GON4L Drives Cancer Growth through a YY1-Androgen Receptor-CD24 Axis

Abstract

In principle, the inhibition of candidate gain-of-function genes defined through genomic analyses of large patient cohorts offers an attractive therapeutic strategy. In this study, we focused on changes in expression of CD24, a well-validated clinical biomarker of poor prognosis and a driver of tumor growth and metastasis, as a benchmark to assess functional relevance. Through this approach, we identified GON4L as a regulator of CD24 from screening a pooled shRNA library of 176 candidate gain-of-function genes. GON4L depletion reduced CD24 expression in human bladder cancer cells and blocked cell proliferation in vitro and tumor xenograft growth in vivo Mechanistically, GON4L interacted with transcription factor YY1, promoting its association with the androgen receptor to drive CD24 expression and cell growth. In clinical bladder cancer specimens, expression of GON4L, YY1, and CD24 was elevated compared with normal bladder urothelium. This pathway is biologically relevant in other cancer types as well, where CD24 and the androgen receptor are clinically prognostic, given that silencing of GON4L and YY1 suppressed CD24 expression and growth of human lung, prostate, and breast cancer cells. Overall, our results define GON4L as a novel driver of cancer growth, offering new biomarker and therapeutic opportunities. Cancer Res; 76(17); 5175-85. ©2016 AACR.

Figure 1

Identification of GON4L as a regulator of CD24 expression. A, schematic diagram showing the screening strategy. MGHU4 shPool cells shifted towards low FITC-CD24 intensity as indicated by the arrow and cells in box were FACS sorted, clonally expanded, screened for low CD24 expression and shRNAs present were identified by sanger sequencing. Cells were transfected with non-target control (siCTL) or GON4L siRNAs and after 96h, B, mRNA levels of GON4L were measured; C, mRNA levels of CD24 and D, representative western blot images showing CD24 protein (densitometric analysis was done using ImageJ software, values were normalized to α-actinin) in cells from (B). Results are shown as mean ± S.D., *p<0.05 by Student’s t-test.

Figure 2

GON4L and CD24 depletion inhibit growth of bladder cancer cells. After 48h of transfection with control and GON4L siRNAs, cells were processed for (A) cell proliferation. Graphs show fold change in CyQuant dye fluorescence intensity compared to day 0 of siCTL and siGON4L, respectively. B, cells from (A) split onto soft agar plates. Graphs show % change in number of soft agar colonies. C and D, cells depleted of CD24 by siRNA knockdown were processed for (C) cell proliferation, CD24 western blot (inset) and (D) soft agar assays. E and F, TCCSUP cells treated with increasing concentrations of GON4L siRNA have dose-dependent reduction in (E) soft agar colonies, CD24 protein levels (inset) and (F) cell proliferation rate. G and H, TCCSUP cells treated with increasing concentrations of CD24 siRNA have dose-dependent reduction in (G) soft agar colonies, CD24 protein levels (inset) and (H) cell proliferation rate. Results are shown as mean ± S.D., *p<0.05 by Student’s t-test.

Figure 3

YY1 interacts with GON4L and regulates CD24 expression and growth of bladder cancer cells. A, Co-IP for the GON4L-YY1 interaction in UMUC3 cells stably expressing Flag-GON4L and transiently transfected for HA-YY1. Cells were lysed with NP-40 lysis buffer after 96h of transfection. Cells were transfected with siCTL and two different siRNAs for YY1 (YY1-1 and YY1-2), after 96h were processed for (B) YY1 mRNA expression, (C) Protein levels of CD24 and (D) CD24 mRNA expression. After 48h of transfection, cells were processed for cell proliferation and soft agar colony formation. E, fold changes in CyQuant dye fluorescence intensity as compared to Day 0 of siCTL and siYY1 cells. F, percent change in soft agar colonies. Results are shown as mean ± S.D., *p<0.05 by Student’s t-test.

