Table 1.
Primers sequences for ChIP-qPCR analysis.
Table 2.
Primers for RT-qPCR analysis.
Fig 1.
ErbB3 expression and localization in normal and tumour prostate tissues.
Staining with ErbB3 N-ter antibody in PCa tissues was located at the membrane, without nuclear expression (a). No ErbB3 C-ter nuclear staining was detected in “normal” prostate tissues (b). Nuclear ErbB3 expression was observed in PCa cells, and more often in CRPC advanced tumours when compared to clinically localized cancers. In CRPC, high nuclear staining was associated with high CCND1 expression and tended to be associated with higher proliferation (Ki67) (CRPC 1, c-e), whereas most tumours without nuclear staining displayed low CCND1 expression and tended to show lower proliferation (CRPC 2, f-h).
Fig 2.
Characterization of an endogenous nuclear ErbB3 variant in prostate cell lines.
(A) Both RWPE and PC3 cells display cytoplasmic and membrane ErbB3 localization. In addition, native PC3 cells exhibit a strong nuclear localization detected by the ErbB3 C-ter antibody (sc-285, Santa-cruz Biotechnology). (B) In total protein extracts, the ErbB3 C-ter antibody detects the full length ErbB3185kDa protein in all cell lines and the 80kDa protein in the LNCaP and PC3 tumour cell lines. (C) The full length ErbB3185kDa protein is almost only detected in the non-nuclear fractions that include plasma membrane and cytoplasmic proteins whereas a strong 80kDa signal is observed in the nuclear fractions of the tumour lines. (D) A 719 bp amplification product is detected in the three cell lines with the primers designed to amplify both the full length mRNA (NM_001982.3) and the AK300909.1 variant (PCR1), whereas PCR2 and PCR3 primers specific to AK124710.1 and AK1250281 respectively, failed to amplify any sequence. (E) Quantitative analysis with specific primers for ErbB3 transcripts indicate that NM_001982.3 and AK300909.1 are expressed at comparable levels in LNCaP and PC3 cell lines whereas AK300909.1 is slightly detectable in RWPE cells.
Fig 3.
ChIP-on-chip workflow and Venn diagram.
(A) Chromatin immunoprecipitation was performed upon endogenous conditions. Nuclear lysates were prepared from native cells grown in complete medium and without any androgen. ChIP-DNA and total DNA (Input) labelled with Cy5 and Cy3 fluorophores respectively, were co-hybridized on Agilent promoters tilling arrays. (B) Data analysis with CoCAS software led to select 1840 ErbB3-target promoters in the three cell lines. (C) Agilent Worbench 7.0 software analysis illustrating the distribution of positive probes (ErbB3-bound DNA/red dots) and negative probes (Input/ green dots) throughout the genes sequence (X-axis). Signal intensity is shown on the Y-axis. CCND1 and MMP7 correspond to positive and negative controls, respectively. (D) Gene ontology analysis of ErbB3-targets with DAVID.
Fig 4.
On the basis of biocomputing data, 16 randomly selected ErbB3-target promoters from LNCaP cells (A) and 19 randomly selected ErbB3-target promoters from PC3 cells (B) were tested. All showed a significant ErbB3 enrichment compared to the negative control ChIP-IgG (value normalized to 1). The CCND1 promoter was used as a positive control whereas non-enriched promoters ERBB3, EBP1, PSA, and MMP7 were used as negative controls. The data are representative of 3 independent experiments and are the means of triplicate samples. *P<0.05, **P<0.01, ns = non-significant, Student’s t test.
Fig 5.
(A) Two siRNAs were used to specifically inhibit the NM_001982.3 transcript (siErbB3A) or both the NM_001982.3 and AK300909.1 transcripts (siErbB3B) and validated by western blotting using the ErbB3 C-ter antibody. (B) LNCaP and PC3 cells were transiently transfected with siErbB3A or siErbB3B before mRNAs were reverse transcribed and amplified. RT-qPCR was performed on a selection of ErbB3-target genes. Expression fold change was normalised to control siRNA transfected samples. Transfections were done in triplicate, and the RT-qPCR results reported are from at least three independent experiments. (C) Expression fold change consecutive to the inhibition of the ErbB380kDa isoform in each cell line and for each target gene is calculated by the ratio (relative gene expression upon siErbB3B treatment)/(relative gene expression upon siErbB3A treatment). ErbB380kDa-dependent transcriptional activation of GATA2, RUNX2, MAP3K14, ErbB2 and CCND1 and inactivation of PPIG, MXI1 were similar in LNCaP and PC3 whereas ARID1A, TBCC, FYN, SIN3A, TOR3A, ACE, RAD52, CXCR3A appeared to be differentially regulated in the two cell lines. *P<0.05, **P<0.01, ns = non-significant, Student’s t test.
Fig 6.
Proposed model for ErbB380kDa functions in prostate cancer progression.
Blocking RA-dependent signalling by androgen withdrawal therapy (AWT) first inhibits tumour growth, but soon alternative proliferative pathways are reactivated inducing tumour progression. Increased ErbB3 expression and subsequent reactivation of the PI3K pathway in response to AWT is a major mechanism of tumour resistance. Alternatively, we report here the nuclear accumulation of ErbB380kDa, a variant lacking the ligand-binding and transmembrane domain of ErbB3180kDa, in advanced PCa resistant to therapy (CRPC). As a co-regulator for the transcription of genes associated with cell proliferation, differentiation, apoptosis, oncogenesis, ErbB380kDa is likely to be strongly involved in therapy resistance and progression to fatal PCa. DHT: dihydrotestosterone; AR: androgen receptor; ARE: androgen responsive elements.