CD24 expression is important in male urothelial tumorigenesis and metastasis in mice and is androgen regulated

Abstract

Overexpression of CD24, a glycosyl phosphatidylinositol-linked sialoglycoprotein, is associated with poor outcome in urothelial carcinoma and contributes to experimental tumor growth and metastasis. However, the requirement for CD24 (Cd24a in mice) in tumorigenesis and spontaneous metastasis from the orthotopic site remains uncharacterized. Using N-butyl-N-(4-hydroxybutyl) nitrosamine induction of invasive and metastatic bladder cancer, we show that Cd24a-deficient male mice developed fewer bladder tumors than C57BL/6 control male mice. Evaluating only mice with evidence of primary tumors, we observed that Cd24a-deficient male mice also had fewer metastases than wild-type counterparts. In parallel observations, stratification of patients based on CD24 immunohistochemical expression in their tumors revealed that high levels of CD24 are associated with poor prognosis in males. In female patients and mice the above observations were not present. Given the significant role of CD24 in males, we sought to assess the relationship between androgen and CD24 regulation. We discovered that androgen receptor knockdown in UM-UC-3 and TCCSUP human urothelial carcinoma cell lines resulted in suppression of CD24 expression and cell proliferation. Androgen treatment also led to increased CD24 promoter activity, dependent on the presence of androgen receptor. In vivo, androgen deprivation resulted in reduced growth and CD24 expression of UM-UC-3 xenografts, and the latter was rescued by exogenous CD24 overexpression. These findings demonstrate an important role for CD24 in urothelial tumorigenesis and metastasis in male mice and indicate that CD24 is androgen regulated, providing the foundation for urothelial bladder cancer therapy with antiandrogens.

Fig. 1.

Germline Cd24a loss is associated with reduced incidence of OH-BBN–induced bladder cancer. (A) Schematic of the experimental design for assessing bladder tumor incidence. WT and Cd24a-deficient (Cd24a^−/−) mice were started on OH-BBN ∼4 wk after weaning. Successive mCT imaging of pilot study animals suggested the use of assessment points at 16, 24, and 28 wk for best assessment of tumor development, so cohorts were analyzed for the presence of bladder tumors at those time points. (B) Corresponding mCT scans (Top), necropsy images (Middle), and histological preparations (Bottom) collected at 24 wk. Images i, iii, and v are of a superficial (i.e., noninvasive) bladder tumor; ii, iv, and vi are of invasive tumor. Arrows indicate the bladder wall in i–iv and the basement membrane in v. L, lumen of bladder. (C) Comparison of tumor incidence over time in populations of WT and Cd24a-deficient mice using binary and logistic regression models revealed that the incidence of malignancy was significantly higher in WT mice than in Cd24a-deficient mice. *P < 0.05. N.S., not significant. (D) Representative H&E-stained bladder tissues displayed no differences in histology between grade-matched WT and Cd24a^−/− tissues.

Fig. 2.

OH-BBN–induced bladder tumors have high levels of Cd24a mRNA and protein expression. (A) C57BL/6 WT mice (n = 21) were provided with normal drinking water until age 4 wk; then 12 mice were killed, and the remaining nine mice were exposed to drinking water containing OH-BBN ad libitum for an additional12 wk. Bladders from each cohort were processed for histological and qRT-PCR analysis. Cd24a mRNA analysis revealed that cancerous urothelium from the cohort exposed to OH-BBN had significantly more Cd24a than comparative control normal urothelium, regardless of the sex of the mice. **P < 0.01, *P < 0.05; Mann–Whitney test; N.S., not significant. (B) The expression of Cd24a protein increases as a function of tumor progression. After exposure to OH-BBN, bladders from WT mice with cancer were found to express higher levels of Cd24a protein than dysplastic counterparts (n = 17). Cancerous urothelial samples from Cd24a-deficient mice were stained similarly to control for antibody specificity (n = 4).

Fig. 3.

