Research ArticleCancer

Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer

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Science Translational Medicine  04 Dec 2019:
Vol. 11, Issue 521, eaax0428
DOI: 10.1126/scitranslmed.aax0428
  • Fig. 1 CXCR2 is a surface marker for NE cells in human PCa and is associated with disease progression.

    (A) Representative hematoxylin and eosin (H&E) staining (left) and IHC for NE marker CHGA (right) of human primary PCa. Nuclei (DAPI staining) are shown in blue. (B) Representative immunofluorescence images of CXCR2 (red) and NE marker CHGA (green) staining in human primary PCa. White arrowheads point to CXCR2+ CHGA+ NE tumor cells. (C) Immunofluorescence of CXCR2 (red) and luminal marker KRT8 (green) in human primary prostate adenocarcinoma. White arrowheads indicate CXCR2+ NE cells (red), which are negative for KRT8 (green). (D and E) Representative images (D) and quantification (E) of CXCR2 IHC staining on TMAs. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and interquartile range. (F) Analysis of CXCR2 expression among primary and metastatic PCa tumors from the dataset of Taylor et al. (11). Logistic regression analysis was performed using t test; lines represent means ± SD.

  • Fig. 2 Distinct signaling was observed between CXCR2+ NE and CXCR2 luminal cells isolated from human primary PCa.

    (A) Experimental scheme for gene expression and cellular function analysis of human PCa CXCR2+ NE and CXCR2 luminal populations. (B) Volcano plot of differentially expressed genes between CXCR2+ NE and CXCR2 luminal cells isolated from fresh primary human PCa tissue. (C) Heat map of genes from sorted benign basal cells (ITGA6+), NE tumor cells (CXCR2+), and luminal-type tumor cells (CXCR2). (D and E) Tumor organoids generated by CXCR2+ NE cells (right) and CXCR2 luminal cells (left). Representative images (D) and quantification (E) of three independent patients’ samples are shown. 3D, three-dimensional. (F) Signaling pathways associated with CXCR2 expression in PCa. Gene expression of 459 primary PCa was obtained from TCGA prostate adenocarcinoma database (TCGA-PRAD). Ingenuity pathway analysis was performed using 682 genes that were correlated with CXCR2 expression (P < 0.05, Pearson’s correlation). (G and H) The enrichment scores of NE gene sets (G) and prostate adenocarcinoma gene sets (H) in CXCR2+ NE cells isolated from primary human PCa tissue (18). FDR, false discovery rate; NES, normalized enrichment score. (I) Heat map of gene expression among CXCR2+ NE and CXCR2 luminal tumor cells from fresh primary human PCa tissue, SCNC/neuroendocrine prostate cancer (NEPC), and prostate adenocarcinoma (18).

  • Fig. 3 CXCR2-mediated phenotypic switch drives therapy resistance in PCa.

    (A) Flow cytometric quantification of CXCR2+ cells in parental LNCaP cell line (left) and LNCaP cells cultured in charcoal-stripped (androgen-deprived) medium for 2 weeks (right). Representative images of three independent experiments are shown. (B) Flow cytometric quantification of CXCR2+ cells in parental C4-2B cell line (left) and C4-2B cells treated with enzalutamide (20 μM) for 2 weeks (right). Representative images of three independent experiments are shown. (C and D) Parental LNCaP cells and LNCaP cells overexpressing CXCR2 (LNCaP-CXCR2) were treated without (left) or with (right) enzalutamide (ENZA) for 2 weeks. Representative image (C) and quantification (D) of colony formation assay are shown. cDNA, complementary DNA. (E and F) CXCR2 expression induces a lineage switch from the luminal phenotype (AR+, KLK3+, and CHGA) to the NE phenotype (AR, KLK3, and CHGA+) in an in vivo xenograft model. Representative images (E) and quantification (F) are shown. (G) Heat map of selected differentially expressed genes between LNCaP cells and LNCaP-CXCR2 cells. (H) Enrichment for binding sites for luminal-defining transcription factors in LNCaP cells measured by ChIP-seq analyses. The red dot represents the query signature of 1000 up- and down-regulated genes between LNCaP and LNCaP-CXCR2 cells. The blue dot represents one of a total of 100,000 randomly sampled gene lists of equal size to the query signature. The rank of the query gene list divided by the total number of resample instances was then used as the P value for the probability of enrichment by chance. (I) mRNA expression of luminal markers in LNCaP cells with/without CXCR2 overexpression or C4-2B cells with/without CRISPR-Cas9 knockout of CXCR2. (J) Growth curves of C4-2B/MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 (CRISPR-Cas9 knockout of CXCR2) with or without enzalutamide (20 μM) treatment. (K) The enrichment score of NE gene sets (18) in LNCaP-CXCR2 cell. (L) A model demonstrating how CXCR2 drives NE phenotype in PCa cells and renders CXCR2+ NE cells resistant to hormonal therapy [androgen deprivation therapy or enzalutamide treatment (ADT/Enza)]. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and interquartile range. ns, nonsignificant; *P < 0.05.

  • Fig. 4 CXCR2 expression in NE cells drives the secretion of proangiogenic factors and promotes the formation of premetastatic niche in the tumor environment.

