1. Cancer Biology
  2. Chromosomes and Gene Expression
Download icon

Co-regulation and function of FOXM1/RHNO1 bidirectional genes in cancer

  1. Carter J Barger
  2. Linda Chee
  3. Mustafa Albahrani
  4. Catalina Munoz-Trujillo
  5. Lidia Boghean
  6. Connor Branick
  7. Kunle Odunsi
  8. Ronny Drapkin
  9. Lee Zou
  10. Adam R Karpf  Is a corresponding author
  1. Eppley Institute for Cancer Research and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, United States
  2. Departments of Gynecologic Oncology, Immunology, and Center for Immunotherapy, Roswell Park Comprehensive Cancer Center, United States
  3. Penn Ovarian Cancer Research Center, University of Pennsylvania Perelman School of Medicine, United States
  4. Massachusetts General Hospital Cancer Center, Harvard Medical School, United States
Research Article
Cite this article as: eLife 2021;10:e55070 doi: 10.7554/eLife.55070
21 figures, 1 table and 4 additional files

Figures

The 12p13.33 amplicon in high-grade serous carcinoma (HGSC).

(A) Recurrent copy number amplifications in The Cancer Genome Atlas HGSC data (n = 579), as determined by GISTIC. Black vertical line indicates the threshold for significance, corresponding to an false discovery rate (FDR) q-value <0.25. The 12p13.33 genomic amplicon is indicated with an arrow. (B) The 33 genes in the 12p13.33 amplicon, listed top to bottom in genomic order from centromere to telomere. FOXM1 and RHNO1 are shown in red.

Figure 2 with 2 supplements
FOXM1/RHNO1 expression correlation in high-grade serous carcinoma (HGSC) and genomic characteristics.

(A) Hierarchical clustering dendrogram of The Cancer Genome Atlas HGSC mRNA expression correlations of the 33 genes in the 12p13.33 amplicon. FOXM1 and RHNO1 are boxed. (B) Genomic configuration and characteristics of FOXM1 and RHNO1. Transcript structure, CpG islands, epigenetic marks, and vertebrate genome conservation data are shown. Data were obtained from the UCSC Genome Browser and ENCODE.

Figure 2—figure supplement 1
The FOXM1/RHNO1 bidirectional promoter (F/R-BDP) region is hypomethylated in normal and cancer tissues.

(A) Distribution of DNA methylation at the F/R-BDP in The Cancer Genome Atlas normal and tumor tissues and Cancer Cell Line Encyclopedia cancer cell lines. Beta values represent the level of DNA methylation, averaged across seven CpG sites within the F/R-BDP CpG island. A value of 1 corresponds to fully methylated, while a value of 0 corresponds to fully unmethylated. (B) F/R-BDP regional DNA methylation as determined by sodium bisulfite clonal sequencing. Gene transcriptional start sites are indicated by the red broken arrows. Filled and open circles indicate methylated and unmethylated CpG sites, respectively, and each row represents one sequenced allele. Four separate epithelial ovarian cancer (EOC) samples with patient identifiers are shown. A mixture of normal ovary samples was analyzed as a control.

Figure 2—figure supplement 2
DNA sequence of the FOXM1/RHNO1 bidirectional promoter (F/R-BDP).

Transcriptional start sites predicted by NCBI are shown with broken arrows. E2F and MYC sites, known to regulate BDPs and FOXM1, are indicated.

FOXM1 and RHNO1 expression correlates with genomic copy number in high-grade serous carcinoma (HGSC).

(A) FOXM1 and RHNO1 mRNA expression (RNA-seq) vs. GISTIC copy number in The Cancer Genome Atlas HGSC data (N = 157). (B) FOXM1 and RHNO1 mRNA expression (microarray) vs. copy number (GISTIC) in Cancer Cell Line Encyclopedia HGSC cell lines (N = 23). p-values for ANOVA with post-test for linear trend are shown, and lines represent group medians. p-value designation: **p<0.01, ****p<0.0001. (C) FOXM1 and RHNO1 mRNA expression in FT282 immortalized Fallopian tube epithelium cells and HGSC cell lines (RT-qPCR). (D) FOXM1 and RHNO1 nuclear protein levels (western blot). Two nuclear protein loading controls are shown. FOXM1/RHNO1 GISTIC copy number values for the cell lines are shown (−1: het loss; 0: diploid; 1: gain; 2: amp).

FOXM1 and RHNO1 expression correlates in high-grade serous carcinoma (HGSC) and in HGSC precursor cells.

