13 research outputs found
Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells
Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and ?III tubulin (?III tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1?M AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer
Up-regulated expression of LAMP2 and autophagy activity during neuroendocrine differentiation of prostate cancer LNCaP cells
Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer
Modulation of immune response gene expression by echinacea extracts: results of a gene array analysis
Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models
Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa
Autophagy inhibition decrease LAMP2 levels and survival of neuroendocrine differentiated LNCaP cells.
<p>LNCaP cells were transfected with negative control siRNA (siC) or siRNA targeting Beclin1 (siBeclin1) or si RNA targeting Atg5 (SiATG5) and grown in serum containing medium (C, control cells) or serum-free medium (NE, neuroendocrine cells) during 6 days. <b>(A)</b> Cells were lysed and Beclin1 (left), Atg5 (right) and LAMP2 were measured in whole lysates by Western blot. GAPDH was used as a loading control. Densitometric analysis of the Western blot bands are shown below. <b>(B)</b> Cell viability of control and NE cells treated with siC, siBeclin1 (left) or SiATG5 (right) was monitored by MTT assay. Results are the mean ± S.D. of three independent experiments (* p<0.05 versus siC transfected control cells and # p<0.05 and ## p<0.01 versus siC transfected neuroendocrine cells, compared by the Student’s t test).</p
Pharmacological inhibition of AKT prevents autophagy, LAMP2 overexpression and neuroendocrine differentiation of LNCaP cells.
<p>LNCaP cells were grown in serum-containing medium (C, control cells) or serum-free medium (NE, neuroendocrine cells) during 6 days. Cells where treated with vehicle (vhc) or 1 μM AKT inhibitor IV for 6 days. When indicated, cells were treated with 100 nM bafilomycin A<sub>1</sub> for the last three hours in culture. Thereafter cells were lysed and autophagy markers, LC3 and p62, LAMP2 and neuroendocrine markers, NSE and βIII Tub, were measured in whole lysates by Western blot. GAPDH was used as a loading control. Densitometric analysis of the Western blot bands are shown on the right. Results are the mean ± S.D. of at least three independent experiments (* p<0.05 and **p<0.01 versus not treated control cells and # p<0.05 versus not treated neuroendocrine cells, compared by the Student’s t test).</p
LAMP2 knockdown prevents autophagy, neuroendocrine differentiation and survival of LNCaP cells.
<p>LNCaP cells were transfected with negative control siRNA (siC) or siRNA targeting LAMP2 (siLAMP2) and grown in serum-containing medium (C, control cells) or serum-free medium (NE, neuroendocrine cells) during 6 days. <b>(A)</b> Cells were lysed and LAMP2, autophagy markers, LC3 and p62, and neuroendocrine markers, NSE and βIII Tub, were measured in whole lysates by Western blot. GAPDH was used as a loading control. Densitometric analysis of the Western blot bands are shown on the right. <b>(B)</b> Cell viability of control and NE cells treated with siC or siLAMP2 was monitored by MTT assay. Results are the mean ± S.D. of at least three independent experiments (* p<0.05 and ** p<0.01 versus siC transfected control cells and # p<0.05 and ## p<0.01 versus siC transfected neuroendocrine cells, compared by the Student’s t test).</p
Heat map showing enrichment in membrane components from DEGs between NE and control cells.
<p>DEGs between NE and control cells were functionally assessed using GSEA. Gene Ontology gene sets were downloaded from the Broad Institute’s Molecular Signature Database (version 4.0). Enrichment on gene set “intrinsic _to_membrane” is shown. Up-regulated (red) and down-regulated (blue) genes in NE and control cells are shown.</p
Lysosomal-associated membrane protein 2 (LAMP2) is over-expressed in neuroendocrine differentiated LNCaP cells.
<p>LNCaP cells were grown in serum-containing medium (C, control cells) or serum-free medium (NE, neuroendocrine cells) during 6 days. <b>(A)</b> LAMP2 was measured in whole lysates of C and NE cells by western blot along with neuroendocrine markers neuron-specific enolase (NSE) and βIII tubulin (βIII Tub). GAPDH was used as a loading control. Densitometric analysis of the Western blot bands are shown on the right. <b>(B)</b> Quantification of LAMP2 mRNA in control and NE cells by real time qRT-PCR. Results are the mean ± S.D. of at least three independent experiments (* p<0.05 versus control cells compared by the Student’s t test). <b>(C)</b> Detection of LAMP2 by immunofluorescence (green). Nuclei are stained with DAPI (blue). Immunofluorescence was analyzed by confocal microscopy. Quantitative analysis of lysotracker and LAMP2 was performed using ImageJ software (NIH).</p