    (A and B) Representative images of immunofluorescence for CXCR2 (red) and vascular endothelial cell marker PECAM1 (green) in TMAs of human primary PCa are shown in (A). Nuclei (DAPI staining) are shown in blue. Spearman correlation between CXCR2 expression and blood vessel density in a panel of 77 PCa cases is shown in (B). (C and D) Representative IHC images (C) and quantification (D) of PECAM1 expression in LNCaP and LNCaP-CXCR2 xenograft tumors. (E to H) Representative images (E) and quantification (F) of human cytokine antibody arrays of LNCaP or LNCaP-CXCR2. Representative images (G) and quantification (H) of human cytokine antibody arrays of C4-2B/MDVR-sgCtrl or C4-2B/MDVR-sgCXCR2. FBS, fetal bovine serum. (I to N) Representative images (I) and quantification (J) of transwell Matrigel invasion assay of LNCaP and LNCaP-CXCR2 cells. The data were normalized to average invasion. Representative images (K) and quantification (L) of transwell Matrigel invasion assay of C4-2B/MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 cells. The data were normalized to average invasion. Representative images (M) and quantification (N) of spheroid invasion of C4-2B/MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 cells at days 0 and 10, with leading edges indicated by the red arrows. Invasion distance normalized to day 0 distance for each sphere. (O) A model of CXCR2+ NE cells remodeling the tumor microenvironment. MMP, matrix metalloproteinase. VEGF, vascular endothelial growth factor. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and interquartile range. *P < 0.05.

  • Fig. 5 Advanced and therapy-resistant PCa is sensitive to CXCR2 inhibition.

    (A to C) Tumorigenesis of C4-2B/MDVR cells with/without CRISPR-Cas9 knockout of CXCR2. Representative images (A), quantification of colony-forming efficiency (B), and colony size (C) of C4-2B/MDVR cells with/without the CXCR2 gene deleted. Cells were cultured for 2 weeks. (D to F) Effect of navarixin on enzalutamide-resistant PCa C4-2B/MDVR cells’ growth in vivo. Images (D), weights (E), and volumes (F) of C4-2B/MDVR tumors in mice treated with navarixin (70 mg/kg) or vehicle control. (G) Representative images of IHC staining for AR and prostate-specific antigen in control and navarixin-treated C4-2B/MDVR xenografts. (H to J) Representative images (H) and quantification (I) of immunostaining for terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) in tumors from mice treated with vehicle or CXCR2 inhibitor navarixin for 3 weeks. Representative images (J) of BCL2 associated X, apoptosis regulator (BAX) immunostaining of xenograft tumors in mice treated with/without navarixin for 3 weeks. (K and L) Representative images (K) and quantification (L) of blood vessel marker CD31 in mice treated with vehicle or navarixin (70 mg/kg) for 3 weeks. (M) Quantification of C4-2B tumor burden in mice treated with vehicle, enzalutamide, navarixin, or both enzalutamide and navarixin. (N and O) Quantification (N) and representative images (O) of immunostaining for TUNEL in mice treated with vehicle, enzalutamide, navarixin, or both enzalutamide and navarixin. ANOVA, analysis of variance. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and interquartile range.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/521/eaax0428/DC1

    Materials and Methods

    Fig. S1. CXCR2 is a surface marker for NE cells at all stages of PCa evolution.

    Fig. S2. CXCR2+ NE cells from human primary PCa show distinct gene expression signatures from CXCR2 luminal cell subpopulation.

    Fig. S3. CXCR2 drives NE phenotype and therapeutic resistance.

    Fig. S4. CXCR2 drives the NE secretion of proangiogenic factors and contributes to premetastatic niche formation.

    Fig. S5. CXCR2 inhibitor inhibits CRPC growth in preclinical model.

    Table S1. Gene set enriched in CXCR2+ NE cells.

    Table S2. Gene set enriched in CXCR2 luminal cells.

    Table S3. Oncogenic gene set enrichment in CXCR2 NE (CXCR2+ malignant versus basal benign).

    Table S4. Six hundred eighty-two–gene signature in CXCR2 NE cells.

    Table S5. Primers for qRT-PCR.

    Table S6. Targeting sequences for CXCR2 sgRNA.

    Data file S1. Primary data.

    References (3133)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. CXCR2 is a surface marker for NE cells at all stages of PCa evolution.
    • Fig. S2. CXCR2+ NE cells from human primary PCa show distinct gene expression signatures from CXCR2 luminal cell subpopulation.
    • Fig. S3. CXCR2 drives NE phenotype and therapeutic resistance.
    • Fig. S4. CXCR2 drives the NE secretion of proangiogenic factors and contributes to premetastatic niche formation.
    • Fig. S5. CXCR2 inhibitor inhibits CRPC growth in preclinical model.
    • Table S1. Gene set enriched in CXCR2+ NE cells.
    • Table S2. Gene set enriched in CXCR2 luminal cells.
    • Table S3. Oncogenic gene set enrichment in CXCR2 NE (CXCR2+ malignant versus basal benign).
    • Table S4. Six hundred eighty-two–gene signature in CXCR2 NE cells.
    • Table S5. Primers for qRT-PCR.
    • Table S6. Targeting sequences for CXCR2 sgRNA.
    • References (3133)

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    Other Supplementary Material for this manuscript includes the following:

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