Integrated and Functional Genomics Analysis Validates the Relevance of the Nuclear Variant ErbB380kDa in Prostate Cancer Progression.

The EGF-family of tyrosine-kinase receptors activates cytoplasmic pathways involved in cell proliferation, migration and differentiation in response to specific extracellular ligands. Beside these canonical pathways, the nuclear localization of the ErbB receptors in primary tumours and cancer cell lines led to investigate their role as transcriptional regulators of cancer genes. The nuclear localization of ErbB3 has been reported in various cancer tissues and cell lines but the nuclear functions and the putative correlation with tumour progression and resistance to therapy remain unclear. We first assessed ErbB3 expression in normal and tumour prostate tissues. The nuclear staining was mainly due to an isoform matching the C-terminus domain of the full length ErbB3185kDa receptor. Nuclear staining was also restricted to cancer cells and was increased in advanced castration-resistant prostate cancer when compared to localized tumours, suggesting it could be involved in the progression of prostate cancer up to the terminal castration-resistant stage. ChIP-on-chip experiments were performed on immortalized and tumour cell lines selected upon characterization of endogenous nuclear expression of an ErbB380kDa isoform. Among the 1840 target promoters identified, 26 were selected before ErbB380kDa-dependent gene expression was evaluated by real-time quantitative RT-PCR, providing evidence that ErbB380kDa exerted transcriptional control on those genes. Some targets are already known to be involved in prostate cancer progression even though no link was previously established with ErbB3 membrane and/or nuclear signalling. Many others, not yet associated with prostate cancer, could provide new therapeutic possibilities for patients expressing ErbB380kDa. Detecting ErbB380kDa could thus constitute a useful marker of prognosis and response to therapy

Primers sequences for ChIP-qPCR analysis.

<p>Primers sequences for ChIP-qPCR analysis.</p

Characterization of an endogenous nuclear ErbB3 variant in prostate cell lines.

<p>(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 ErbB3_185kDa protein in all cell lines and the 80kDa protein in the LNCaP and PC3 tumour cell lines. (C) The full length ErbB3_185kDa 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.</p

ErbB3 expression and localization in normal and tumour prostate tissues.

<p>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).</p

ChIP-on-chip workflow and Venn diagram.

<p>(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. <i>CCND1</i> and <i>MMP7</i> correspond to positive and negative controls, respectively. (D) Gene ontology analysis of ErbB3-targets with DAVID.</p

Proposed model for ErbB3_80kDa functions in prostate cancer progression.

<p>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 ErbB3_80kDa, a variant lacking the ligand-binding and transmembrane domain of ErbB3_180kDa, in advanced PCa resistant to therapy (CRPC). As a co-regulator for the transcription of genes associated with cell proliferation, differentiation, apoptosis, oncogenesis, ErbB3_80kDa is likely to be strongly involved in therapy resistance and progression to fatal PCa. <i>DHT</i>: <i>dihydrotestosterone; AR</i>: <i>androgen receptor; ARE</i>: <i>androgen responsive elements</i>.</p

ChIP-qPCR validation.

<p>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 <i>CCND1</i> promoter was used as a positive control whereas non-enriched promoters <i>ERBB3</i>, <i>EBP1</i>, <i>PSA</i>, and <i>MMP7</i> 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 <i>t</i> test.</p

Integrated and Functional Genomics Analysis Validates the Relevance of the Nuclear Variant ErbB3_80kDa in Prostate Cancer Progression

<div><p>The EGF-family of tyrosine-kinase receptors activates cytoplasmic pathways involved in cell proliferation, migration and differentiation in response to specific extracellular ligands. Beside these canonical pathways, the nuclear localization of the ErbB receptors in primary tumours and cancer cell lines led to investigate their role as transcriptional regulators of cancer genes. The nuclear localization of ErbB3 has been reported in various cancer tissues and cell lines but the nuclear functions and the putative correlation with tumour progression and resistance to therapy remain unclear. We first assessed ErbB3 expression in normal and tumour prostate tissues. The nuclear staining was mainly due to an isoform matching the C-terminus domain of the full length ErbB3_185kDa receptor. Nuclear staining was also restricted to cancer cells and was increased in advanced castration-resistant prostate cancer when compared to localized tumours, suggesting it could be involved in the progression of prostate cancer up to the terminal castration-resistant stage. ChIP-on-chip experiments were performed on immortalized and tumour cell lines selected upon characterization of endogenous nuclear expression of an ErbB3_80kDa isoform. Among the 1840 target promoters identified, 26 were selected before ErbB3_80kDa-dependent gene expression was evaluated by real-time quantitative RT-PCR, providing evidence that ErbB3_80kDa exerted transcriptional control on those genes. Some targets are already known to be involved in prostate cancer progression even though no link was previously established with ErbB3 membrane and/or nuclear signalling. Many others, not yet associated with prostate cancer, could provide new therapeutic possibilities for patients expressing ErbB3_80kDa. Detecting ErbB3_80kDa could thus constitute a useful marker of prognosis and response to therapy.</p></div

Gene expression profiling.

<p>(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 ErbB3_80kDa 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). ErbB3_80kDa-dependent transcriptional activation of <i>GATA2</i>, <i>RUNX2</i>, <i>MAP3K14</i>, <i>ErbB2</i> and <i>CCND1</i> and inactivation of <i>PPIG</i>, MXI1 were similar in LNCaP and PC3 whereas <i>ARID1A</i>, <i>TBCC</i>, <i>FYN</i>, <i>SIN3A</i>, <i>TOR3A</i>, <i>ACE</i>, <i>RAD52</i>, <i>CXCR3A</i> appeared to be differentially regulated in the two cell lines. *P<0.05, **P<0.01, ns = non-significant, Student’s <i>t</i> test.</p