Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma

Abstract

Advanced upper urinary tract urothelial carcinoma (UTUC) is often associated with poor oncologic outcomes. The secreted protein acidic and rich in cysteine-like 1 (SPARCL1) protein, belongs to the SPARC-related family of matricellular proteins. Much literature has been published describing the role of SPARCL1 in the prognosis many cancers. In this study, methylated promoter regions in high-grade and high-stage upper urinary urothelial tumours compared with normal urothelium were analyzed and revealed that SPARCL1 was the most significantly hypermethylated gene in UTUC tissues. Then we prospectively collected UTUC samples and adjacent normal urothelium for pyrosequencing validation, identifying significant CpG site methylation in UTUC tissues. In addition, SPARCL1 RNA levels were significantly lower in UTUC samples. Multivariate Cox regression analysis from 78 patients with solitary renal pelvic or ureteral pT3N0M0 urothelial carcinomas revealed that only negative SPARCL1 expression and nonpapillary tumour architecture were independently associated with systemic recurrence (p = 0.011 and 0.008, respectively). In vitro studies revealed that the behaviour of BFTC-909 cells was less aggressive and more sensitive to radiation or chemotherapy after SPARCL1 overexpression. Thus, SPARCL1 could be considered as a prognostic marker and help decision-making in clinical practice.

Keywords: SPARCL1; upper urinary tract urothelial carcinoma.

Figure 1

DNA methylation profiles between the high-grade and high-stage urothelial tumor and normal urothelium sets. We conducted methylation microarray assays on 3 UTUC and 3 normal urothelium samples and the raw data generated was analyzed with Partek. (A) Hypermethylated promoter region of each chromosome. (B) Principal component analysis plot based on methylation profiles in UTUC (red) and normal urothelium (blue). (C) The top-10 hypermethylated genes in UTUC compared with normal urothelium samples.

Figure 2

SPARCL1 associated with hyper-DMR was down-regulated in the upper urinary tract urothelial carcinoma. (A) Schematic view of the SPARCL1 locus. There were four CpG sites in the SPARCL1 promoter region. (B) Four methylation sites in the SPARCL1 promoter region identified by bisulphite pyrosequencing. (C,D) SPARCL1 CpG site methylation levels in the control (adjacent urothelium, n = 25) and the paired tumor samples. (E) Quantitative polymerase chain reaction from UTUC (n = 55 patients) and their matched adjacent normal tissues.

Figure 3

SPARCL1 was down-regulated in aggressive UTUC via DNA methylation. (A) The IHC staining of SPARCL1 in human UTUC TMA samples with triplicate cores per case. (B) A significantly reduced expression of SPARCL1 in UTUC with pathologic tumor stages 3 and 4 compared to those with stages 0, 1, and 2 was determined by IHC staining. * p < 0.05 between the indicated groups. (C) Immunoreactivity score of SPARCL1 protein expression in M0 and M1 tumor of human UTUC. * p < 0.05 between the indicated groups. (D) Significantly worse systemic UTUC recurrence free survival in SPARCL1 negative UTUCs. (E) RT-qPCR and (F) Western blots of SPARCL1 expression in an immortalized human urothelial cells (SV-HUC-1), a low grade urothelial carcinoma cell line (RT4) and 3 high grade urothelial carcinoma cell lines (J82, BFTC909, and T24). β-actin was used as a loading control. The predicted molecular weight of SPARCL1 is ~75kDa and we did not detect SPARCL1 monomer bands (~75 kD). SPARCL1 molecular weight was approximately 150 kDa, suggesting that it may be present in the homodimer form. (G) SPARCL1 CpG site methylation levels in UC cell lines. (H) The mRNA expression of SPARCL1 was detected by RT qPCR in SV-HUC-1, RT4 and BFTC909 cell lines treated with 5-aza-2′-deoxycytidine. Error bars represent the mean ± S.E.M., ** p < 0.01; ns indicates no significance.

Figure 4

SPARCL1 decreases proliferation and migration of BFTC909 cells and improves the anti-tumor effects of radiation or chemotherapy. (A) SPARCL1 overexpression inhibited clonogenicity in vitro. Magnification: 1×. (B) Transwell assays were performed to evaluate the effects of SPARCL1 expression on migration of BFTC909 cells. Magnification: 100×. Values are mean ± S.E.M. of three independent experiments. (C) SPARCL1 expression is correlated with EMT marker expression. (D) SPARCL1 overexpression increased the efficacy of radiotherapy on BFTC909 cells in colony formation assays. Magnification: 1×. (E) SPARCL1 overexpression augmented cisplatin effects on cell viability evaluated using MTT assays.