(A) FOXM1 vs. RHNO1 expression in primary HGSC tumors, as determined by RT-qPCR. (B) FOXM1 vs. RHNO1 expression in Fallopian tube epithelium (N = 5), ovarian surface epithelium (N = 3), and HGSC (N = 10) cell lines, as determined by RT-qPCR. (C) FOXM1 vs. RHNO1 expression in FT282 cells, as determined by scRNA-seq. (D) FOXM1 vs. RHNO1 expression in OVCAR8 HGSC cells, as determined by scRNA-seq. (AD) show the Spearman r value, p-value, and the number of samples analyzed. (E) FOXM1 and RHNO1 expression in FT282 and OVCAR8 cells, plotting the median values from scRNA-seq data. Mann–Whitney p-values are shown.

Figure 5 with 1 supplement
Distribution of correlation coefficients for bidirectional gene pairs (n = 2172) from scRNA-seq data.

Data are shown for (A) FT282 and (B) OVCAR8 cell lines. The x-axis plots the degree of co-expression (Pearson's coefficient) between bidirectional gene pairs, and y-axis plots the frequency of bidirectional pairs. The rank percentile of the FOXM1/RHNO1 correlation in each cell type is shown and is indicated by the dashed vertical line.

Figure 5—figure supplement 1
Principal component analyses (PCA) of FT282 (n = 1440) and OVCAR8 (n = 1454) single cells, using scRNA-seq data.

PCA analyses were performed using Python.

FOXM1 and RHNO1 mRNA expression and expression ratio in normal and cancer tissues.

(A) FOXM1 and (B) RHNO1 expression (RNA-seq) in normal tissues (from Genotype-Tissue Expression and The Cancer Genome Atlas [TCGA]) vs. different types of TCGA tumor tissues (i.e., primary, metastatic, and recurrent). The number of samples in each group is shown. All comparisons of normal vs. tumor samples were highly significant (p<0.0001, t-tests). (C) FOXM1/RHNO1 mRNA expression ratio in normal and cancer tissues, for the data shown in (AB). The FOXM1/RHNO1 ratio for the samples to the right of the dashed line was normalized to MKI67 (a.k.a. Ki67), the canonical proliferation marker.

FOXM1 and RHNO1 are overexpressed in high-grade serous carcinoma (HGSC).

(A) FOXM1 and RHNO1 expression in Genotype-Tissue Expression normal Fallopian tube (FT; N = 5) vs. The Cancer Genome Atlas HGSC tissues (RNA-seq) (N = 427). (B) FOXM1 and RHNO1 mRNA expression in Fallopian tube epithelium/ovarian surface epithelium cells vs. HGSC cell lines (RT-qPCR). t-test p-values are shown. p-value designation: ****<0.0001, ***<0.001, **<0.01, *<0.05. The key shown at right indicates the cell types for each data point.

FOXM1 and RHNO1 expression in primary and recurrent high-grade serous carcinoma (HGSC).

(A) FOXM1 vs. RHNO1 expression (RNA-seq data) in primary (left) and recurrent (right) HGSC samples from Kreuzinger et al., 2017. (B) FOXM1 vs. RHNO1 expression (RNA-seq data) in primary (left) and recurrent (right) HGSC samples from Patch et al., 2015. For (AB) , the number of samples (n), Spearman r value, and p-value are shown. (C) FOXM1 (top) and RHNO1 (bottom) expression (RNA-seq) in patient-matched primary vs. recurrent HGSC samples from Patch et al., 2015. Asterisks indicate patients showing coordinately increased expression of both FOXM1 and RHNO1 in recurrent tumors (5/11 = 45% of patients).

Figure 9 with 2 supplements
F/R-BDP promoter activity assays.

(A) Schematic of the F/R-BDP dual luciferase reporter construct. The FOXM1 promoter direction drives Renilla luciferase, and the RHNO1 promoter direction drives firefly luciferase. Secreted embryonic alkaline phosphatase (SEAP) was used as a normalization control for transfection efficiency. (B) Correlation of FOXM1 and RHNO1 promoter activity in Fallopian tube epithelium (FTE) (FT282) and high-grade serous carcinoma (HGSC) cell lines (OVCAR5, COV318, OVCAR4, Kuramochi, SNU119). (C, D) Correlation of FOXM1 or RHNO1 promoter activity vs. endogenous expression of the cognate gene in FTE (FT282) and HGSC (OVCAR5, COV318, Kuramochi, SNU119) cell lines, as measured by RT-qPCR. In (B–D), error bars represent standard deviations. (E) Schematic of the F/R-BDP dual fluorescence reporter construct. The FOXM1 promoter direction drives red fluorescent protein (RFP) expression, and the RHNO1 promoter direction drives green fluorescent protein (GFP) expression. (F) Fluorescence-activated cell sorting analysis of GFP and RFP fluorescence in 293T cells transfected with the F/R-BDP reporter shown in (E). Quadrant 2 (RFP + GFP + cell population) correlation statistics are shown.

