# Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer
## 1 Introduction  
FOXM1 is a member of the Forkhead box (FOX) transcription factor family, which is unified by a conserved winged helix DNA binding motif [1]. FOXM1 plays a key role in proliferation and cell cycle progression through transcriptional activation of a G2/M-specific gene network [2; 3; 4]. Increased FOXM1 gene expression and its transcriptional signature are detected in many cancer types [5; 6; 7; 8; 9]. Notably, pan-cancer analysis identified FOXM1 as the top gene associated with poor prognosis [10]. FOXM1 is also a promising therapeutic target for cancer treatment [11; 12; 13; 14; 15]. While increased FOXM1 expression is reported in many cancers, we lack an understanding of how FOXM1 copy number, mRNA, and protein expression compare and are associated in pan-cancer.  
Human _FOXM1_ has ten exons, with alternative splicing of exons Va and VIIa, giving rise to three variants: FOXM1a, FOXM1c, and FOXM1b. FOXM1b and -c are transcriptional activators, while FOXM1a was reported to be transcriptionally inactive [16]. While other cancer-specific transcriptshave been reported for FOXM1 [17; 18], only FOXM1a, -b and -c have been consistently investigated for their expression and function [19; 20; 21; 22; 23; 24; 25; 26]. Previous studies of _FOXM1_ isoform expression in cancer have reported that either _FOXM1b_ or _FOXM1c_ has the highest level of expression; however, these studies use different methods and have small sample sizes. Additionally, studies of _FOXM1_ isoform expression have focused on a single or limited number of cancer types, warranting a need for a comprehensive analysis using normal and cancer tissues. In this regard, the first comprehensive comparison of _FOXM1_ isoform expression employed genotype-tissue expression (GTEx) normal, The Cancer Genome Atlas (TCGA) cancer, and Target Cancer datasets [27], and validated the findings of others showing that _FOXM1c_ has the highest expression followed by _FOXM1b_ and _FOXM1a_[25; 26; 28]. However, it is still uncertain if FOXM1 isoforms show a differential increase in expression between paired normal and tumor samples. Furthermore, FOXM1 isoforms have been assigned various functions and transcriptional activities but these studies were limited to luciferase promoter assays or targeted gene expression studies [16; 21; 29; 30].  
Herein, we: 1) investigated changes in and links between FOXM1 copy number, mRNA, and protein expression across TCGA cancers; 2) compared _FOXM1_ expression in cancer vs. normal tissues; 3) identified key genomic features that contribute to increased FOXM1 expression using both pan-cancer analyses and engineered cell models; and 4) determined the association between FOXM1 expression and cancer genomic instability. Furthermore, we determined _FOXM1_ isoform expression in GTEx and TCGA normal and TCGA cancer tissues, TCGA paired normal and tumor samples, and human immortalized and cancer cell lines. Finally, we characterized the transcriptional activity of the three major FOXM1 isoforms in a newly developed FOXM1 knockout cell line, using luciferase assays and RNA-sequencing. Together, these data provide significant new understanding of the nature of FOXM1 deregulation in cancer, including its causes and potential consequences. The findings presented are also relevant for targeting FOXM1 in cancer, both by revealing vulnerable cancer types and by identifying potential biomarkers for therapeutic studies.