Stathmin 1 expression in neuroendocrine and proliferating prostate cancer

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related mortality among men in the United States. While PCa initially responds to androgen deprivation therapy, a significant portion progresses to castration-resistant PCa. Approximately 20-25% of these cases acquire aggressive neuroendocrine (NE) features, ultimately leading to neuroendocrine prostate cancer (NEPC). In this study, we investigated the expression of stathmin 1 (STMN1) across PCa subtypes using bioinformatics, western blotting, and immunohistochemical staining analyses in human and murine models. We found that elevated STMN1 expression correlated with high Gleason Scores, increased cell proliferation, and poor clinical outcomes in PCa patients. Notably, STMN1 expression was significantly higher in NEPC compared to prostate adenocarcinoma, suggesting its role in NEPC progression. Findings from TRAMP tumors, a murine NEPC model, further supported these results. In conclusion, STMN1 expression is elevated in advanced PCa, particularly in NEPC, suggesting its involvement in the progression of aggressive forms of PCa. While STMN1 shows potential as a diagnostic and prognostic marker for aggressive PCa, further studies are necessary to establish its clinical utility.

Fig. 1

Expression profile of stmn1 in prostate cancer (PCa). The mRNA expression data of STMN1 in human PCa patients were extracted from Beltran Nat Med 2016 dataset (A, B) and SU2C PNAS 2019 study (C, D). The mRNA expression of STMN1 was significantly higher in neuroendocrine prostate cancer (NEPC) compared to adenocarcinoma prostate cancer (AdPC) (P < 0.01). Heatmaps were generated to illustrate the expression of STMN1, androgen receptor (AR) and cell proliferation markers including PCNA, TOP2A and E2F1 in NEPC versus AdPC. E STMN1 expression positively correlates with NEPC markers including CHGA, NCAM1 and SYP in human PCa (SU2C dataset, PNAS 2019, NEPC score > 0.4) (p < 0.02). F, G The mRNA expression data of STMN1 across various PCa cell lines were extracted from the CTPC collection. H Western blot analysis confirmed the protein expression of STMN1 and E2F1 in PCa cell lines, with relative quantification of western blot results shown in the right panel

Fig. 2

Association of STMN1 expression with tumor grade and clinical outcomes in prostate cancer (PCa). Gene expression and clinical data were extracted from the TCGA-PRAD (A, B) and DFKZ (C, D) datasets. A, C STMN1 expression levels were significantly higher in high-grade tumors (GS ≥ 8) compared to low-grade (GS 6) and intermediate-grade tumors (GS 7) in both TCGA-PRAD and DFKZ datasets. B, D STMN1 expression was significantly higher in the GS 4 + 3 group compared to the GS 3 + 4 group in both datasets. E, F Kaplan–Meier survival analysis of the SU2C (PNAS, 2019) and TCGA (Firehose Legacy) datasets showed significantly shorter overall survival and disease-free survival, respectively, in patients with high STMN1 expression compared to those with low STMN1 expression (p < 0.01). G No significant difference in overall survival was observed between high and low STMN1 expression groups in the TCGA (Firehose Legacy) dataset

Fig. 3

The expression of STMN1 protein in human prostatic tissues. A–H Representative immunohistochemical staining of STMN1 in human prostatic tissues. A Positive STMN1 staining was observed in basal epithelial cells in benign prostatic tissue. B–G Varying intensity of STMN1 staining were present in luminal and/or basal epithelial cells in adenocarcinoma tissues. The # symbol and arrowhead indicated basal epithelial cells and luminal epithelial cells, respectively. H and I Serial sections of a NEPC tumor showed STMN1 expression in the NEPC area (highlighted by the expression of NEPC marker, INSM1) but no expression was detected in adjacent AdPC cells. J Distribution of STMN1 expression (Allred score) among prostate specimens, demonstrating an association between STMN1 expression and Gleason Score of PCa. (Spearman correlation: 0.73, p < 0.01). K Dual immunofluorescence staining to access the co-expression STMN1 with NEPC marker Chromogranin A (CHGA) in AdPC tumor with NE differentiation. STMN1 expression was detected in both NE (arrowheads) and non-NE (arrows) cells. Scale bars = 20 μm

Fig. 4

Stmn1 expression in normal neuroendocrine (NE) cells. A Stmn1 expression in normal NE cells in murine prostate. Single-cell RNA sequencing data from prostates of wild-type mice were obtained from Sawyers’ study collected in MoPSA [17, 21]. Specific markers used to identify different cell types include Nkx3-1, Hoxb13, and Pbsn for luminal epithelial cells; Krt5, Krt14, and Trp63 for basal epithelial cells; and Chga, Syp, and Ncam1 for NE cells. B, C Dual immunofluorescence staining showing co-expression of STMN1 with NE markers CHGA or SYP in human benign prostate specimens. STMN1 expression was detected in cells positive for SYP or CHGA expression. Arrowhead in (B) indicated a STMN1-negative NE cell. Scale bars = 20 μm

Fig. 5

Correlation of STMN1 expression with cell proliferation in PCa. A Gene ontology (GO) analysis revealed pathways enriched in STMN1-positive PCa. B, C STMN1 expression positively correlates with Ki67 (B) and E2F1 (C) in human PCa (SU2C dataset, PNAS 2019) (p < 0.01). D STMN1 expression was significantly higher in RB1-deleted PCa group compared to the diploid group, based on data extracted from SU2C 2019 dataset. E E2F1 binding peaks were identified near the transcription start site of STMN1 through analysis of publicly available ChIP-seq datasets F Dual immunofluorescence staining to show the co-expression of STMN1 in Ki67-positive cells in AdPC. The arrowhead denoted cells co-expressed Ki67 and STMN1. Scale bar = 20 μm

Fig. 6

Immunofluorescence (IF) staining of STMN1 in TRAMP tumors from intact (A–C) and castrated mice (D–G). A–C In intact TRAMP tumors, high STMN1 expression was detected in both NEPC areas (approximately 100% of NEPC cells, indicated by the co-expression of INSM1) and adjacent PIN lesions (over 50% of PIN cells, which lacked INSM1 expression). While TAg was expressed in both PIN and NEPC cells, notable AR protein was detected in PIN cells but not in NEPC cells (C). In castrated TRAMP tumors, STMN1 expression was detected in almost all NEPC cells but was largely absent in PIN cells (D–F). Rare Stmn1-positive cells in the PIN lesions did not express Ki67 (D), SYP (E), or TAg (F), indicating the presence of non-proliferating, non-NE cells that express Stmn1. G Triple IF staining for STMN1/SYP/Ki67 revealed co-expression of STMN1 with Ki67 and SYP in NEPC. Some rare STMN1-positive cells in the PIN lesions did not express SYP nor Ki67. The arrowhead denotes a STMN1-positive cell that was neither proliferating nor NE. N > 3 for each group. Scale bar = 20 μm

Fig. 7

Differential expression of STMN family members in PCa. A, B Heatmap showing the expression levels of STMN family members in patient samples. STMN1 was the predominant isoform, followed by STMN3. Both STMN1 and STMN3 mRNA levels were significantly higher in NEPC compared to AdPC (p < 0.01). Gene expression data were extracted from two RNA-seq datasets SU2C (A) and Beltran (B). C Heatmap of STMN family members in PCa cell lines. RNA-seq data were extracted from CTPC collection. D Copy number alterations of STMN family members in PCa, based on SU2C cohorts