Publicly available datasets

The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma dataset was accessed via cBioPortal (http://www.cbioportal.org) [28,29,30], for ovarian cancer patient RNA-seq data. This dataset was last accessed 31 January 2023. The TCGA PanCancer Atlas Ovarian Serous Cystadenocarcinoma dataset was accessed via cBioPortal, for ovarian cancer patient CPTAC data. This dataset was last accessed 6 Sept 2023. DepMap was used to correlate CASC4 dependencies or EGFR protein levels with sensitivities to EGFR inhibitors in ovarian cancer lines (https://depmap.org/portal) [44, 45]. DepMap was last accessed 30 May 2022. CASC4 molecular score was accessed through CanSar Black (https://cansarblack.icr.ac.uk) [46] on 30 May 2022.

Cell culture

Human ovarian cancer cell lines PEO1 and OVCAR3 (obtained from the GTFB) and CaOV3 (obtained from Dr. Katherine Aird’s lab, University of Pittsburgh) were cultured in RPMI 1640 supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (referred to as “Complete RPMI”). Murine ovarian cancer cell line ID8 (obtained from Dr. Iain McNeish’s lab, Imperial College London) were cultured in high glucose DMEM supplemented with 1% penicillin/streptomycin, 4% fetal bovine serum, and 1% ITS (insulin-transferrin-selenium). ID8 cells were Trp53-null, Brca2-null, GFP+, and luciferase-tagged. Packaging cells (293FT; obtained from The University of Colorado Functional Genomics Shared Resource) were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. For suspension experiments, cells were cultured as previously described [19]. Cells were authenticated at The University of Arizona via small tandem repeat analysis. Mycoplasma detection was performed regularly, using the MycoLookOut Mycoplasma PCR detection kit, from Sigma.

CASC4 knockdowns and overexpression

All constructs were obtained from the University of Colorado Functional Genomics Facility (control shRNA: SHC001, pLKO.1-puro Empty Vector; RRID:Addgene_8453; shCASC4 #1: TRCN0000133832; shCASC4 #2: TRCN0000136384; empty vector: pLX304 control; CASC4 overexpression: ccsbBroad304_13019) and packaged using 293FT cells as previously described [18]. After 2 days, viruses were harvested from filtered 293FT media. Cells were transduced with the virus and treated with 1 mg/ml of hexadimethrine bromide (polybrene). After 24 h, media was refreshed. The cells used for experiments were a heterogenous mixture of antibiotic selected cells. For shRNA expressing cells, selection was performed with 1 μg/ml of puromycin. For CASC4 overexpression or empty vector expressing cells, selection was performed with 5 μg/ml of blasticidin.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

RNA extraction was performed using the RNAeasy Plus Mini Kit (Qiagen) as according to the manufacturer’s instructions. RT-qPCR was performed using the Luna Universal One-step qRT-PCR kit (New England BioLabs) on a BioRad thermocycler using primers for specific target transcripts. 18S rRNA was amplified as housekeeping genes. The following primer sequences were used:

18S (F: 5’- AACTTTCGATGGTAGTCGCCG-3’, R: 5’- CCTTGGATGTGGTAGCCGTTT-3’)

CASC4 (F: 5’-CAGAATCCTTCCAGTCCTCTTC-3’, R: 5’-CCTTGGTAGCCTGCTTTAGTAT-3’)

EGFR (F: 5’-AACACCCTGGTCTGGAAGTACG -3’, R: 5’- TCGTTGGACAGCCTTCAAGACC-3’).

Actb (F: 5’-TGTACCCAGGCATTGCTGAC-3’, R: 5’-AACGCAGCTCAGTAACAGTCC-3’)

Casc4 (F: 5’-TCCCCATGGGAAAGAACAACT-3’, R: 5’-GCTAACACAGGGGGCTTCTT-3’)

EGFR recycling assay

Cells were plated in suspension in complete RPMI for 48 h, then switched over to serum-free RPMI for 24 h. Recombinant EGF (R&D systems; Cat. 236-EG-200) was then added to the cells to a final concentration of 20 ng/ml, and the cells were incubated on ice for 10 min. Cells were centrifuged for 5 min at 4 °C and 1000 rpm, resuspended in 1x PBS, centrifuged again for 5 min at 4 °C, then resuspended in serum-free RPMI, and incubated in suspension at 37 °C for the desired timepoints. Cells were then processed for flow cytometry analysis or immunofluorescence, as described below.

