Analysis of STAT3 post-translational modifications (PTMs) in human prostate cancer with different Gleason Score

Abstract

Prostate Cancer (PCa) is a complex and heterogeneous disease. The androgen receptor (AR) and the signal transducer and activator of transcription 3 (STAT3) could be effective targets for PCa therapy. STAT3, a cytoplasmatic latent transcription factor, is a hub protein for several oncogenic signalling pathways and up-regulates the expression of numerous genes involved in tumor cell proliferation, angiogenesis, metastasis and cell survival. STAT3 activity can be modulated by several Post-Translational Modifications (PTMs) which reflect particular cell conditions and may be implicated in PCa development and progression. The aim of this work was to analyze STAT3 PTMs at different tumor stages and their relationship with STAT3 cellular functions. For this purpose, sixty-five prostatectomy, Formalin-fixed paraffin-embedded (FFPE) specimens, classified with different Gleason Scores, were subjected to immunoblotting, immunofluorescence staining and RT-PCR analysis. All experiments were carried out in matched non-neoplastic and neoplastic tissues. Data obtained showed different STAT3 PTMs profiles among the analyzed tumor grades which correlate with differences in the amount and distribution of specific STAT3 interactors as well as the expression of STAT3 target genes. These results highlight the importance of PTMs as an additional biomarker for the exactly evaluation of the PCa stage and the optimal treatment of this disease.

Keywords: PTMs; STAT3; biomarkers; prostate cancer; signaling pathways.

Figure 1. Analysis of pY⁷⁰⁵-STAT3 distribution in prostate cancer FFPE tissues Immunofluorescence staining of representative FFPE tissue sections with different Gleason score

pY⁷⁰⁵-STAT3: phosphorylated at tyrosine 705, PI: Propidium Iodide, G: Gleason Score, FFPE: Formalin Fixed and Paraffin Embedded.

Figure 2. Analysis of STAT3 PTMs in FFPE samples with different Gleason score

(A) Representative western blot analysis of protein extracted from FFPE corresponding to Gleason Scores (G) 6, 7, 8 and 9. T: prostate carcinomas; N: normal tissue, adjacent to tumor. (B) Cumulative results of STAT3 PTMs levels in tumor prostate tissues from 65 matched prostate carcinomas. Values are presented as the means, standard deviations (boxes) and min/max values (bars). Data were analyzed by One Way Anova test and all differences between Gleason score groups in each data set were statistically significant (p < 0,01%) with the exception of pS727 G6 vs G7. pY⁷⁰⁵-STAT3: phosphorylated at tyrosine 705, pS⁷²⁷-STAT3: phosphorylated at serine 727, acK⁶⁸⁵-STAT3: acetylated at lysine 685, glut-STAT3 or GSH: glutathionylated STAT3.

Figure 3. Analysis of STAT3 interactors in FFPE samples with different Gleason score

(A) Representative Immunoblotting of selected STAT3 protein interactors expressed in each Gleason Score. (B) Average level of expression of the selected STAT3 protein interactors matching to the same Gleason Score normalized to the corresponding normal samples. Values are presented as the means, standard deviations (boxes) and min/max values (bars). Data were analyzed by One Way Anova test and all differences between Gleason score groups in each data set were statistically significant (p < 0,01%). CBP/p300: CREB-binding protein/Histone Acetyltransferase p300 interactor. PDIA3/ERp57: Protein Disulfide-isomerase A3/Endoplasmic Reticulum Protein 57, APE1/Ref-1: Apurinic/apyrimidinic endonuclease 1/redox factor-1.

Figure 4. Expression analysis of STAT3 dependent genes in FFPE samples with different Gleason score

Quantitative RealTime-PCR for PSA, BIRC5, CRP, SOD2, SRD5A2 and MMP2 genes in FFPE tissues with different Gleason Score. Each gene expression value was normalized to housekeeping gene (β-actin) and to a control sample (normal matching tissue). Values are presented as the means, standard deviations (boxes) and min/max values (bars). Data were analyzed by One Way Anova test and all differences between Gleason score groups in each data set were statistically significant (p < 0,01%). PSA: Prostate Specific Antigen, BIRC5: Baculoviral Inhibitor of Apoptosis Repeat-containing 5, CRP: C-reactive Protein, SOD2: Superoxide Dismutase 2, SRD5A2: Steroid-5-Alpha-Reductase, Alpha Polypeptide 2, MMP2: Matrix Metalloproteinase-2.