Carter J. Barger

FOXM1 is frequently overexpressed in cancer, but this has not been studied in a comprehensive manner. We utilized genotype-tissue expression (GTEx) normal and The Cancer Genome Atlas (TCGA) tumor data to define FOXM1 expression, including its isoforms, and to determine the genetic alterations that promote FOXM1 expression in cancer. Additionally, we used human fallopian tube epithelial (FTE) cells to dissect the role of Retinoblastoma (Rb)-E2F and Cyclin E1 in FOXM1 regulation, and a novel human embryonic kidney cell (HEK293T) CRISPR FOXM1 knockout model to define isoform-specific transcriptional programs. FOXM1 expression, at the mRNA and protein level, was significantly elevated in tumors with FOXM1 amplification, p53 inactivation, and Rb-E2F deregulation. FOXM1 expression was remarkably high in testicular germ cell tumors (TGCT), high-grade serous ovarian cancer (HGSC), and basal breast cancer (BBC). FOXM1 expression in cancer was associated with genomic instability, as measured using aneuploidy signatures. FTE models confirmed a role for Rb-E2F signaling in FOXM1 regulation and in particular identified Cyclin E1 as a novel inducer of FOXM1 expression. Among the three FOXM1 isoforms, FOXM1c showed the highest expression in normal and tumor tissues and cancer cell lines. The CRISPR knockout model demonstrated that FOXM1b and FOXM1c are transcriptionally active, while FOXM1a is not. Finally, we were unable to confirm the existence of a FOXM1 auto-regulatory loop. This study provides significant and novel information regarding the frequency, causes, and consequences of elevated FOXM1 expression in human cancer.

Abstract A hallmark of human cancer is global DNA hypomethylation (GDHO), but the mechanisms accounting for this defect and its pathological consequences have not been defined in human epithelial ovarian cancer (EOC). In EOC, GDHO was associated with advanced disease and reduced overall and disease-free survival. GDHO(+) EOC was enriched for a proliferative gene expression signature, including CCNE1 and FOXM1 overexpression. DNA hypomethylation preferentially occurred within genomic blocks (hypomethylated blocks) overlapping late-replicating, lamina-associated domains, PRC2 binding, and H3K27me3. Increased proliferation coupled with hypomethylated block formation at late replicating regions suggested passive hypomethylation, which was further supported by the observation that cytosine DNA methyltransferases ( DNMTs) and UHRF1 showed significantly reduced expression in GDHO(+) EOC, after normalization to proliferation markers. Importantly, GDHO(+) EOC showed elevated chromosomal instability (CIN), and copy number alterations (CNA) were enriched at hypomethylated blocks. Together, these findings implicate a passive demethylation mechanism for GDHO that promotes genomic instability and poor prognosis in EOC.

A hallmark of human cancer is global DNA hypomethylation (GDHO), but the mechanisms accounting for this defect and its pathological consequences have not been investigated in human epithelial ovarian cancer (EOC). In EOC, GDHO was associated with advanced disease and reduced overall and disease-free survival. GDHO (+) EOC tumors displayed a proliferative gene expression signature, including FOXM1 and CCNE1 overexpression. Furthermore, DNA hypomethylation in these tumors was enriched within genomic blocks (hypomethylated blocks) that overlapped late-replicating regions, lamina-associated domains, PRC2 binding sites, and the H3K27me3 histone mark. Increased proliferation coupled with hypomethylated blocks at late-replicating regions suggests a passive hypomethylation mechanism. This hypothesis was further supported by our observation that cytosine DNA methyltransferases (DNMTs) and UHRF1 showed significantly reduced expression in GDHO (+) EOC after normalization to canonical proliferation markers, including MKI67. Finally, GDHO (+) EOC tumors had elevated chromosomal instability (CIN), and copy number alterations (CNA) were enriched at the DNA hypomethylated blocks. Together, these findings implicate a passive DNA demethylation mechanism in ovarian cancer that is associated with genomic instability and poor prognosis.

Forkhead box M1 (FOXM1) is a member of the conserved forkhead box (FOX) transcription factor family. Over the last two decades, FOXM1 has emerged as a multifunctional oncoprotein and a robust biomarker of poor prognosis in many human malignancies. In this review article, we address the current knowledge regarding the mechanisms of regulation and oncogenic functions of FOXM1, particularly in the context of ovarian cancer. FOXM1 and its associated oncogenic transcriptional signature are enriched in >85% of ovarian cancer cases and FOXM1 expression and activity can be enhanced by a plethora of genomic, transcriptional, post-transcriptional, and post-translational mechanisms. As a master transcriptional regulator, FOXM1 promotes critical oncogenic phenotypes in ovarian cancer, including: (1) cell proliferation, (2) invasion and metastasis, (3) chemotherapy resistance, (4) cancer stem cell (CSC) properties, (5) genomic instability, and (6) altered cellular metabolism. We additionally discuss the evidence for FOXM1 as a cancer biomarker, describe the rationale for FOXM1 as a cancer therapeutic target, and provide an overview of therapeutic strategies used to target FOXM1 for cancer treatment.

// Wa Zhang 1, 10 , Carter J. Barger 1 , Kevin H. Eng 4 , David Klinkebiel 2 , Petra A. Link 3 , Angela Omilian 5 , Wiam Bshara 5 , Kunle Odunsi 6, 7, 8 , Adam R. Karpf 1, 9 1 Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE, USA 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA 3 Department of Pharmacology, Roswell Park Cancer Institute, Buffalo, NY, USA 4 Department of Biostatistics and Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY, USA 5 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, USA 6 Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA 7 Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA 8 Center for Immunotherapy, Roswell Park Cancer Institute, Buffalo, NY, USA 9 Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA 10 Current address: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Correspondence to: Adam R. Karpf, email: adam.karpf@unmc.edu Keywords: PRAME, cancer testis antigens, epithelial ovarian cancer, high grade serous cancer, DNA methylation Received: May 02, 2016      Accepted: May 29, 2016      Published: June 13, 2016 ABSTRACT PRAME is a cancer-testis antigen (CTA) and potential immuno-therapeutic target, but has not been well-studied in epithelial ovarian cancer (EOC) or its high grade serous (HGSC) subtype. Compared to normal ovary, PRAME expression was significantly increased most EOC, regardless of stage and grade. Interestingly, PRAME mRNA expression was associated with improved survival in the HGSC subtype. The PRAME locus was a frequent target for copy number alterations (CNA) in HGSC but most changes were heterozygous losses, indicating that elevated PRAME expression is not typically due to CNA. In contrast, PRAME promoter DNA hypomethylation was very common in EOC and HGSC and correlated with increased PRAME expression. PRAME expression and promoter hypomethylation both correlated with LINE-1 hypomethylation, a biomarker of global DNA hypomethylation. Pharmacologic or genetic disruption of DNA methyltransferase (DNMT) enzymes activated PRAME expression in EOC cells. Immunohistochemistry (IHC) of PRAME in EOC revealed frequent, but low level, protein expression, and expression was confined to epithelial cells and localized to the cytoplasm. Cytoplasmic PRAME expression was positively associated with PRAME mRNA expression and negatively associated with promoter methylation, but the latter correlation was not statistically significant. PRAME protein expression did not correlate with EOC clinicopathology or survival. In summary, PRAME is frequently expressed in EOC at the mRNA and protein levels, and DNA methylation is a key mechanism regulating its expression. These data support PRAME as an immunotherapy target in EOC, and suggest treatment with DNMT inhibitors as a means to augment PRAME immunotherapy.