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
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 …

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 …

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 …

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 …

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 =…

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 …

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, …

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 …

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 …

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 …

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 …

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 + …

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

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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

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 …

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 …

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 …

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 …

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 …

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 …

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

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