Loss of Cd24a decreases metastasis in male mice. (A) Schematic of the experimental design for assessing metastatic incidence. WT (n = 102) and Cd24a-deficient (n = 88) mice were provided OH-BBN until they reached previously established surrogates of death (cachexia >20% of control animal weight, lethargy/behavioral changes, or overt respiratory/general distress). (B) Lung metastatic tissues from Cd24a-deficient and WT mice were stained by H&E and showed similar histological architecture. (C) Of animals with documented primary tumors, WT males had higher rates of metastasis (21.7%) than WT females (3.1%). Additionally, Cd24a-deficient males had decreased metastatic incidence (7.0%) relative to their WT counterparts. *P = 0.039; one-tailed Wald test. Comparisons of WT and Cd24a-deficient females showed no statistically significant difference (N.S.) in metastasis. (D) Evaluation of a previously reported tissue microarray with immunohistochemical staining for CD24 (4). Stratification of disease-free survival data from human bladder cancer patients as a function of patient sex and CD24 expression level. The red line indicates a score of 1 or 2; the blue line indicates a score of 3. [Scoring was described previously (4)]. Kaplan–Meier analysis suggests that CD24 is an independent prognostic factor for disease-free survival in males (P < 0.05).

Fig. 4.

Tumor growth promoted by androgen signaling is dependent on CD24 in vitro and in vivo. (A) Relative growth of UM-UC-3 cells as assessed by alamarBlue 72 h after indicated treatments and siRNA transfections. Treatment with R1881 increases growth compared with DMSO. Transfection of cells with either AR or CD24 siRNA inhibits R1881-induced growth. *P < 0.05. (B) Xenograft in vivo tumorigenesis model. Normal and castrated male nude mice (n = 10) were injected s.c. with 5 × 10⁵ UM-UC-3 cells. After 4 wk, tumors were resected and evaluated for CD24 protein expression with human CD24-specific antibodies. Tumors isolated from normal male mice expressed higher levels of CD24 than tumors resected from castrated male mice. (C) Normal and castrated male nude mice (n = 10) were injected s.c. with 5 × 10⁵ UM-UC-3 cells that had been vector transfected or CD24 transfected as described previously (5). Graph represents tumor size over time. Data show that castration can reduce growth of UM-UC-3 tumors and that stable exogenous expression of CD24 in UM-UC-3 cells can rescue this growth reduction. Error bars indicate SEM.

Fig. 5.

CD24 mRNA and protein expression is regulated by androgen. (A) UM-UC-3, TCCSUP, and HTB9 cells were transfected with control (CTL si) or AR siRNA (AR si) for 48 h before being treated with DMSO, R1881 (1 nM), or Casodex (30 µM). After 24 h, lysates were collected and analyzed for CD24 and AR protein expression. OD values obtained with Alpha Innotech software (normalized for protein loading using β-actin staining) are plotted below the corresponding immunoblots. Representative blots from three separate experiments are shown. (B and C) Total RNA extracted from UM-UC-3 (B) and TCCSUP (C) cells in A was analyzed for CD24 expression by qRT-PCR. Treatment with R1881 increases CD24 expression, an effect that is abrogated when cells are transfected first with AR siRNA. *P < 0.05.

Fig. 6.

Androgen signaling promotes activation of the CD24 promoter. (A) Two CD24 promoters of different length fused to a luciferase reporter were tested for sensitivity to androgen treatment. The plasmids were transiently transfected into UM-UC-3 or TCCSUP cells and subsequently were incubated with or without R1881 for 24 h. The PSA promoter reporter was used as a positive control, and the GL4 empty vector was used as a negative control for response to R1881. *P < 0.05. (B) The ARE in the 1,896-bp promoter was mutated (pCD24-1896m); this mutation blocks R1881-induced activity of the CD24 promoter. The precise base pairs of the ARE in the CD24 promoter are shown. (C) The 1,896-bp reporter no longer responds to R1881 when cells are treated with AR siRNA. (D) ChIP with AR antibody confirms that AR associates with the CD24 promoter at the aforementioned ARE. Positive controls included histone H3 antibody and PSA ARE-based primers. Mouse IgG and primers targeting a non-ARE element in the CD24 promoter were used as negative controls.

Fig. P1.

The loss of the Cd24a gene in mice reduces tumor formation in a sex-specific manner. Comparison of tumor incidence over time revealed that male WT mice had a significantly higher incidence of tumor formation than female mice. Interestingly, Cd24a deficiency in males dramatically reduced this incidence in males but had no impact in female mice. The studies presented here suggest that this difference in tumor formation in the sexes is driven by androgen. This male-specific hormone is shown to increase directly expression of CD24, a known tumor-promoting protein. Thus, the presence of androgen in male mice leads to an increase in CD24 and an increase in tumor formation. However, the ability of androgen to induce tumor formation is reduced dramatically if CD24 is eliminated (Cd24a^−/−). AR, androgen receptor.