# Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer
## 3 Discussion  
While FOXM1 over-expression is reported in many different cancers, there has not been a comprehensive pan-cancer analysis of FOXM1 status. To address this, we performed a pan-cancer analysis of FOXM1 across 32 TCGA cancer types compared to TCGA normal and GTEx normal tissues. Importantly, in TCGA, increased \(\mathit{FOXM1}\) mRNA expression corresponded to both increased FOXM1 protein expression and increased target gene expression, showing that FOXM1 is functionally active in these tumors. Another notable finding was that \(\mathit{FOXM1}\) copy number alterations contribute to FOXM1 overexpression. For example, TGCT (i.e., testicular germ cell tumors) showed the highest FOXM1 expression level and \(\mathit{FOXM1}\) amplification frequency among TCGA cancer types. Our analyses revealed cancer types with distinct levels of \(\mathit{FOXM1}\) copy number alterations and expression, including HGSC and BBC, which have highly elevated FOXM1, and THCA and PCPG, which show remarkably lower FOXM1. Notably, within BRCA, only BBC showed increased FOXM1 expression, emphasizing the importance of genomics in classifying tumors for therapeutic targeting. Consistent with this, a recent study identified FOXM1 as a valuable therapeutic target in BBC [57], a disease, like HGSC, that is in need of new therapeutic strategies. The data presented here provide useful knowledge for prioritizing the study of FOXM1 function and therapeutic targeting in different types of human cancer. Pan-cancer analyses demonstrated that FOXM1 expression is increased in TCGA tumors with p53 mutations and Rb loss of function, and we validated these findings using genetic modeling of p53, Rb/E2F1, and Cyclin E1 in FTE cells. In particular, we identified Cyclin E1 as an upstream activator of FOXM1 expression. In cell models, Cyclin E1 induced FOXM1 but the reverse was not true, and TCGA data sets showed significant associations between Cyclin E1 and FOXM1 expression. Cyclin E1/CDK2 complexes inactivate Rb by phosphorylation, leading to E2F activation [58], which may mechanistically explain why Cyclin E1 induces FOXM1 expression, but this requires testing. Moreover, TP53 mutations are an early or initiating event in HGSC, and Cyclin E1 and FOXM1 expression are detected in HGSC precursor lesions [59; 60; 52]. Our data thus suggest that FOXM1 expression may contribute to early oncogenic events in HGSC development and progression, alone or in combination with Cyclin E1, potentially including the induction of genomic instability. In support of this idea, forced overexpression of FOXM1 contributes to genomic instability in cell line models [43; 61]. Pan-cancer analyses performed here also demonstrated a significant association between both FOXM1 mRNA and protein expression and aneuploidy. Importantly, these associations were retained in tumors with diploid FOXM1, suggesting that increased FOXM1 expression promotes genomic instability, rather than just representing a by-product of it. FOXM1 has three predominant splice variants: FOXM1a, b, and c, which encode proteins with varying activities [1]. Considering the differential expression and functional potential of differentFOXM1 isoforms, it is important to determine which variants are responsible for oncogenic activity. We performed a comprehensive analysis of _FOXM1_ isoform expression in GTEx and TCGA normal, TCGA pan-cancer, and CCLE cell lines to determine the FOXM1 isoform expression profiles. We found that the predominant _FOXM1_ isoform expressed in all contexts was FOXM1c. However, analysis of TCGA paired normal and tumor showed that all three _FOXM1_ isoforms have relatively equivalent increases in paired cancer vs. normal tissues. We further investigated FOXM1 isoform transcriptional activity using cell models, FOXM1 reporter assays, endogenous target gene expression, and RNA-seq analyses. Our data confirmed that FOXM1c and FOXM1b are transcriptionally active but that FOXM1a is not, or has highly reduced activity.