# Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer
## 2 Results
### FOXM1 isoforms b and c Have Similar Transcriptional Activity  
FOXM1 encodes several transcripts, two of which (b and c) are the predominant isoforms used in most experimental studies. To understand how increased FOXM1 isoform expression in cancer relates to function, we used CRISPR to knock out _FOXM1_ in 293T cells (Figure 9A). We validated two FOXM1 knockout cell clones using the 6X-FOXM1 luciferase reporter assay, and observed the knockout clones showed significantly decreased reporter activity compared to parental cells (Figure 9B). We next tested the ability of FOXM1 isoforms to rescue FOXM1 reporter activity and observed that FOXM1 isoforms b and c were capable of rescue, but FOXM1a and a FOXM1c DNA binding domain mutant (DBD MT) were not (Figure 9C). We additionally confirmed the transcriptional activity of FOXM1 isoforms in FT282 cells, which have low endogenous FOXM1 expression (Figure 6), and observed that FOXM1b and FOXM1c activated the 6X-FOXM1 reporter, while FOXM1a and a FOXM1c DBD MT did not (Figure S5). To extend these observations, we reconstituted a 293T FOXM1 knockout clone with FOXM1a, b, or c (Figure 9D) and performed RNA-seq (Table S3). We compared transcriptional  
Figure 8: _FOXM1_ isoform expression in GTEx normal, TCGA normal, and TCGA cancer tissues. (**A**) _FOXM1_ isoform expression (RNA-seq RSEM, log2(norm count +1)) in GTEx (\(n\) = 843) and TCGA normal (\(n\) = 175) and cancer tissues (\(n\) = 6309). (**B**–**E**) _FOXM1_ isoform expression (RNA-seq RSEM, log2(norm count +1)) in TCGA paired normal and tumor tissues (\(n\) = 135). (**B**) _FOXM1a_ mRNA expression (RNA-seq RSEM, log2(norm count +1)). (**C**) _FOXM1b_ mRNA expression (RNA-seq RSEM, log2(norm count +1)). (**D**) _FOXM1c_ mRNA expression (RNA-seq RSEM, log2(norm count +1)). (**E**) _FOXM1_ isoform mRNA expression (RNA-seq RSEM, log2(norm count+1)) ratio between paired tumor and normal tissue (\(n\) = 135).  
changes, including both significant increases and decreases (False Discovery Rate (FDR) \(<\) 0.05), associated with FOXM1 isoform reconstitution and observed that FOXM1c and b produced the greatest overlap in transcriptional targets (Figure 9E; Table S4). When comparing only genes that showed a significant increase in expression (FDR \(<\) 0.05), we again observed greatest overlap between FOXM1c and FOXM1b, and minimal overlap with FOXM1a (Figure 9F; Table S5). Collectively, these data confirm that FOXM1c and FOXM1b are transcriptionally active, but that FOXM1a is either inactive or shows substantially less activity.  
Figure 9: FOXM1 isoform transcriptional activity in HEK293T (293T) FOXM1 knockout cells. **(A)** PCR genotype of two _FOXM1_ CRISPR knockout clones. **(B)** 6X-FOXM1 reporter assay in 293T _FOXM1_ knockout clones. **(C–F)** 293T _FOXM1_ knockout clone 7 transiently reconstituted with FOXM1 isoforms a, b and c. **(C)** 6X-FOXM1 reporter assay. **(D)** FOXM1 Western blot related to RNA-seq. **(E–F)** Venn diagrams of gene transcripts showing significant changes in expression for each FOXM1 isoform. **(E)** Overlap of gene transcripts showing those with both significant increases and decreases (FDR \(<\) 0.05) in expression. **(F)** Overlap of transcriptional targets showing only those with significant (FDR \(<\) 0.05) increases in expression. Bars represent mean \(\pm\) SD. Student’s \(t\) test \(p\) values are shown. \(p\) value designations: *** \(<\) 0.001.