Figure 4

GON4L and YY1 affect tumor formation in vivo and their expression is associated with bladder cancer. UMUC3 cells transduced with non-target, GON4L and YY1 shRNAs and selected with puromycin for 4 days before use. A, knockdown of GON4L and YY1 confirmed by qRT-PCR, inset showing CD24 protein levels after GON4L and YY1 knockdown. Results are shown as mean ± S.D. B, differences in tumor incidence with the bars representing percentage of tumors formed in nude mice injected with 500,000 cells/site (n = 15). C, subcutaneous tumor growth over time. Results are shown as mean ± S.E., *p<0.05 by Student’s t-test. D, genomic alterations in GON4L in 4 human bladder cancer patient datasets (indicated), as reported in cBioPortal (http://www.cbioportal.org). CNA-copy number alterations. E and F, GON4L, YY1 and CD24 mRNA expression in human urothelial tumors compared to normal human bladder tissue. F, normal tissues were “matched” to tumors in the same patient in TCGA set (gray). Results in (E) and (F) were analyzed using different gene expression platforms as mentioned in the Materials and Methods section, statistical comparisons were done using the Wilcoxon rank sum test. Horizontal lines in boxes represent median of gene expression. Box and whisker represents the first and third quartiles..

Figure 5

GON4L and YY1 regulate CD24 transcription via AR. A, diagram of 3400bp, 1896bp wild type and 3400bp CD24 promoter with both YY1 binding sites mutated. B, percent change in DLR ratio (firefly/renilla luciferase activity) representing CD24 promoter activity in control and YY1 knockdown cells. After 96h of transfection with (C) GON4L and (D) YY1 siRNA, AR⁻ and AR⁺ cells were blotted for CD24 and α-actinin as loading control. Non-target siRNA was used as control. Densitometric analysis was done using ImageJ software, values were normalized with α-actinin. E, Co-IP in UMUC3 cells overexpressing empty vector (EV) or Flag-AR and HA-YY1 (AR/YY1). F, Co-IP in 293T cells transfected with EV, full length GON4L or deletion mutant GON4L (ΔC3) along with Flag-AR and HA-YY1. G, cells were transfected with control, GON4L and YY1 siRNA, 24h later transfected with wild type or ARE mutant 1896bp CD24 promoter and activity was measured after 48h. Results are shown as mean ± S.D., *p<0.05 by Student’s t-test.

Figure 6

GON4L and YY1 knockdown affect CD24 expression and growth of human lung, prostate and breast cancer cell lines. A, genomic alterations in GON4L in lung, prostate and breast cancer patient datasets, as taken from cBioPortal (http://www.cbioportal.org). CNA-copy number alterations. B–D, cells were transfected with non-target, GON4L and YY1 siRNAs. 48h later, cells were split to measure (B and C) proliferation and (D) soft agar colonies. Cells were also processed for CD24 western blot after 96h of transfection shown in inset. Densitometric analysis was done using ImageJ software, values were normalized with α-actinin. Results are shown as mean ± S.D., *p<0.05 by Student’s t-test.

Figure 7

AR and CD24 independent effect of GON4L and YY1 on cell growth. A, cells transfected with siGON4L or siCTL treated with either DMSO as solvent control or 50µM Enzalutamide (Enza) were processed for cell viability by MTT assay after 72h. Graphs show the percent change in optical density at 530nm. B, UMUC3 and TCCSUP cells stably overexpressing CD24 or empty vector were transfected with non-target or GON4L siRNA and processed for soft agar colony formation. Empty vector + siCTL transfected cells were used as control. Representative CD24 western blots are shown in inset. Densitometric analysis was done using ImageJ software, values were normalized with loading control. Values on the arrows indicate fold decrease in number of colonies formed. C and D, graphs showing changes in (C) cell proliferation rate and (D) percent soft agar colonies formed by siCTL, siGON4L and siYY1 transfected AR⁻ cells. Results are shown as mean ± S.D., *p<0.05 by Student’s t-test. E, proposed mechanism of action of GON4L regulated tumor growth.