Identification of BLCAP as a novel STAT3 interaction partner in bladder cancer

<div><p>Bladder cancer associated protein (Blcap) expression is commonly down-regulated in invasive bladder cancer, and may have prognostic value given that its expression is negatively correlated with patient survival. We have previously investigated the expression patterns and cellular localization of Blcap in bladder cancer, where we found that about 20% of the lesions examined displayed strong nuclear expression of Blcap, and that this phenotype was associated with overall poor disease outcome. Here we report on the analysis of possible functional associations between nuclear expression of Blcap and canonical signaling pathways. We performed serial immunohistochemistry (IHC) analysis of bladder tissue samples, with serial sections stained with phospho-specific antibodies recognizing key signaling intermediates, such as P-Stat3, P-Akt, and P-Erk1/2, among others, in an immunophenotyping approach we have established and reported previously. Using this approach, we found that nuclear localization of Blcap was associated with expression of P-Stat3. A parallel analysis, cytokine profiling of bladder tumor interstitial fluids of samples expressing (or not) Blcap, showed interleukin (IL)-6, IL-8, and monocyte chemotactic protein 1 (MCP-1) to be correlated with nuclear expression of Blcap, independently supporting a role for Stat3 signaling in localization of Blcap. Multiple indirect immunofluorescence analysis of tissue biopsies confirmed that Blcap co-localized with Stat3. Furthermore, we could also demonstrate, using an in situ proximity ligation assay that Blcap and Stat3 are in close physical proximity of each other in bladder tissue, and that Blcap physically interacts with Stat3 as determined by co-immunoprecipitation of these proteins. Our data indicates that Blcap is a novel Stat3 interaction partner and suggests a role for Blcap in the Stat3-mediated progression of precancerous lesions to invasive tumors of the bladder.</p></div

Correlation between Blcap and p-Stat3 expression in 80 UC samples.

<p>Correlation between Blcap and p-Stat3 expression in 80 UC samples.</p

Proximity ligation assay.

<p>Representative images for Blcap and Stat3 PLA assay in bladder cancer samples showing nuclear staining (DAPI) in blue and PLA signals in red. (A) For control experiments, one of the primary antibodies was exchanged for an isotype-matched control antibody. (B) PLA signals in a sample (T#2) with strong scattered nuclear expression of Blcap and Stat3, show that the two proteins are in close proximity of each other (white arrows) in some nuclei, and in more spuriously in the cytoplasm (yellow arrows). Other nuclei had no detectable PLA signals (red arrows). This pattern was consistent with immunofluorescence results for a tandem section (C) that showed colocalization of Blcap and Stat3 in some nuclei (white arrows) and no expression of either protein in other nuclei (red arrow). (D) Higher magnification clearly shows the nuclear PLA signals (white arrow) and occasional cytoplasmic signal (yellow arrow). (E) PLA analysis of a sample that showed moderate cytoplasmic expression of Blcap (panel H) displaying only occasional cytoplasmic PLA signals. (F) PLA analysis of a sample that had no detectable expression of Blcap also lacked PLA signals. (G-I) IHC images of samples used for PLA analysis of panels B, E, and F, respectively.</p

Immunophenotyping of Blcap positive tumor cells.

<p>Tandem sections of a bladder tumor specimen (T#9) were stained with antibodies against Blcap, p-Stat3, p-mTor, p-p44/42, p-Sapk/Jnk, and p-p38 and allowing us to determine which signaling event was associated with Blcap expression. Magnification, 20x. Inset are higher magnifications (40x) of the same region in the consecutive sections.</p

Cytokine profiling of bladder tumor interstitial fluid.

<p>Cytokine-specific antibody arrays (RayBio^® human cytokine array 5.1, RayBioTech Inc, USA) were incubated with 0.5 ml of TIF from four different samples either strongly expressing Blcap (T#3), or devoid of Blcap (T#4, T#5, and T#6), respectively, according to the manufacturer’s instructions. Expression of three cytokines, interleukin (IL)-6, IL-8, and monocyte chemotactic protein 1 (MCP-1), correlated with the Blcap expression status of the samples. Two other factors, VEGF and angiogenin, are also highlighted as examples of seemingly uncorrelated factors predominantly expressed in one of the samples. A map of the cytokines detected by the antibody array is provided on the upper panel.</p

Staining patterns of Blcap and p-Stat3.

<p>(A and B) IHC staining of Blcap and p-Stat3, respectively, in consecutive sections of a non-malignant bladder section demonstrated the expression of the two antigens in urothelial cells with weak cytoplasmic and moderate, scattered nuclear expression. Black arrow points to a region showing expression of Blcap. (C and D) IHC of an UC (T#10) composed of two distinct lesions, one with strong expression of Blcap (black arrow) and another with no expression of this protein (red arrow), displayed the exact same staining pattern for p-Stat3, with the same areas being positive (black arrow) or negative (red arrow) for p-Stat3, respectively. (E and F) an UC sample (T#1) with a very distinctive Blcap interspersed nuclear staining showed an almost identical staining pattern for p-Stat3, with positive (black arrow) and negative cells (red arrow) intermingling. Inset are higher magnifications (40x) of selected representative areas of each section.</p

2D immunoblotting analysis.

<p>Lysates of two tissue specimens determined by IHC to express Blcap (A) or not (B) were resolved by 2D PAGE (IEF) and blotted onto a nitrocellulose membrane. The immunoblot protein patterns show that expression of Blcap followed the IHC analysis and that Stat3 was expressed at similar levels in both cases. Black arrows indicate the primary form- and white arrows a posttranslational modified form of the protein, presumably due to phosphorylation as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188827#pone.0188827.ref001" target="_blank">1</a>]. Actin was used as loading control.</p

Clinicopathological correlation of BLCAP expression in 2,197 sample TMA.

*<p>Kruskal-Wallis one way analysis of variance on ranks.</p>**<p>Mann-Whitney rank-sum test.</p

2D PAGE and 2D Western blot analysis of BLCAP protein spot patterns.

<p>(A) COS-1 cells transfected with pZeoSV2 empty vector and labeled with ³⁵S-methionine. (B) COS-1 cells transfected with pZeoSV2– BLCAP overexpressing construct and labeled with ³⁵S-methionine. Radioactive metabolic labeling (³⁵S-methionine) of COS-1 cells was used to ensure the highest detection sensitivity. (C) 2D Western blot of COS-1 cells transfected with pZeoSV2– BLCAP construct detected with anti-BLCAP antibody (10 sec film exposure). (D) 2D gel of proteins from breast tumor 63 stained with silver. (E) 2D Western blot of protein lysate from breast tumor 63 (see D) reacted with anti-BLCAP antibody (1 min film exposure). The positions of the BLCAP protein in the 2D-PAGE gels and corresponding 2D Western blots, are indicated by black arrows. The positions of several reference proteins are indicated by red arrows: ACTB – beta actin; ENO1 -alpha enolase 1; CANX – calnexin; PDI - Protein disulfide-isomerase; TUBA1A - tubulin alpha-1A chain; YWHAZ - 14-3-3 protein zeta/delta. The identity of all reference spots were confirmed by MS analysis.</p

Clinical characteristics of DCTB tissue specimens.

<p>Clinical characteristics of DCTB tissue specimens.</p