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.
The FOXM1 transcription factor is an oncoprotein and a top biomarker of poor prognosis in human cancer. Overexpression and activation of FOXM1 is frequent in high-grade serous carcinoma (HGSC), the most common and lethal form of human ovarian cancer, and is linked to copy number gains at chromosome 12p13.33. We show that FOXM1 is co-amplified and co-expressed with RHNO1, a gene involved in the ATR-Chk1 signaling pathway that functions in the DNA replication stress response. We demonstrate that FOXM1 and RHNO1 are head-to-head (i.e., bidirectional) genes (BDG) regulated by a bidirectional promoter (BDP) (named F/R-BDP). FOXM1 and RHNO1 each promote oncogenic phenotypes in HGSC cells, including clonogenic growth, DNA homologous recombination repair, and poly-ADP ribosylase inhibitor resistance. FOXM1 and RHNO1 are one of the first examples of oncogenic BDG, and therapeutic targeting of FOXM1/RHNO1 BDG is a potential therapeutic approach for ovarian and other cancers.
The National Institutes of Health and the National Science Foundation have made a compelling call to action not only to strengthen the pipeline of available STEM-trained talent, but in addition to foster students who are members of populations currently under-represented in science. Furthermore, the scientific community must not only increase the accessibility of STEM-related education, but also implement and test evidence-based practices. Presented here, we detail the proceedings of a hands-on, science-focused informal learning opportunity aimed at educating an underrepresented population in cancer biology. Fifteen undergraduate and graduate student volunteer instructors from the University of Nebraska at Omaha and the University of Nebraska Medical Center engaged with 89 high school students, mostly Native American, in an informal learning event called "Cancer Biology and You Day." Throughout the event, students completed two independent lessons focusing on breast cancer and skin cancer and demonstrated strong learning gains associated with the lessons as assessed by KWL charts. Exit surveys of the students indicated high levels of satisfaction with the event, and positive attitudes associated with considering a career in science/research were evident in survey responses. Overall, we report the event as a success and outline how similar experiences may be achieved. Although the rate at which American Indian/Alaska Native (AI/AN) students start an undergraduate degree is Cancer Learning Event for Native American Students -Herek Vol. 2, September 2019 Journal of STEM Outreach METHODSInstitutional Review Board. All protocols and data collections were systematically collected about the activities and outcomes of the specific program event to contribute to continuous improvement and inform future program development. Thus, this study was approved by the UNMC Institutional Review Board as an educational exempt program assessment. All administered surveys were voluntary, and identities anonymized prior to electronic upload and analysis. Journal of STEM OutreachDetails of the Event. Eighty-nine registered student attendants, mostly Native American, attended the event, accompanied by their teachers. The group was comprised of students from five schools on Native American Reservations in Nebraska and South Dakota. The exact number of students who were Native American is not known, since the attendees were not required to identify their race; however, most of the partner schools invited have a student population that is >95% American Indian. The invitation issued to the schools by the UNMC YES Program did not specify any subset of students (e.g., science class students); whether the schools selected specific subsets of students to bring to the event is
We report that the oncogenic transcription factor FOXM1 is arranged in a head-to-head configuration with RHNO1, a gene involved in the ATR/CHK1-dependent DNA replication stress (DRS) response. FOXM1 and RHNO1 are both amplified and upregulated in high-grade serous ovarian cancer (HGSC). FOXM1 and RHNO1 expression are closely associated in normal and cancer tissues, including single cells, and a bidirectional promoter (F/R-BDP) mediates balanced expression. Targeting of FOXM1 and RHNO1 in HGSC cells using shRNA, CRISPR mutagenesis, or CRISPR interference directed to the F/R-BDP reduced DNA homologous recombination repair (HR) capacity, increased DNA damage, reduced clonogenic survival, and sensitized HGSC cells to the poly-ADP ribosylase inhibitor (PARPi) olaparib. Thus, there is functional cooperativity between FOXM1 and RHNO1 in cancer cells, and combinatorial targeting of this bidirectional gene pair may be a novel cancer therapeutic strategy. More broadly, our data provide evidence that bidirectional gene units function in human cancer.Here, we show that FOXM1 and RHNO1 are co-amplified and co-expressed in HGSC, and that their expression in controlled by a bidirectional promoter (named the F/R-BDP). We additionally show that FOXM1 and RHNO1 they cooperatively promote HR, cell survival, and PARPi resistance. Co-regulation and functional cooperativity between FOXM1 and RHNO1 suggests that this bidirectional gene unit is a potential therapeutic target in HGSC and/or other cancers. More generally, our data provide evidence that cooperative bidirectional gene units functionally contribute to cancer. ResultsThe 12p13.33 amplicon contains FOXM1 and RHNO1, co-expressed bidirectional genes FOXM1 is located at 12p13.33, a region with frequent copy number gains in HGSC and several other cancers (Barger et al., 2019;Barger et al., 2015). 12p13.33 amplification was associated with reduced overall survival in HGSC but, unexpectedly, FOXM1 mRNA expression did not (Barger et al., 2015). The 12p13.33 amplicon includes 33 genes (Figure S1). To identify genes at 12p13.33 that might functionally cooperate with FOXM1, we performed expression correlation analyses. FOXM1 expression showed the strongest correlation with RHNO1 expression in HGSC (Figure 1A), pan-cancer (data not shown), and normal human tissues ( Figure S2). In addition, RHNO1 mRNA expression correlated with reduced HGSC survival (data not shown). Genomic analyses of FOXM1 and RHNO1 revealed a head-to-head arrangement with an intervening putative bidirectional promoter (BDP) ( Figure S3). Approximately 10% of human genes are arranged in this manner, and the gene pairs are frequently co-regulated and can function in similar biochemical pathways (Trinklein et al., 2004;Wakano et al., 2012;. The FOXM1/RHNO1 BDP (F/R-BDP) region contains a CpG island (CGI), a common feature of BDPs ( Figure S3) (Antequera, 2003;Takai and Jones, 2004;Wakano et al., 2012).BDPs are often enriched with specific histone modifications H3K4me3, H3K9ac, H3K27ac, and H3K4me2 (Bornelov et al., 20...