Figure 9—figure supplement 1
FOXM1 and RHNO1 transcriptional start sites.

5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) mapping data of the 5′ ends of FOXM1 and RHNO1 mRNA. The arrows indicate the orientation of each gene 5′ to 3′. The bars indicate the sequenced 5′ ends of each transcript, with the names of the individual cell line samples (color coded) at left. NCBI RefSeq transcript assembly data is shown at bottom.

Figure 9—figure supplement 2
Fluorescence-activated cell sorting analyses of red fluorescent protein (RFP) and green fluorescent protein (GFP) expression in 293T cells.

(A, B) Empty vector transfections. (C, D) F/R-BPD dual reporter transfections. The reporter schematic is shown in Figure 9E. (E, F) Spearman correlation analyses of Quadrant 2 (i.e., RFP + GFP + cells) from (CD) as indicated.

Endogenous FOXM1 and RHNO1 are co-regulated by their bidirectional promoter (F/R-BDP).

(A) Schematic of the experimental approach used in (B, C). The position of the CRISPR guide RNAs is indicated with black rectangles. (B) CRISPRa experiment in FT282 cells. Endogenous FOXM1 and RHNO1 expression was measured by RT-qPCR. (C) CRISPRi experiment in OVCAR8 cells. Endogenous FOXM1 and RHNO1 mRNA expression was measured by RT-qPCR. In (BC), NT1 and NT2 indicate non-targeting control guide RNAs, and t-test p-values are shown for the comparison with NT1 control for either FOXM1 or RHNO1. p-values: ****<0.0001, ***<0.001, **<0.01, *<0.05. (D) Genotyping data for 293T cells with knockout (KO) of the 200 bp F/R-BDP (BDP KO) or, as a control, the entire upstream regions (5′ of the translational start sites) of both FOXM1 and RHNO1 (ATG-ATG KO). N: no template control. (E) Endogenous FOXM1 and RHNO1 expression in 293T cells with the indicated genetic configurations. BDP KO and ATG-ATG KO correspond to cells described in (D). KRAB data for 293T cells (i.e., CRISPRi), using the strategy shown in (A), are shown for comparison on the right.

Figure 11 with 2 supplements
FOXM1 and RHNO1 each promote high-grade serous carcinoma cell clonogenic growth.

(A) OVCAR8 and CAOV3 cells engineered for doxycycline (dox)-inducible FOXM1 or RHNO1 shRNA knockdown. Cells were grown in the presence of dox for 72 hr, and protein was extracted for western blot analyses. (B) Cells were seeded into 6-well dishes, in triplicate. Dox was added at the time of seeding, and media containing dox was replenished every 48 hr. Clonogenic growth was measured at 12–14 days, and the number of colonies (>50 cells) was counted. NS: nontargeting shRNA. (C) OVCAR8 and CAOV3 cells were engineered for FOXM1 or RHNO1 CRISPR knockout. Western blot analysis of polyclonal cell populations was performed 72 hr post-lentivirus infection. CTL: control guide RNA. (D) Clonogenic growth was measured as described in (B). t-test p-values vs. control: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 11—figure supplement 1
FOXM1 and RHNO1 each promote high-grade serous carcinoma cell clonogenic growth.

(A) Representative colony formation data for OVCAR8 and CAOV3 cells sustaining FOXM1 or RHNO1 knockdown. NS: nontargeting shRNA. (B) Representative colony formation data for OVCAR8 and CAOV3 cells sustaining FOXM1 or RHNO1 CRISPR knockout. CTL: control guide RNA. (C) Annexin V apoptosis assay results for OVCAR8 cells sustaining FOXM1 or RHNO1 CRISPR knockout. Etoposide-treated cells served as the positive control for apoptosis. t-test p-values: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 11—figure supplement 2
Representative TIDE analyses data for high-grade serous carcinoma cells sustaining FOXM1 or RHNO1 CRISPR gene knockout.

