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. 2020 Jan 9;18(1):17.
doi: 10.1186/s12967-020-02211-4.

Selenium-binding protein 1 transcriptionally activates p21 expression via p53-independent mechanism and its frequent reduction associates with poor prognosis in bladder cancer

Yulei Wang  1   2 , Wenzhen Zhu  3 , Xiaoqing Chen  3 , Guangnan Wei  4 , Guosong Jiang  5 , Guochun Zhang  6   7
Affiliations

Selenium-binding protein 1 transcriptionally activates p21 expression via p53-independent mechanism and its frequent reduction associates with poor prognosis in bladder cancer

Yulei Wang et al. J Transl Med. .

Abstract

Background: Recent studies have shown that selenium-binding protein 1 (SELENBP1) is significantly down-regulated in a variety of solid tumors. Nevertheless, the clinical relevance of SELENBP1 in human bladder cancer has not been described in any detail, and the molecular mechanism underlying its inhibitory role in cancer cell growth is largely unknown.

Methods: SELENBP1 expression levels in tumor tissues and adjacent normal tissues were evaluated using immunoblotting assay. The association of SELENBP1 expression, clinicopathological features, and clinical outcome was determined using publicly available dataset from The Cancer Genome Atlas bladder cancer (TCGA-BLCA) cohort. DNA methylation in SELENBP1 gene was assessed using online MEXPRESS tool. We generated stable SELENBP1-overexpression and their corresponding control cell lines to determine its potential effect on cell cycle and transcriptional activity of p21 by using flow cytometry and luciferase reporter assay, respectively. The dominant-negative mutant constructs, TAM67 and STAT1 Y701F, were employed to define the roles of c-Jun and STAT1 in the regulation of p21 protein.

Results: Here, we report that the reduction of SELENBP1 is a frequent event and significantly correlates with tumor progression as well as unfavorable prognosis in human bladder cancer. By utilizing TCGA-BLCA cohort, DNA hypermethylation, especially in gene body, is shown to be likely to account for the reduction of SELENBP1 expression. However, an apparent paradox is observed in its 3'-UTR region, in which DNA methylation is positively related to SELENBP1 expression. More importantly, we verify the growth inhibitory role for SELENBP1 in human bladder cancer, and further report a novel function for SELENBP1 in transcriptionally modulating p21 expression through a p53-independent mechanism. Instead, ectopic expression of SELENBP1 pronouncedly attenuates the phosphorylation of c-Jun and STAT1, both of which are indispensable for SELENBP1-mediated transcriptional induction of p21, thereby resulting in the G0/G1 phase cell cycle arrest in bladder cancer cell.

Conclusions: Taken together, our findings provide clinical and molecular insights into improved understanding of the tumor suppressive role for SELENBP1 in human bladder cancer, suggesting that SELENBP1 could potentially be utilized as a prognostic biomarker as well as a therapeutic target in future cancer therapy.

