PEG10 is associated with treatment-induced neuroendocrine prostate cancer

in Journal of Molecular Endocrinology
Correspondence should be addressed to A Zoubeidi: azoubeidi@prostatecentre.com
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Neuroendocrine (NE) differentiation of advanced prostate adenocarcinoma following androgen receptor (AR) axis-directed therapy is becoming increasingly recognized. Several models of this transdifferentiation provide insight into its molecular pathogenesis and have highlighted the placental gene PEG10 for further study. Using our unique model of enzalutamide resistance (ENZR) and NE differentiation, we studied PEG10/AR interplay in enzalutamide treatment-resistant cell lines 42DENZR and 42FENZR compared to LNCaP and castration-resistant 16DCRPC cells. ENZR cell lines with positive terminal NE marker status also displayed higher baseline expression of PEG10 compared to LNCaP and 16DCRPC. Antagonism of AR activity increased PEG10 expression followed by an increase in terminal NE markers. Conversely, stimulating AR activity via androgen supplementation reversed PEG10 and NE marker expression in a time and dose-dependent manner. These results were supported by human data showing that PEG10 expression is highest in NEPC and that AR-dependent gene, PSA, is negatively correlated with PEG10 in adenocarcinoma. Further, ChIP assay confirmed binding of activated AR to the PEG10 enhancer, decreasing PEG10 expression. While PEG10 did not drive NEPC, its knockdown reduced NE markers in our cell lines. Moreover, PEG10 knockdown in vitro- and in vivo-attenuated tumor growth. Overall, these observations indicate that PEG10 is an AR-repressed gene which modulates NE markers in ENZR cells and targeting PEG10 in advanced prostate cancer with NE features is a rational and viable option.

Downloadable materials

  • Supplementary Figure 1. Reciprocal PEG10 and PSA expression patterns in response to androgen activity.
  • Supplement Figure 2. AR binds to downstream enhancer region of PEG10 gene.
  • Supplement Figure 3. Bright field images of LNCaP cells upon PEG10 overexpression.
  • Supplement Figure 4. Representative shPEG10 clones demonstrating variable knockdown of PEG10 expression.
  • Supplementary Table 1. PEG10 and PSA mRNA expression pre vs post docetaxel+androgen deprivation therapy from 2 independent cohorts. PEG10 and PSA mRNA fold changes shown in log values accompanied by corresponding p values.
  • Supplementary Table 2. Primer sequences for selected genes used for quantitative PCR.
  • Supplementary Table 3. AR binding site on PEG10 enhancer used to design ChIP primers.

 

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    PEG10 is highly expressed in non-AR driven human NEPC, and ENZR cells feature increased levels of PEG10 and a NE differentiation signature. (A) PEG10 expression in Adeno (n = 37), CRPC (n = 34) and NEPC (n = 15) patients obtained from Beltran cohort (Beltran et al. 2016). (B) Relative mRNA expression of PEG10 and (C) relative mRNA expression of NE markers in V16DCRPC, 42DENZR, 42FENZR and NCIH660 cells compared to LNCaP (=1). (D) Heat map of PEG10 and neuroendocrine-associated genes as well as canonical AR targets PSA, FKBP5 and NKX3-1 in RNA-sequencing data obtained from The Cancer Genome Atlas (TCGA) prostate cancer samples (n = 499). (E) PEG10 expression in CRPC tumors versus patient serum PSA (Grasso et al. 2012). Statistical significance is shown as *P < 0.05; ****P < 0.0001.

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    PEG10 and PEG10-dependent neuroendocrine marker expression are induced by ENZ and suppressed by reintroduction of AR activity. (A) Protein and relative mRNA expression of PEG10 and PSA in LNCaP cells treated ±10 µmol/L ENZ for 2, 4, 7 days compared with day 0 (=1) (Relative PSA expression after treatment is <0.2). (B) Relative mRNA expression of NE markers expression in LNCaP cells treated 10 µmol/L ENZ for 2, 4, 7 days compared with day 0 (=1) in FBS or CSS. (C and D) Relative mRNA expression of PEG10, PSA and NE markers expression in LNCaP, V16DCRPC, 42DENZR and 42FENZR with reintroduction of androgen after 7 days of 10 µmol/L ENZ treatment (=1). Graphs show representative data from three independent experiments. (E) Chromatin immunoprecipitation showing AR binding to the enhancer region of PEG10 in 42DENZR and 42FENZR treated with 10 nmol/L R1881 for 6 h non-treated control (=1); Corresponding PSA binding used as positive control. Graphs show pooled data from three independent experiments.

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    PEG10 is required to maintain NE marker expression across multiple cell lines. (A) Relative mRNA expression of PEG10 in LNCaP after overexpression compared with control (Ctr = 1). (B) (Top) Relative mRNA expression of PEG10 and NE markers in LNCaP and V16DCRPC transfected with PEG10 siRNA (siPEG10) or scrambled siRNA (siCtr) and treated with 10 µmol/L ENZ for 48 h compared to basal levels (basal = 1). Statistics show the decrease from siPEG10 to siCtr, (Bottom) Relative mRNA expression of PEG10 and NE markers and protein expression of PEG10 in 42DENZR and 42FENZR transfected with PEG10 siRNA (siPEG10) or scrambled siRNA. Western blots validate protein levels of PEG10 after knockdown. (C) (Left panel) PEG10 protein expression in stable PEG10 knockdown (shPEG10) compared with control-transfected cells (shCtr) in LNCaP, V16DCRPC, 42DENZR and 42FENZR cells maintained in 10 µmol/L ENZ for 7 days; (Right panel) Corresponding relative mRNA expression of PEG10 and NE markers compared with control (shCtr = 1). Statistical analyses were performed on pooled data from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.

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    PEG10 promotes the proliferation of ENZR cell lines in vitro. Proliferation rate of shCtr and shPEG10 in LNCaP, V16DCRPC, 42DENZR and 42FENZR cells maintained in 10 µmol/L ENZ over the course of 8 days. Statistics were performed on pooled data from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

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    PEG10 knockdown via shPEG10 attenuates tumor growth in vivo. (A) (Left) Tumor volume of 42FENZR shCtr and shPEG10 xenografts grown in vivo (n = 10), with (Right) representation in waterfall plot. Graph represents pooled data from five shPEG10 and five shCtr tumors. (B) Relative mRNA expression of PEG10 and representative NE marker chromogranin A (CHGA) in 42FENZR shPEG10 and shCtr xenografts (=1), harvested at 12 weeks after inoculation. Values are expressed as mean ± s.e.m.

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