OVCAR8 and CAOV3 cells engineered for FOXM1 or RHNO1 CRISPR knockout were assessed for indel frequency among a mixed pool of cells. (A–D) Indel spectra for (A) OVCAR8 LCV2 FOXM1 462 cells; (B) CAOV3 LVC2 FOXM1 462 cells; (C) OVCAR8 LCV2 RHNO1 413 cells; (D) CAOV3 LCV2 RHNO1 413 cells.

Figure 12 with 1 supplement
OVCAR8 cells with dual FOXM1 and RHNO1 shRNA knockdown have diminished clonogenic growth.

OVCAR8 high-grade serous carcinoma cells were engineered for doxycycline (dox)-inducible FOXM1 and/or RHNO1 shRNA knockdown. (A) OVCAR8 cells treated with dox for 72 hr followed by western blot analysis. (B) Representative clonogenic growth data. Cells were seeded into a 6-well dish, in triplicate, at a density of 500 cells. Dox was added at the time of seeding, and media containing dox was replenished every 48 hr. Clonogenic survival was measured at 12 days, and cells were fixed with methanol and stained with crystal violet. (C) Quantification of clonogenic growth data. Colonies containing more than 50 cells were counted, and clonogenic survival was quantified as an average of the replicates. NS: nontargeting shRNA. The ANOVA linear trend p-value is shown.

Figure 12—figure supplement 1
CAOV3 cells with dual FOXM1 and RHNO1 shRNA knockdown have diminished clonogenic growth.

CAOV3 high-grade serous carcinoma cells were engineered for doxycycline (dox)-inducible FOXM1 and/or RHNO1 shRNA knockdown. CAOV3 cells treated with dox for 72 hr followed by (A) western blot confirmation of FOXM1 knockdown or (B) RT-qPCR confirmation of RHNO1 knockdown. (C) Representative clonogenic growth data. Cells were seeded into a 6-well dish, in triplicate, at a density of 500 cells. Dox was added at the time of seeding, and media containing dox was replenished every 48 hr. Clonogenic survival was measured at 12 days, and cells were fixed with methanol and stained with crystal violet. (D) Quantification of clonogenic growth data. Colonies containing more than 50 cells were counted, and clonogenic survival was quantified as an average of the replicates. NS: nontargeting shRNA. The ANOVA linear trend p-value is shown.

RHNO1 functions in the replication stress response in high-grade serous carcinoma cells.

(A) Schematic of RHNO1 wild-type and SWV mutant. The proteins are the same molecular weight but residues 55–61 are converted to alanine in the SWV mutant, disrupting its ability to interact with 9-1-1 proteins. (B) 293T cells were transfected with empty vector, vector expressing HA-RHNO1-WT or SWV mutant. Protein was harvested 24 hr post transfection for Co-IP and western blot analyses. The red asterisk indicates a non-specific band. (C) OVCAR8 cells were seeded for 24 hr, then treated with vehicle or 5 mM hydroxyurea (HU) for 1 hr, and cells were harvested for subcellular fraction into soluble nuclear and chromatin bound proteins and western blot analysis. Histone H3 was used as the chromatin control and Sp1 as the soluble nuclear control. (D) OVCAR8-inducible RHNO1 knockdown cells were engineered for inducible expression of HA-tagged RHNO1-WT or SWV mutant, grown in the presence of doxycycline (dox) for 72 hr, and then protein was harvested for Co-IP/western blot analyses. (E) OVCAR8 western blot measurement of P-Chk1 sustaining RHNO1 shRNA knockdown. (F) OVCAR8 cells were treated with the ATR inhibitor VE-822 or dox to induce RHNO1 shRNA knockdown, and gH2AX-positive cells in different cell cycle phases were quantified by fluorescence-activated cell sorting. (G) COMET analysis of DNA strand breakage in OVCAR8 cells following the treatments described in (F).

Binding to the 9-1-1 complex is required for RHNO1 function in OVCAR8 cell clonogenic growth.

OVCAR8 cells were engineered for doxycycline (dox)-inducible RHNO1 knockdown and simultaneous dox-inducible expression of HA-RHNO1 WT or SWV mutant. (A, B) Cells were seeded in the presence of dox and grown for 72 hr to harvest RNA and protein extracts, followed by (A) RT-qPCR or (B) western blot to confirm knockdown efficiency. (B) also shows HA-RHNO1 protein ectopic expression efficiency. (C) Cells were seeded into 6-well dishes in triplicate, in the presence of dox and at a density of 500 cells. Media containing dox was replenished every 48 hr. Clonogenic survival was measured 12 days later. A representative data set is shown. (D) For the experiment described in (C), colonies containing more than 50 cells were counted and clonogenic survival was quantified as an average of the replicates. V: vector control; NS: nontargeting shRNA. t-test p-values are shown. p-value designation: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Homozygous knockout of RHNO1 in FT282 cells does not impact cell proliferation, and high-grade serous carcinoma (HGSC) cells show elevated expression of replication stress (RS) biomarkers compared to Fallopian tube epithelium (FTE) cells.