Keywords: Bladder cancer; Methylation; STAT1; Selenium-binding protein 1 (SELENBP1); c-Jun; p21; p53.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
SELENBP1 is frequently down-regulated in human bladder cancer, and low expression of SELENBP1 predicts poor clinical prognosis. a Twelve pairs of bladder cancers (T) and adjacent matched normal tissues (N) were extracted and subjected to immunoblotting assay for determining the levels of SELENBP1 protein expression. GAPDH was used as a loading control. b Relative intensity of SELENBP1 in a was determined using Quantity One software, and normalized to GAPDH. The relative intensity of SELENBP1 in normal tissues was set to 1.0. Paired t test was performed and p value was indicated in top panel. cSELENBP1 mRNA levels were extracted from TCGA bladder cancer (TCGA-BLCA) cohort, including 19 of normal bladder tissues and 406 of bladder cancer tissues. Non-paired t test was performed and p value was indicated in the panel. d Kaplan–Meier 10-year survival analysis among patients with high and low SELENBP1 expression in TCGA-BLCA cohort. The log-rank test was performed and p value was indicated in the panel. Hazard ration (HR) and 95% confidence interval (CI) were also calculated
Fig. 2
Fig. 2
Visualization of the association between SELENBP1 expression and DNA methylation in TCGA-BLCA cohort using the MEXPRESS tool. SELENBP1 gene together with its transcripts is indicated in the left bottom panel. Next to the gene, blue line plots show the methylation data for the location of each probe (Infinium Human Methylation 450 microarray data; infinium 450 k), whose relative location in SELENBP1 gene is also indicated in the far right panel. The yellow line plot displays the RNA-seq-derived expression data, and the samples are ordered by SELENBP1 expression value. This view shows how SELENBP1 expression and DNA methylation in different parts of SELENBP1 gene are correlated, which is confirmed by the Pearson correlation test. The correlation coefficients (r) are indicated in the right panel. The symbol (***) shows a significant correlation (p < 0.001). TSS, transcription start site; UTR, untranslated region
Fig. 3
Fig. 3
Overexpression of SELENBP1 inhibits bladder cancer cell growth. a Cell lysates of human bladder cancer cell lines were extracted and subjected to immunoblotting assay for determining the levels of SELENBP1 protein expression. GAPDH was used as a loading control. b UMUC3 cells were stably transfected with empty vector (Vector) or construct encoding HA-tagged SELENBP1 (HA-SELENBP1). Cell lysates of these stable transfectants were extracted and subjected to immunoblotting assay by using anti-HA antibody. GAPDH was used as a loading control. c The number of 1 × 104 stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells per well was seeded in 6-well plates. The number of viable cells at the indicated time points was determined by trypan blue exclusion assay. The values were shown as mean ± standard deviation from at least three independent experiments. The asterisk (*) indicates a significant difference in comparison to that of control group (p < 0.05). d Anchorage-independent cell growth of UMUC3 cells stably transfected with empty vector or construct encoding HA-SELENBP1 was determined by soft agar assay. The colony formation was observed and captured under an inverted microscope (×40 magnification; left panel) and numbers of colonies were scored on day 15, and presented as colonies (×100)/104 cells (right panel). The values were shown as mean ± standard deviation from at least three independent experiments. The asterisk (*) indicates a significant difference in comparison to that of control transfectants (p < 0.05)
Fig. 4
Fig. 4
Induction of p21 protein is crucial for SELENBP1-mediated G0/G1 phase cell cycle arrest. a UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were collected at day 4 as described in “Fig. 3c”, and subjected to flow cytometry assay following the staining of propidium iodide. b Cell lysates of stable transfectants UMUC3 (Vector), UMUC3 (HA-SELENBP1), T24T (Vector) and T24T (HA-SELENBP1) were extracted and subjected to immunoblotting assay for determining expression levels of G0/G1 phase arrest-associated regulatory proteins. GAPDH was used as a loading control. Densitometric quantification of p21 (relative to GAPDH) is shown under each blot. c HCT116 WT and HCT116 p21−/− cells were transfected with empty vector or HA-SELENBP1, each along with pGIPZ, and stable transfectants were established under the selection of puromycin. Cell lysates of these stable transfectants were extracted and then subjected to immunoblotting assay with anti-SELENBP1, anti-p21 or anti-GAPDH antibodies. d Anchorage-independent cell growth of stable transfectants described in “c” was determined by soft agar assay. Representative images of colonies were visualized under an inverted microscope (×40 magnification; left panel) and relative numbers of colonies were calculated on day 12 (right panel). The asterisk (*) indicates a significant difference in comparison to that of control transfectants (p < 0.05)
Fig. 5
Fig. 5
SELENBP1 transcriptionally stimulates p21 expression in a p53-independent manner. a Total RNA was isolated from indicated stable transfectants, and then subjected to RT-PCR assay for determining p21 mRNA levels. The mRNA levels of GAPDH were used as a loading control. b The potential binding sites of multiple transcription factors in the p21 promoter are shown in upper panel, and full-length (2.4 Kb), 1.8 Kb, 1.3 Kb and 200 bp of p21 promoter-driven luciferase reporters are schematically shown in lower panel. c Stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were transiently co-transfected with pRL-TK and p21 promoter-driven luciferase reporters as described in “b”. Following 36 h of transient transfection, luciferase reporter activity was determined and normalized to that of corresponding UMUC3-Vector. The asterisk (*) indicates a significant difference in comparison to that of corresponding control transfectants (p < 0.05). d Cell lysates of HCT116 WT and HCT116 p53−/− were subjected to immunoblotting assay for identification of p53 expression (left panel). Following 48 h of transient transfection with HA-SELENBP1 or empty vector, HCT116 p53−/− cells were extracted and then subjected to immunoblotting assay with anti-SELENBP1, anti-p21 or anti-GAPDH antibodies (right panel). Densitometric quantification of p21 (relative to GAPDH) is shown
Fig. 6
Fig. 6
The restoration of SELENBP1 leads to the suppression of c-Jun and STAT1 phosphorylation, both of which are required for SELENBP1-mediated transcriptional induction of p21 protein. a Cell lysates of stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were subjected to immunoblotting assay with indicated antibodies that might be involved in transcriptional regulation of p21 expression. b UMUC3 cells stably transfected with dominant-negative mutant form of c-Jun (TAM67) or pcDNA3.1(Vector) were extracted and then subjected to immunoblotting analysis with indicated antibodies. Densitometric quantification of p21 (relative to GAPDH) is shown. c UMUC3 cells were stably transfected with dominant-negative mutant STAT1 (DN-STAT1) or pEGFP-C1 construct (Vector), and were then extracted for immunoblotting assay with indicated antibodies. Densitometric quantification of p21 and SOCS1 (relative to GAPDH) is shown under each blot. d TAM67 (1 μg) and pcDNA3.1 (1 μg) plasmids were transiently transfected into stable UMUC3 (DN-STAT1) and UMUC3 (pEGFP-C1), respectively. Following 48 h of transient transfection, cells were collected and then subjected to immunoblotting assay. Densitometric quantification of p21 (relative to GAPDH) is shown. e Indicated amounts of empty vector (pcDNA3.1) and TAM67 was transiently co-transfected into stable UMUC3 (DN-STAT1) or UMUC3 (Vector) cells, together with 1.3 Kb of p21 promoter-driven luciferase reporter and pRL-TK, as an internal control. Thirty-six hours post transfection, luciferase reporter activity was determined. Luciferase activities of UMUC3 (Vector) and UMUC3 (DN-STAT1) groups are normalized to those of corresponding control group that transfected with 0.6 μg of pcDNA3.1, respectively. The asterisk (*) indicates a significant difference as compared to UMUC3 (DN-STAT1) group transfected with 0.6 μg of pcDNA3.1 (p < 0.05). The symbol (#) indicates p < 0.05 when compared with UMUC3 (Vector) group transfected with 0.6 μg of pcDNA3.1. The values are shown as mean ± standard deviation from four independent experiments. f Following 48 h of transient transfection with indicated plasmids, UMUC3 cells were collected and then subjected to flow cytometry assay
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