Low ANXA10 expression is associated with disease aggressiveness in bladder cancer

Abstract

Background: Markers for outcome prediction in bladder cancer are urgently needed. We have previously identified a molecular signature for predicting progression in non-muscle-invasive bladder cancer. ANXA10 was one of the markers included in the signature and we now validated the prognostic relevance of ANXA10 at the protein level.

Methods: We investigated ANXA10 expression by immunohistochemistry using a tissue microarray with 249 Ta and T1 urothelial carcinomas. The expression of ANXA10 was also investigated in an additional set of 97 more advanced tumours. The functional role of ANXA10 in cell lines was investigated by siRNA-mediated ANXA10 knockdown using wound-healing assays, proliferation assays, and ingenuity pathway analysis.

Results: Low expression of ANXA10 correlated with shorter progression-free survival in patients with stage Ta and T1 tumours (P<0.00001). Furthermore, patients with more advanced tumours and low ANXA10 expression had an unfavourable prognosis (P<0.00001). We found that ANXA10 siRNA transfected cells grew significantly faster compared with control siRNA transfected cells. Furthermore, a wound-healing assay showed that ANXA10 siRNA transfected cells spread along wound edges faster than control transfected cells.

Conclusion: We conclude that ANXA10 may be a clinical relevant marker for predicting outcome in both early and advanced stages of bladder cancer.

Figure 1

Survival as function of ANXA10 expression. (A) Kaplan–Meier survival plot with progression-free survival as function of ANXA10 expression measured by microarray analysis (patient cohort 1). The patients were divided into two groups based on ANXA10 mean expression. (B) Kaplan–Meier plot of progression-free survival as a function of the percentage of nuclear ANXA10 expression (n=249; patient cohort 2). (C) Kaplan–Meier plot of progression-free survival as a function of ANXA10 expression in combination with p53 nuclear immunostaining (patient cohort 2). (D) Kaplan–Meier plot of metastasis-free survival as a function of ANXA10-positive regions (patient cohort 3). (E) Kaplan–Meier plot of metastatic-free survival as a function of both ANXA10-positive regions and S100A4 focal staining (patient cohort 3). (F) Kaplan–Meier plot of metastasis-free survival as a function of ANXA10-positive regions, nuclear staining of p53, and nuclear staining of pRB (patient cohort 3).

Figure 2

ANXA10 expression in tumour tissue and CIS lesions. (1–4) Non-muscle-invasive bladder tumours with either high (1, 2) or low (3, 4) expression of ANXA10. (5–8) Muscle-invasive bladder tumours with either high (4–6) or low (7, 8) expression of ANXA10. (9, 10) ANXA10 expression in CIS lesions.

Figure 3

Phenotypic effects of siRNA-mediated knockdown of ANXA10. (A) ANXA10 knockdown in the bladder cancer cell line SW780 was validated by RT–pPCR (left) and western blotting analysis (right) at 48 h post-transfection. Cells were transfected with 5, 10, 50 nM of ANXA10 (ANXA10), 10 nM Control siRNA (Control), no siRNA with transfection reagent (Mock+Lipo), or no siRNA or transfection reagent (Mock). A concentration of 10 nM siRNA was used for western blotting (right). Both experiments were performed in duplicate. (B) The SW780 bladder cancer cell line was transfected with 50 nM siRNA against either ANXA10 (purple) and 50 nM control siRNA (red). The cell index (CI) describes the degree of cell confluence. (C) Wound-healing experiments were performed by wounding confluent SW780 cells 96 h post-transfection with either anti-ANXA10 siRNA or control siRNA (left). ANXA10 and S100A4 expression were measured by RT–qPCR and normalised to Ubiquitin B expression at 72 and 96 h post-transfection (right). Control samples were normalised to 1. ANXA10 knockdown was validated by western blotting analysis at 72 and 96 h post-transfection.