and OS, respectively, in the cohort who had standard chemotherapy (pGSEA ≤6 × 10−3). Conclusions: We have identified SNPs in three lncRNAs that might be important targets for novel EOC therapies. Clin Cancer Res; 21(23); 5264–76. ©2015 AACR.