PURPOSE: The FOXM1 transcription factor is canonically involved in cell cycle progression, but also promotes DNA repair and drug resistance, via its transcriptional activity. We previously reported that FOXM1 is located within the Ch.12p13.33 amplicon, which sustains copy number gains in ~60% of high-grade serous ovarian cancer (HGSC). More recently, we discovered that FOXM1 is arranged in a head-to-head configuration with RHNO1, a gene involved in the ATR/CHK1-dependent DNA replication stress (DRS) response. By virtue of its role in the DRS response, RHNO1 promotes homologous recombination (HR) DNA repair. We hypothesized that FOXM1 and RHNO1 are co-expressed in HGSC due to their bidirectional configuration, and that they functionally cooperate to promote HGSC cell survival, HR repair, and PARPi resistance. METHODS: We used 5' RACE, reporter constructs, and CRISPR-Cas9 (CRISPR) to characterize the FOXM1-RHNO1 bidirectional promoter. We correlated FOXM1 and RHNO1 mRNA expression and copy number in normal tissue and cancer data, including single cell RNA sequencing (scRNA-seq) of immortalized fallopian tube epithelial (FTE) cells and HGSC cells. We used RNAi and CRISPR to disrupt FOXM1 and/or RHNO1 in HGSC cells, and determined the resulting impact on gene expression, cell cycle, cell survival, HR repair, and olaparib (PARPi) sensitivity. RESULTS: FOXM1 and RHNO1 mRNA expression significantly correlated in normal tissues, including FTE, and in HGSC tumors and cell lines, both in bulk cells/tissues and at the single cell level. Co-expression appeared to result from the FOXM1-RHNO1 bidirectional promoter, which showed similar activity in each direction in cell models and correlated with endogenous mRNA expression. FOXM1 knockdown in HGSC cells led to enriched gene expression signatures for the G2/M checkpoint and reduced clonogenic survival. RHNO1 knockdown attenuated ATR/CHK1 signaling, reduced HR repair efficiency, and reduced HGSC clonogenic survival. Dual targeting of FOXM1 and RHNO1 using shRNA caused an additive effect on HR repair and clonogenic survival and caused synergistic sensitization of HGSC cells to olaparib treatment. Critically, bidirectional promoter targeting via a CRISPR-KRAB repressor recapitulated the effects of shRNA knockdown. Finally, FOXM1/RHNO1 bidirectional promoter repression led to olaparib sensitization in an in vitro HGSC model of acquired olaparib resistance. CONCLUSIONS: We demonstrate that FOXM1 and RHNO1 are frequently co-expressed in HGSC, most likely due to coordinated activity from their bidirectional promoter. Importantly, the FOXM1/RHNO1 bidirectional unit exhibits functional cooperativity for HR DNA repair and cell survival, and synergistically promotes olaparib resistance. Based on our data, we suggest that functionally interactive bidirectional gene modules are an under-appreciated oncogenic mechanism. Finally, we suggest that FOXM1 and RHNO1, or their associated pathways, are promising combinatorial therapeutic targets in HGSC. Citation Format: Carter J Barger, Linda Chee, Connor Branick, Kunle Odunsi, Lee Zou, Adam R. Karpf. CO-REGULATION AND FUNCTIONAL COOPERATIVITY BETWEEN FOXM1 AND RHNO1 BIDIRECTIONAL GENES IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-034.
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