RHNO1 knockout was achieved using CRISPR-Cas9 with guide RNAs that targeted near the start and stop codon of RHNO1. (A, B) RHNO1 homozygous knockout was confirmed by (A) genomic DNA PCR and (B) RHNO1 mRNA expression measured by end-point RT-PCR. NTC: no template control. (C) FT282 parental and RHNO1 clonal knockout cells were seeded in quadruplicate and grown in culture for a period of 1 week. Cell growth/viability was measured by the AlamarBlue assay every 24 hr. (D) Western blot analyses of two canonical phosphoprotein biomarkers of RS in FTE and HGSC cell lines. Cell line sample names are shown below.

FOXM1 and RHNO1 promote homologous recombination (HR).

DR-GFP cell lines were treated with doxycycline (dox) to induce shRNA expression, and mRNA knockdowns were measured by RT-qPCR. (A, B) FOXM1, RHNO1, and RAD51 mRNA expression in U2OS-DR-GFP cells sustaining mRNA knockdown. (C) HR repair of I-SceI-induced DSBs in U2OS cells after the indicated mRNA knockdowns. Values are expressed relative to the percentage of green fluorescent protein (GFP)+ cells in I-SceI-transfected control cells. RAD51 knockdown was used as a robust positive control for HR impairment. (D, E) FOXM1, RHNO1, and RAD51 mRNA expression in OVCAR8-DR-GFP cells. (F) HR repair of I-SceI-induced DSBs in OVCAR8 cells after the indicated mRNA knockdowns. Values are expressed relative to the percentage of GFP+ cells in I-SceI-transduced control cells. RAD51 knockdown was used as a robust positive control for HR impairment. All results are shown as mean ± SE from three independent replicates. (C) and (F) show p-values for the linear trend (excluding the RAD51 control).

Figure 17 with 1 supplement
FOXM1 and RHNO1 knockdown-sensitize OVCAR8 cells to olaparib.

OVCAR8 cells engineered for doxycycline (dox)-inducible FOXM1 and/or RHNO1 shRNA knockdown were grown in dox for 72 hr prior to seeding cells for experiments. (A) Cells were seeded in 96-well plates in quadruplicate in the presence of dox at a density of 500 cells per well. 24 hr later, cells received media containing dox + vehicle control or olaparib, and this was repeated every 48 hr. Cell viability was measured at 8 days post-olaparib treatment using AlamarBlue assays. The ANOVA linear trend p-value is shown. (B) Cells were seeded into 6-well dishes in triplicate in the presence of dox at a density of 5000 cells per well. 24 hr later, cells received media containing dox and vehicle or olaparib, and this was repeated every 48 hr. After 8 days of growth, cells were fixed with methanol and stained with crystal violet for clonogenic assays. A representative result is shown. NS/CTL: nontargeting shRNA. (C) Western blot analysis of γ-H2AX in chromatin extracts from cells with dox-inducible control shRNA (NS), FOXM1 shRNA (FX), RHNO1 shRNA (RH), or both (FX/RH). After 48 hr in dox, cells were treated with vehicle control dimethyl sulfoxide (DMSO) or 1 µM olaparib for 24 hr, and protein extracts were harvested. Histone H3 served as a loading control. (D) Cells were treated as in (C), and cell cycle analyses were conducted 24 hr post-olaparib treatment.

Figure 17—figure supplement 1
Quantification of γ-H2AX in OVCAR8 chromatin extracts following FOXM1 and/or RHNO1 knockdown and DMSO or olaparib treatment.

Western blots are shown in Figure 17C.

CRISPRi repression of the F/R-BDP sensitizes OVCAR8 cells to olaparib.

