N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells

Abstract

MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.

Figure 1. N-Myc and myrAKT1 initiate NEPC from human prostate basal epithelial cells

(A) Schematic of a human prostate regeneration and transformation assay (UbC=human ubiquitin C promoter, CMV=cytomegalovirus promoter, SIN=self-inactivating). The red square outlines the Trop2⁺CD49f^hi basal epithelial cell population. (B) Grafts transduced with N-Myc, myrAKT1, and N-Myc/myrAKT1 harvested after 8 weeks (scale bar=2 mm). (C) H&E-stained sections of N-Myc/myrAKT1 tumors derived from individual patient prostatectomy samples (scale bar=100 µm). (D) Immunoblot of the human NEPC cell lines PC-3 and NCI-H660 and an N-Myc/myrAKT1 tumor with antibodies against N-Myc, p-AKT (Ser473), and p84 as a loading control. (E) H&E and immunohistochemical stains of an N-Myc/myrAKT1 tumor for CK8 and p63 (scale bar=100 µm). See also Figure S1 and Table S1.

Figure 2. Prostate tumors initiated by N-Myc and myrAKT1 lack AR expression and exhibit neuroendocrine markers

(A–C) Photomicrographs of N-Myc/myrAKT1 tumor sections containing regions of neuroendocrine carcinoma with H&E stains and immunohistochemical staining for AR and NCAM1, CHGA and SYP, and TTF-1 and FOXA2 (scale bar=100 µm). (D) Summary of the immunohistochemical staining for neuroendocrine markers in the regenerated tumors derived from five independent patient prostate samples. Positive staining represents visible staining in at least 5% of the tumor cells. (E) Immunoblot of the AR-positive human prostate cancer cell line LNCaP, five different N-Myc/myrAKT1 (NA) tumors, and the AR-null human prostate cancer cell line DU145 with antibodies against AR, N-Myc, AKT, and GAPDH as a loading control.

Figure 3. N-Myc/myrAKT1 prostate tumors are castration-resistant and metastasize widely

(A) LNCaP and transplanted N-Myc/myrAKT1 (NA) tumor volumes +/− SD (n=4 for each condition) in intact and surgically castrate mice over time. (B) Average percentage of neuroendocrine carcinoma identified by smart image segmentation +/− SEM in sections of primary, secondary, and tertiary N-Myc/myrAKT1 prostate tumors. p-values were calculated from an one-way analysis of variance. (C) Bioluminescent imaging of mice 21 days after tail vein injection with the LNCaP-Luc or N-Myc/myrAKT1-Luc cell lines (signal intensity is represented by radiance, p/sec/cm²/sr). (D) Gross tumor deposits marked by closed arrows localized to the sacrum and liver in N-Myc/myrAKT1-Luc injected mice (scale bar=2 mm). (E) Bioluminescent imaging of mice 74 days after orthotopic injection with the LNCaP-Luc or N-Myc/myrAKT1-Luc cell lines. (F) Gross metastatic tumors marked by closed arrows involving the liver, mesenteric lymph nodes (LNs), and kidney of N-Myc/myrAKT1-Luc mice (scale bar=1 cm). (G) H&E-stained sections of metastatic N-Myc/myrAKT1-Luc tumors in the pelvis and liver with M=marrow space, B=bone, L=liver, and T=tumor (scale bar=100 µm). (H) H&E-stained sections of metastatic N-Myc/myrAKT1-Luc tumors in a mesenteric LN and kidney with K=kidney and T=tumor (scale bar=100 µm). See also Figure S2.

Figure 4. Transcriptome profiling of the N-Myc/myrAKT1 tumors demonstrates similarity to human NEPC

(A) Schematic of the laser capture microdissection of matched regions of adenocarcinoma and neuroendocrine carcinoma in an N-Myc/myrAKT1 tumor and the workflow for whole transcriptome shotgun sequencing. (B) Gene set enrichment analysis for genes differentially expressed (>4-fold) in the adenocarcinoma or neuroendocrine carcinoma from the N-Myc/myrAKT1 tumor derived from Patient 1. (C) Neuroendocrine prostate cancer signature scores +/− SD for Beltran et al. neuroendocrine prostate cancer (NEPC, n=7), N-Myc/myrAKT1 adenocarcinoma (NA ADCA, n=3), N-Myc/myrAKT1 neuroendocrine carcinoma (NA NECA, n=3), and Beltran et al. adenocarcinoma (ADCA, n=30). (D) Heatmap of a selection of genes in NA ADCA, NA NECA, Beltran et al. NEPC, and Beltran et al. ADCA samples (contrast=+/−2⁵). See also Figure S3 and Tables S2–S4.

