Pathobiological implications of the expression of EGFR, pAkt, NF-κB and MIC-1 in prostate cancer stem cells and their progenies

Abstract

The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse. The present study was undertaken to establish the possibility of using a combination of specific oncogenic products, including epidermal growth factor receptor (EGFR), pAkt, nuclear factor-kappaB (NF-κB) and macrophage inhibitory cytokine-1 (MIC-1) as biomarkers and therapeutic targets for optimizing the management of patients with localized PC at earlier disease stages. The immunohistochemical and immunofluorescence data have revealed that the expression levels of EGFR, Ser(473)-pAkt, NF-κB p65 and MIC-1 proteins were significantly enhanced in the same subset of 76 cases of prostatic adenocarcinoma specimens during the disease progression and these biomarkers were expressed in a small subpopulation of CD133(+) PC cells and the bulk tumor mass of CD133(-) PC cells. Importantly, all of these biomarkers were also overexpressed in 80-100% of 30 PC metastasis bone tissue specimens. Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells. Of therapeutic interest, the targeting of EGFR, pAkt, NF-κB or MIC-1 was also effective at suppressing the basal and EGF-promoted prostasphere formation by SP WPE1-NB26 cells, inducing disintegration of SP cell-derived prostaspheres and decreasing the viability of SP and non-SP WPE1-NB26 cell fractions. Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel. These findings suggest that the combined use of EGFR, pAkt, NF-κB and/or MIC-1 may represent promising strategies for improving the accuracy of current diagnostic and prognostic methods and efficacy of treatments of PC patients in considering the disease heterogeneity, thereby preventing PC progression to metastatic and lethal disease states.

Figure 1. Immunohistochemical analyses of the expression levels of EGFR, Ser⁴⁷³-pAkt, NF-κB p65 and MIC-1 in non-malignant prostate and prostatic adenocarcinoma tissues.

Microarray sections of non-malignant and malignant prostate tissue specimens were probed with an anti-EGFR, -Ser⁴⁷³-pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained tissue specimens of normal prostate, non-malignant adjacent tissues of prostatic tumor and prostatic adenocarcinoma obtained for (a) EGFR, (b) Ser⁴⁷³-pAkt, (c) NF-κB p65 and (d) MIC-1 are shown at original magnifications of ×100 and ×400. The arrows indicate the localization of basal cells in normal and non-malignant prostate epithelium and immunostaining detected for these biomarkers in prostatic adenocarcinoma tissue specimen. Moreover, the positive immunostaining detected for secreted MIC-1 protein in the stromal compartment adjacent to prostatic tumor tissue is also indicated.

Figure 2. Comparison of the composite scores of expression levels of EGFR, Ser⁴⁷³-pAkt, NF-κB p65 and MIC-1 in non-malignant and malignant tissues from PC patients.

Box plots showing the expression levels of (a) EGFR, (b) Ser⁴⁷³-pAkt, (c) NF-κB p65 and (d) MIC-1 during PC progression to metastatic disease stages. *, P<0.0001, indicates a significant increase between the composite scores obtained for prostatic adenocarcinoma and PC bone metastasis specimens relative to composite scores obtained for normal prostatic tissues.

Figure 3. Immunohistochemical analyses of the expression levels of EGFR, Ser⁴⁷³-pAkt, NF-κB p65 and MIC-1 in PC bone metastasis tissues.

Microarray sections of PC bone metastasis tissue specimens were probed with anti-EGFR, -Ser⁴⁷³-pAkt, -NF-κB p65 or -MIC-1 antibody after blocking with serum. All sections were examined under a microscope and the immunoreactivity was judged by dark brown staining. Representative pictures of stained PC bone tissue specimens obtained for EGFR, Ser⁴⁷³-pAkt, NF-κB p65 or MIC-1 are shown at original magnifications of ×100 and ×400.

Figure 4. Immunofluorescence analyses of expression levels of EGFR, Ser⁴⁷³-pAkt, NF-κB p65 and MIC-1 signaling elements and their co-localization with a CD133 stem cell-like marker in non-malignant and malignant prostatic tissues.

The double immunofluorescence analyses of the co-localization of the expression of markers in normal prostate and prostatic adenocarcinoma specimens from patients was simultaneously done with fluorescein-labeled anti-EGFR, -Ser⁴⁷³-pAkt, -NF-κB p65 or -MIC-1 antibody (green) plus phycoerythrin-labeled anti-CD133 antibody (red) after blocking with goat serum as described in Materials and Methods. The arrows indicate a double staining (yellow/purple) detected by confocal analyses, which is indicative of the co-localization of these markers. Representative pictures are shown at the original magnification of ×630.