(A) OVCAR8 cells expressing the KRAB transcriptional repressor and a guide RNA targeting the F/R-BDP, and the corresponding changes in mRNA expression as measured by RT-qPCR. NT: nontargeting guide RNA. (B) Cells were seeded in 96-well plates in quadruplicate at a density of 500 cells per well. 24 hr later, cells received media containing vehicle or olaparib, and this was repeated every 48 hr. Cell viability was measured at 8 days using AlamarBlue, and the IC50 for olaparib was determined. An F-test was used to compare IC50 values. (C) Cells were seeded into a 6-well dish, in triplicate, at a density of 5000 cells per well. 24 hr later, cells received media containing vehicle or olaparib, and this was repeated every 48 hr. After 8 days of growth, cells were fixed with methanol and stained with crystal violet to visualize colonies. (D) The indicated cells were treated with olaparib for 48 hr, and apoptosis was measured using Annexin V staining/fluorescence-activated cell sorting (FACS) analysis. (E) Cell cycle profiles were measured using propidium iodide staining/FACS analysis. ANOVA linear trend p-values are shown.

CRISPRi repression of the F/R-BDP in olaparib-resistant cells increases olaparib sensitivity.

Parental and olaparib-resistant UWB1 cells were engineered to express the KRAB transcriptional repressor and a control guide RNA or guide RNA targeting the F/R-BDP (sg130). (A) Cells were seeded and grown for 72 hr, and RNA was harvested for RT-qPCR to measure CRISPRi efficiency. (B, C) UWB1 parental KRAB and (B) SyR12 KRAB or (C) SyR13 KRAB cells were seeded in 96-well plates in quadruplicate at a density of 750 cells per well. 24 hr later, cells received media containing vehicle or olaparib, and this was repeated every 48 hr. Cell viability was measured at 8 days post-treatment using AlamarBlue assays, and the IC50 for olaparib was determined. NT1: nontargeting control guide RNA. FOXM1/RHNO1: sg130 targeting the F/R-BDP. In (BC), ANOVA linear trend statistics for the IC50 values are shown. Linear trends were ordered – SyR12/13 NT, SyR12/13 FOXM1/RHNO1 sg130, parental NT.

FOXM1 and RHNO1 knockdown enhances OVCAR8 cell sensitivity to carboplatin.

OVCAR8 cells were treated with doxycycline (dox) for 48 hr and seeded into triplicate wells of 6-well dishes in single-cell suspension. Cells were treated with carboplatin and dox the day after seeding and every 48 hr thereafter, and allowed to form colonies for 7–21 days. After incubation, cells were fixed in methanol and stained using 0.5% crystal violet; colonies containing >~50 cells were counted. (A) Representative clonogenic growth data. (B) Quantified data for experimental groups. Each data set was normalized to the DMSO-only treatment data point. The ANOVA p-value for the group comparison is shown.

Model for FOXM1/RHNO1 bidirectional gene (BDG) function and cooperativity.

FOXM1 and RHNO1 are BDGs regulated by a bidirectional promoter (BDP) and are co-amplified and co-expressed in high-grade serous carcinoma (HGSC), including single cells. Both genes are overexpressed in HGSC and other cancers. FOXM1, via the activation of specific gene targets, promotes oncogenic phenotypes, including cell cycle progression, proliferation, replication stress (RS), genomic instability, and metastatic progression; the latter phenotype may reflect activation of a distinct set of transcriptional targets later in tumor progression. In contrast, RHNO1 promotes ATR-Chk1 signaling via its binding to 9-1-1 and TOPBP1 and promotes the RS response. This protects HGSC cells from excessive RS and DNA damage and enhances cell survival. RHNO1 co-expression may help to mitigate excessive FOXM1-induced RS and thus facilitate other FOXM1 oncogenic functions. FOXM1 and RHNO1 co-expression may increase tumor resiliency by allowing tumor cells to gain a fitness advantage of increased growth and proliferation without accumulating excessive DNA damage. Consistently, FOXM1-expressing tumors are highly enriched for biomarkers of RS (Li et al., 2020). Therapeutic targeting of the FOXM1/RHNO1 bidirectional gene unit may be accomplished using inhibitors of FOXM1 and/or ATR/Chk1/WEE1. RHNO1 inhibitors have not been reported (dashed line).

