# Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer
## 2 Results
### FOXM1 Overexpression in Pan-Cancer Correlates with TP53 and RB1 Alterations
As hypothesized, BBC and HGSC showed similarly increased frequency of _FOXM1_ amplifications compared to other molecular subtypes of breast cancer (Figure 5A). In addition, there was a striking similarity in _FOXM1_ copy number alteration frequency between BBC and HGSC (Figure 5B). BBC and HGSC showed consistently increased FOXM1 mRNA and protein expression compared to other breast cancer molecular subtypes and normal breast tissue (Figure 5C,D). These data reveal a similar spectrum of FOXM1 alterations in BBC and HGSC, further underscoring the similar molecular phenotype of these tumors [6; 47]. Deregulation of the Rb-E2F Pathway, Including Cyclin E1 Expression, Promotes FOXM1 Overexpression in Fallopian Tube Epithelial (FTE) Cells, An HGSC Precursor Cell Model  
We show that FOXM1 expression increases with _TP53_ and _RB1_ genetic alterations in pan-cancer (Figure 4). We previously reported that FOXM1 expression increases in p53 and Rb disrupted ovarian surface epithelial (OSE) cells, and observed that _FOXM1_ expression decreases following E2F1 knockdown [25]. To validate these findings in a more relevant genetic context and precursor cell model for HGSC, we utilized hTERT-immortalized fallopian tube epithelial (FTE) cells [48; 49]. We used FTE cells engineered to overexpress mutant p53-R175H (FT282), large T antigen (TAg) (FT190), or mutant p53-R175H + Cyclin E1 (FT282-E1). Rb-E2F deregulation via either TAg or Cyclin E1 expression in FTE cells increased FOXM1 mRNA and protein expression compared to FTE cells expressing mutant p53 alone (Figure 6A,B). Notably, Cyclin E1 expressing cells showed the highest expression of FOXM1,  
Figure 5: _FOXM1_ copy number alterations (CNA) and expression in TCGA OV (HGSC) vs. BRCA (Breast) molecular (PAM50) subtypes. (**A**) _FOXM1_ amplification frequency in HGSC vs. breast molecular subtypes. (**B**) _FOXM1_ copy number status in HGSC vs. basal breast. (**C,D**) FOXM1 expression in HGSC vs breast molecular subtypes (**C**) _mRNA_ expression (RNA-seq) and (**D**) protein expression (RPPA, pan-can normalized). Lines represent group medians. Low and high dotted lines across the graph represent median expression for Her2, LumA and LumB breast molecular subtypes, and HGSC, respectively. revealing Cyclin E1 as a novel upstream regulator of FOXM1. Next, we introduced temporal genetic modifications to the Rb-E2F pathway into FT282 cells. We modified the lenti-CRISPR vector into an inducible system and engineered FTE cells for _RB1_ knockout using a guide RNA to human _RB1_[50; 51]. We doxycycline (dox) treated the resulting cells for 72 h, then sorted cells based on positive green fluorescent protein (GFP) expression to enrich for _RB1_ knockout. We expanded the cells without dox for one week prior to harvesting. Importantly, we observed a significant increase in FOXM1 mRNA and protein expression with RB knockout (Figure 6C,D). In addition, we observed increased expression of both FOXM1 mRNA and protein when E2F1 was inducibly overexpressed (Figure 6E,F). Figure 6: FOXM1 expression in fallopian tube epithelial (FTE) cells engineered for deregulation of the Rb-E2F pathway. (**A,B**) FOXM1 mRNA and protein expression were measured by RT-qPCR and Western blot, respectively, in the indicated FTE cell lines. \(\beta\)-actin is shown as a loading control. (**C,D**) FOXM1 expression by RT-qPCR and Western blot, respectively, in RB1 CRISPR KO FT282 cells. (**E,F**) FOXM1 expression by RT-qPCR and Western blot, respectively, in E2F1 inducible FTE cells. (**G,H**) FOXM1 expression by RT-qPCR and Western blot, respectively, in CCNE1 inducible FTE cells. Bars represent mean \(\pm\) SD. Student’s \(t\) test \(p\) values are shown. \(p\) value designations: *** \(<\) 0.001, ** \(<\) 0.01, * \(<\) 0.05. _CCNE1_ is a frequently amplified oncogene in HGSC, resulting in its overexpression [52].