Figure 5. Establishment of a human NEPC cell line LASCPC-01 with cancer stem cell-like features

(A) Photomicrograph of the LASCPC-01 cell line growing in suspension (scale bar=100 µm). (B) Immunoblot analysis of the parental N-Myc/myrAKT1 tumor from which LASCPC-01 was derived and the LASCPC-01 cell line with antibodies against N-Myc, p-AKT (Ser473), AURKA, ASCL1, NSE, and GAPDH as a loading control. (C) Conventional karyotyping of the LASCPC-01 cell. (D) Gross tumors generated from the subcutaneous xenotransplantation of serially diluted LASCPC-01 cells after five weeks (scale bar=5 mm). (E) Representative H&E-stained section of LASCPC-01 xenograft tumors with regions of neuroendocrine carcinoma (NECA) and adenocarcinoma (ADCA) (black scale bar=200 µm, white scale bar=100 µm). (F) Top panel, photomicrographs of LASCPC-1 cells in culture after single cell sorting, deposition, and culture (scale bar=50 µm). Bottom panel, gross tumors from the subcutaneous xenotransplantation of eleven clonal LASCPC-01 sublines after 4 weeks (scale bar=5 mm). (G) Representative H&E-stained section of a xenograft tumor derived from a clonal LASCPC-01 subline (scale bar=100 µm). See also Figure S4.

Figure 6. N-Myc expression is required for tumor maintenance in the N-Myc/myrAKTl tumors

(A) Lentiviral constructs used for doxycycline-inducible expression of N-Myc and constitutive expression of myrAKT1 (TRE=tetracycline response element, rtTA=reverse tetracycline-controlled transactivator). (B) H&E-stained section of an inducible N-Myc/myrAKT1 tumor (scale bar=100 µm). (C) Average tumor volume of passaged inducible N-Myc/myrAKT1 tumors +/− SD over time under conditions of no doxycycline (No dox, n=13), continuous doxycycline (Dox continuous, n=11), and doxycycline withdrawal on day 21 after initial doxycycline (Dox on/off, n=11). p-values were calculated from a paired t-test. (D) Immunoblot analysis of inducible N-Myc/myrAKT1 tumors after continuous doxycycline or initial doxycycline followed by withdrawal using antibodies against N-Myc and GAPDH as a loading control. (E) Representative H&E-stained sections of inducible N-Myc/myrAKT1 tumors after continuous doxycycline and after doxycycline withdrawal (scale bar=100 µm).

Figure 7. Therapeutic targeting of N-Myc dependence in the N-Myc/myrAKT1 model of NEPC

(A) Immunoblot analysis of LASCPC-01 cells treated with a dose range of CD532 with antibodies against N-Myc, AURKA, phosphorylated histone H3 (p-H3), histone H3, cleaved PARP (cPARP), and GAPDH as a loading control. (B) Immunoblot of LASCPC-01 treated with DMSO or 500 nM of CD532, MLN8237, or VX-680 for 3 hours with antibodies against, N-Myc, AURKA, p-H3, H3, cPARP, and GAPDH as a loading control. (C) LASCPC-01 cell viability +/− SD after 3 hours of treatment with 1 µM of CD532, MLN8237, or cabazitaxel relative to treatment with DMSO (n=6 for each condition). (D) Immunoblot of LASCPC-01 cells treated with 1 µM of CD532 over a time course with antibodies against N-Myc, cleaved caspase-3, and GAPDH as a loading control. (E) Cell cycle analysis of LASCPC-01 cells after 3 hours of treatment with DMSO or 1 µM of CD532, MLN8237, or cabazitaxel. Quantification of the sub-G1, G1, and S/G2/M fractions is shown. (F) Dose response of CD532 +/− SD (normalized to DMSO treatment only) at 48 hours using the CellTiter-Glo cell viability assay in LASCPC-01 cells. (G) Immunoblot analysis of N-Myc/myrAKT1 tumors after treatment with vehicle or CD532 for 24 hours with antibodies against N-Myc and GAPDH as a loading control. (H) Average tumor volume of LASCPC-01 subcutaneous xenografts +/− SD with vehicle (n=6) or CD532 (n=7) treatment initiated on day 11. p-values were calculated from a paired t-test. See also Figure S5 and Table S5.