Immunoblotting

For cell line experiments, cells were lysed with Radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, supplemented with complete EDTA-free protease inhibitors (Roche) and phosphatase inhibitors sodium fluoride (10 mM) and sodium orthovanadate (1 mM), briefly sonicated, and incubated on ice for 30 min. Lysates were then centrifuged at 4 °C, 15,000 rpm, for 10 min, and the supernatant was isolated. Protein concentrations in the supernatant were measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher), and the SpectraMaxM2e spectrophotometer (Molecular Devices). A similar procedure was followed for both human and mouse tissues, which were first homogenized with beads before sonication.

Protein lysates were run on SDS-Page gels and transferred onto methanol-activated PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were then dried at room temperature for 30 min, re-hydrated with methanol, and then incubated in blocking buffer (Intercept® Blocking Buffer (Li-Cor)) for 30 min and incubated with primary antibody overnight. Membranes were then washed thrice with TBS-T (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20), for 5 min each, incubated with secondary for 30 min, then washed thrice more with TBS-T. Membranes were imaged using the LI-COR Odyssey Imaging System. The antibodies used for immunoblotting can be found in Supplementary Table 2 (Table S2).

Co-immunoprecipitation

EGFR recycling was performed as described above. Cells were cultured in suspension for 30 min after EGF was washed away, lysed using IP lysis buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Triton X-100, 150 mM NaCl, 10 mM N-ethylmaleimide, and the protease and phosphate inhibitors described above), and incubated on ice for 30 min. N-ethylmaleimide served as a deubiquitinase inhibitor. In total, 60 μl of Protein A magnetic beads (New England Biosciences) were washed with lysis buffer twice, and then used to pre-clear the protein lysate. In total, 1.35 mg of pre-cleared lysate was incubated with EGFR antibody (Cell Signaling Technologies, 7 μl of 4267S and 3 μl of 54359S) or IgG antibody (R&D Systems, AB-105-C) and rocked overnight at 4 °C. The next day, lysate-antibody mixtures were incubated with 30 μl of washed Protein A beads and rocked for 30 min at 4 °C. Supernatant was removed, and the beads were washed with lysis buffer and rocked for 5 min at 4 °C, thrice. Beads were then eluted with sample loading buffer, and boiled at 95 °C prior to the immunoblot. The antibodies used for IP can be found in Supplementary Table 2 (Table S2).

Colony formation

In total, 25,000 cells were plated in suspension for 2 days, and then the media (containing the cells in suspension) was transferred to an adherent plate. After 7 days, cells were washed with PBS, fixed (10% acetic acid, 10% methanol in PBS) for 5 min, and then stained with crystal violet (0.4% crystal violet in ethanol). Plates were washed with deionized water and left to dry overnight. Crystal violet was then dissolved for 10 min in the fixation solution, and absorbance (at 590 nm) was measured using a SpectraMaxM2e spectrophotometer (Molecular Devices).

Proliferation

In total, 15,000 cells were plated in suspension in 96 well plates, which were placed in a S3 Incucyte (Essen BioScience) for 7 days, with images of each well being taken every 4 h, at ×10 magnification. The GFP signal of each well was measured using the Incucyte Zoom (Essen BioScience) software, as a proxy for cell count.

Flow cytometry

For flow cytometric detection of cell surface-localized EGFR, cells were grown in suspension for 2 days, then trypsinized for 5 min at 37 °C to obtain a single-cell suspension. Cells were then stained with a fluorophore-conjugated anti-EGFR antibody (see antibody chart for details) on ice in the dark for 30 min. Cells were washed with PBS and then stained with DAPI or Fixable Viability Stain 520 (BD Horizon™, Cat. # 564407) to differentiate between live and dead cells. Flow cytometry was performed using the Gallios 561 (Beckman Coulter) flow cytometer and data analysis was performed using FlowJo software. The EGFR intensity of DAPI− (live cells) was measured. For flow cytometric detection of live cells, a similar protocol was performed, without the addition of the anti-EGFR antibody. The antibodies used for flow can be found in Supplementary Table 2 (Table S2).