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)FOXM1GenBankGene ID: 2305
Gene (Homo sapiens)RHNO1GenBankGene ID: 83695
AntibodyFOXM1 (rabbit monoclonal)Cell Signaling Technology5436(1:1000)
Antibodyβ-Actin (mouse monoclonal)Santa Cruzsc-47778(1:200)
Antibodyα-Tubulin (rabbit polyclonal)Cell Signaling Technology2144(1:1000)
AntibodyLamin B (goat polyclonal)Santa Cruzsc-6217Discontinued
AntibodyHistone H3 (goat polyclonal)Santa Cruzsc-8654Discontinued
AntibodyCHEK1 (mouse monoclonal)Santa Cruzsc-8408(1:200)
AntibodyP-CHEK1 S345 (rabbit monoclonal)Cell Signaling Technology2348(1:1000)
AntibodyP-CHEK1 S317 (rabbit monoclonal)Cell Signaling Technology12302(1:1000)
AntibodyRPA32/RPA2 (rabbit monoclonal)Abcamab76420(1:1000)
AntibodyRPA32 (S33) (rabbit polyclonal)BethylA300-246A-M(1:1000)
AntibodyH2AX (goat polyclonal)BethylA303-837A(1:2000)
AntibodyP-H2AX S139 (rabbit monoclonal)Cell Signaling Technology9718(1:1000)
AntibodyFlag (rabbit monoclonal)Cell Signaling Technology14793(1:1000)
AntibodyHA (rabbit monoclonal)Cell Signaling Technology3724(1:1000)
AntibodyRHNO1 (rabbit polyclonal)NovusNBP1-93694Discontinued
AntibodyRHNO1 (rabbit polyclonal)SigmaHPA038682Discontinued
AntibodyTOPBP1 (rabbit monoclonal)Cell Signaling Technology14342(1:1000)
AntibodyRAD9 (rabbit polyclonal)Santa Cruzsc-8324Discontinued
AntibodyRAD1 (rabbit monoclonal)Invitrogen702149(1–2 µg/mL)
AntibodyHUS1 (rabbit monoclonal)Cell Signaling Technology16416(1:1000)
AntibodyAnti-HA magnetic beads (mouse monoclonal)Pierce88837
Cell line (Homo sapiens)COV362Sigma7071904
Cell line (Homo sapiens)COV318Sigma7071903
Cell line (Homo sapiens)KURAMOCHIJCRBJCRB0098
Cell line (Homo sapiens)OVSAHOJCRBJCRB1046
Cell line (Homo sapiens)SNU-119Korean Cell Line Bank00119
Cell line (Homo sapiens)OVCAR4NCI
Cell line (Homo sapiens)OVCAR8NCI
Cell line (Homo sapiens)OVCAR3ATCCHTB-161
Cell line (Homo sapiens)CAOV3Anirban Mitra
Cell line (Homo sapiens)OVCAR5Anirban Mitra
Cell line (Homo sapiens)UWB1.289Lee Zou
Cell line (Homo sapiens)UWB1-SyR12Lee Zou
Cell line (Homo sapiens)UWB1-SyR13Lee Zou
Cell line (Homo sapiens)hOSEScienCell7310
Cell line (Homo sapiens)FT190Ronny Drapkin
Cell line (Homo sapiens)FT282-E1Ronny Drapkin
Cell line (Homo sapiens)FT282Ronny Drapkin
Cell line (Homo sapiens)IOSE-21Francis Balkwill
Cell line (Homo sapiens)IOSE-121Nelly Auersperg
Cell line (Homo sapiens)283TATCCCRL-3216
Cell line (Homo sapiens)U2OS-DR-GFP (282C)Jeremy Stark
Cell line (Homo sapiens)OVCAR8-DR-GFPLarry Karnitz and Scott Kaufmann
Chemical compound, drugBerzosertibSelleckChemVE-822
Chemical compound, drugOlaparibSelleckChemABT-888
Chemical compound, drugHydroxyureaSigmaH8627
Chemical compound, drugCarboplatinSigmaC2538
Chemical compound, drugEtoposideSigmaE1383
Chemical compound, drugDoxycyclineSigmaD9891
Commercial assay or kitDirect-zol RNA Purification KitZymo ResearchR2072
Commercial assay or kitComet Assay KitTrevigen4250-050-K
Commercial assay or kitiScript cDNA Synthesis KitBio-Rad1708890
Commercial assay or kitDNA-free kitAmbionAM1906
Commercial assay or kitNE-PER Nuclear and Cytoplasmic Extraction KitPierce78833
Commercial assay or kitBCA protein assayPierce23225
Commercial assay or kitEnhanced chemiluminescencePierce32106
Commercial assay or kitPuregene Tissue KitQIAGEN158667
Commercial assay or kitEZ DNA Methylation KitZymo ResearchD5001