Figure 5. Characterization of phenotypic features of SP and non-SP cell fractions from parental tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye efflux technique and FACS.

a) Representative data of the Hoechst dye efflux profile obtained after staining of parental WPE1-NB26 cell line with fluorescent Hoechst dye showing the SP cell subpopulation (green) and non-SP fraction (blue) detected in the total mass of parental WPE1-NB26 cells. b) FACS profiles obtained after staining of parental WPE1-NB26 cells with phycoerythrin -labeled anti-CD133 antibody showing the percentage of CD133⁺ and CD133⁻ PC cells detected in the total mass of parental WPE1-NB26 cells. c) Comparative Western blot analyses of expression levels of prostatic stem cell-like markers (CD133 and CD44), multidrug transporter ABCG2, EGFR, Ser⁴⁷³-pAkt, NF-κB p65 subunit and secreted MIC-1 proteins detected in the SP and non-SP cell fractions isolated from parental WPE1-NB26 cell line. d) Immunofluorescence staining of methanol-fixed prostaspheres derived from SP cells and the adherent non-SP cell fraction isolated from the parental WPE1-NB26 cell line were done with anti-EGFR plus Tyr¹¹⁷³-pEGFR, Ser⁴⁷³-pAkt, NF-κB p65 or MIC-1 primary antibody plus fluorescein (green) and/or Texas red secondary antibody (red) and 4′,6-diamidino-2-phenylindole (nuclear blue) after blocking with goat serum. Representative pictures showing the expression level and cellular localization obtained for the stem cell-like markers including CD133 (red), CD44 (green) and ABCG2 (red) as well as overlaps of EGFR/Tyr¹¹⁷³-pEGFR (red/green, hybrid yellow), Ser⁴⁷³-pAkt (red), NF-κB p65 (red) and MIC-1 (red) are shown at the original magnification of ×630.

Figure 6. Determination of the effects induced by different drugs on the prostasphere-forming ability of the SP cell fraction from highly invasive and tumorigenic WPE1-NB26 cell line.

The SP and non-SP cell fractions from the WPE1-NB26 cell line were subjected to the prostasphere formation culture on an ultra-low attachment plate in serum-free keratinocyte medium. The representative pictures of the dense prostaspheres formed by SP WPE1-NB26 cells (a) without or (b) after a treatment with exogenous EGF as compared to diffuse, abortive and very small aggregates formed by non-SP WPE1-NB26 cells are shown at a similar magnification of ×200. Moreover, the representative pictures of the prostaspheres formed by the SP WPE1-NB26 cell fraction (a) without or (b) after a treatment with exogenous EGF in the presence of different drugs, including a specific inhibitory agent of EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide), MIC-1 (anti-MIC-1 antibody) or docetaxel, are also shown at a similar magnification of ×200. The quantitative data of the number of prostaspheres formed by the SP WPE1-NB26 cell fraction (c) without or (d) after a treatment with exogenous EGF in the absence (control) or presence of different inhibitory agents obtained from at least 3 separate experiments are shown.

Figure 7. Cytotoxic effects induced by different tested drugs on SP and non-SP cell fractions isolated from highly tumorigenic and invasive WPE1-NB26 cells.

The SP and non-SP WPE1-NB26 cell fractions were untreated or treated with the indicated concentrations of gefitinib, LY294002, pAkt inhibitor VIII, partenolide, anti-MIC-1 antibody or docetaxel for 72 hours and the cell viability was analyzed by MTT tests. The data are the means of at least three different experiments done in triplicate. *, P<0.05, indicates a significant difference between the cytotoxic effects induced by individual drugs on the SP cells versus the non-SP WPE1-NB26 cell fraction.

Figure 8. Estimation of the disintegration effects induced by different tested drugs on prostaspheres derived from SP WPE1-NB26 cell-and implication of the mitochondrial and caspase pathways.

a) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P<0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.

Figure 9. Quantitative analyses of apoptotic effects induced by different tested drugs, alone or in combination with docetaxel, on SP cells isolated from highly tumorigenic and invasive WPE1-NB26 cells.

The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P<0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.