Immunofluorescence (IF)

Cells were grown in suspension for 2 days, washed with PBS, and fixed with 4% paraformaldehyde. Fixed cells were then spun onto charged slides using the Thermo Shandon Cytospin 2 (Thermo Fisher Scientific). Slides were washed again in PBS, permeabilized with 0.5% Triton X-100 for 5 min, washed with PBS three more times, and then blocked with blocking buffer (2% BSA in PBS) for 1 h. Slides were then incubated with primary antibody overnight at 4 °C. The next day, slides were washed with PBS thrice, incubated with DAPI and secondary antibodies (diluted in blocking buffer) for 30 min in the dark, and then washed thrice again in PBS, in the dark. Mounting medium (20 mg/mL O-phenylenediamine, dissolved in 1 M pH 8.5 Tris, and then diluted 1:10 in glycerol) was added to the slides, which were then covered with a glass coverslip and sealed with nail polish. The antibodies used for IF can be found in Supplementary Table 2 (Table S2). Confocal microscopy was performed using LSM780 (Zeiss).

Co-localization analysis was performed using the FIJI software. As the two proteins of interest were stained for using secondary antibodies conjugated to either AlexaFluor488 (represented as green) or Cy3 (represented as red), we performed thresholding to identify the “yellow” areas (as defined by a threshold value between 23–46), and normalized the “yellow” area to the number of cells (nuclei) for each image.

Immunohistochemistry (IHC)

Immunohistochemistry on fixed mouse omentum for cleaved caspase 3 and Ki67 was performed, and slides were analyzed using the QuPath software, as previously described [47, 48]. The antibodies used for IHC can be found in Supplementary Table 2 (Table S2).

Reverse phase protein array (RPPA)

One million PEO1 control or shCASC4 cells were cultured, each in triplicate, in suspension in a 6-well dish for 48 h, centrifuged, washed with PBS, and centrifuged again. Pellets were then flash-frozen at −80 °C and shipped to the MD Anderson Reverse Phase Protein Array Core Facility. Lysis and subsequent RPPA was performed by the core facility as outlined here (https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core/education-and-references.html). Fold-changes (FC) for each antibody were calculated using the NormLinear values, relative to the average of the control (PEO1 shCTRL) cells. Significance was determined by performing a 2 tailed Student’s t-test between the NormLinear values for each antibody, with an FDR of 0.15.

In vivo mouse experiments

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the University of Colorado IACUC (Protocol #00569). ID8 cells (Trp53-null, Brca2-null, GFP- and luciferase-tagged) were transduced with a control shRNA (CTRL), or one out of two shCasc4 constructs (labeled shCasc4 #1 and shCasc4 #2). In total, 5 × 106 cells, suspended in 100 μl PBS, were injected intraperitoneally into 6–8 week-old female mice. Starting 7 days after initial cell injection, tumor burden was measured weekly using an In Vivo Imaging System, and analyzed using Live Imaging 4.0 software (PerkinElmer), as previously described [47]. Five weeks post-injection, mice were euthanized using a CO2 chamber followed by a cervical dislocation. The peritoneal cavity and harvested omentum tissue and tumors were exposed to UV light. Omentum and tumors were weighed. Each omentum was cut in half; one half was fixed in 4% paraformaldehyde for IHC, and the other was snap-frozen for immunoblot experiments.

Statistical analysis and considerations

Statistical analyses and p value calculations were performed using GraphPad Prism 9 for MacOS. Quantitative data are expressed as mean ± standard error of the mean unless otherwise noted. Analysis of variance was used to identify significant differences in multiple comparisons. We performed F-tests for all tests. For all statistical analyses, the level of significance was set at 0.05. Outliers were identified using Grubbs’ outlier test with a power of 0.05, and excluded. For animal studies, no blinding or randomization was performed. A population size of 7 for each group was determined through a power analysis, using colony formation data as expected means.