Commercial assay or kitQIAquick Gel Extraction KitQIAGEN28704
Commercial assay or kitTOPO TA Cloning KitInvirogenK457501
Commercial assay or kitFirstChoice RLM-RACE KitAmbionAM1700
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaE1910
Commercial assay or kitPhospha-Light SEAP Reporter Gene Assay SystemThermo ScientificT1015
Commercial assay or kitTRIzol reagentThermo Scientific15596026
Commercial assay or kitLipofectamine 2000 reagentLife Technologies11668019
Commercial assay or kitQuickExtractLucigenQE09050
Commercial assay or kitM-PERPierce78501
Commercial assay or kitHalt Protease and Phosphatase CocktailPierce78440
Commercial assay or kitTurbo NucleaseSigmaT4330-50KU
Commercial assay or kitAlamarBlueBio-RadBUF012A
Commercial assay or kitVectashield with DAPIVector LaboratoriesH-1200-10
Genetic reagent (Homo sapiens)pCBASceIAddgene26477Plasmid
Genetic reagent (Homo sapiens)pCW57-GFP-P2A-MCSAddgene71783Plasmid
Genetic reagent (Homo sapiens)pCW57-GFP-P2A-MCS-(Neo)Addgene89181Plasmid
Genetic reagent (Homo sapiens)pCW57-MCS1-P2A-MCS2-(Hygro)Addgene80922Plasmid
Genetic reagent (Homo sapiens)pCW57-MCS1-P2A-MCS2-(Blast)Addgene80921Plasmid
Genetic reagent (Homo sapiens)pCW57-RFP-P2A-MCSAddgene78933Plasmid
Genetic reagent (Homo sapiens)pCW57-RFP-P2A-MCS-(Neo)Addgene89182Plasmid
Genetic reagent (Homo sapiens)pTRIPZ Non-silencingDharmaconNC0257175Plasmid
Genetic reagent (Homo sapiens)pGL4 Luc Rluc EmptyAddgene64034Plasmid
Genetic reagent (Homo sapiens)pCMV6-SEAPAddgene24595Plasmid
Genetic reagent (Homo sapiens)pTurbo-GFP-RFP-emptyCustomPlasmid
Genetic reagent (Homo sapiens)pTurbo-GFP-PGKCustomPlasmid
Genetic reagent (Homo sapiens)pTurbo-RFP-PGKCustomPlasmid
Genetic reagent (Homo sapiens)pTurboGFP-RFP-FOXM1-RHNO1CustomPlasmid
Genetic reagent (Homo sapiens)pLentiCRISPRv2Addgene52961Plasmid
Genetic reagent (Homo sapiens)PX458-WT-Cas9-emptyAddgene48138Plasmid
Genetic reagent (Homo sapiens)lenti-SAMv2-PuroAddgene92062Plasmid
Genetic reagent (Homo sapiens)lenti-MPHv2-NeoAddgene92065Plasmid
Genetic reagent (Homo sapiens)pLV hU6-sgRNA hUbC-dCas9-KRAB-T2A-Puro (KRAB)Addgene71236Plasmid
Genetic reagent (Homo sapiens)psPAX2Addgene12260Plasmid
Genetic reagent (Homo sapiens)pMD2.GAddgene12259Plasmid
Genetic reagent (Homo sapiens)pOTB7-RHNO1Harvard PlasmID RepositoryHsCD00326310Plasmid
Genetic reagent (Homo sapiens)pENTR-MGC RHNO1 siRStephen ElledgePlasmid
Genetic reagent (Homo sapiens)pENTR-MGC RHNO1 SWVStephen ElledgePlasmid
Genetic reagent (Homo sapiens)pCMV6 AN-HA emptyOrigenePS100013Plasmid
Genetic reagent (Homo sapiens)AdNGUS24iFrank Graham and Phillip NgAdenovirus
Software, algorithmPrism 8 statistical softwareGraphPad
Software, algorithmLasergene SeqMan ProDNASTAR
Software, algorithmComet Analysis SoftwareTrevigen
Software, algorithmR packageR Project
Software, algorithmCell Ranger Single Cell Software10X Genomics
Software, algorithmGSEA software version 3Broad Institute
Software, algorithmCufflinks (v2.1.1)https://doi.org/10.1038/nbt.1621
Software, algorithmTrim Galore software packageThe Babraham Institute
Software, algorithmTopHat (v2.0.8)https://doi.org/10.1186/gb-2013-14-4-r36
Software, algorithmCuffdiff (v2.1.1)https://doi.org/10.1038/nbt.2450
Software, algorithmPerlThe Perl Foundation
Software, algorithmscImputehttps://doi.org/10.1038/s41467-018-03405-7

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)