Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells

PLoS One. 2016 Sep 14;11(9):e0162977. doi: 10.1371/journal.pone.0162977. eCollection 2016.

Abstract

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.

MeSH terms

  • Autophagy / physiology*
  • Blotting, Western
  • Cell Line, Tumor
  • Cell Transformation, Neoplastic / metabolism
  • Gene Expression Regulation, Neoplastic / physiology
  • Humans
  • Lysosomal-Associated Membrane Protein 2 / metabolism*
  • Lysosomal-Associated Membrane Protein 2 / physiology
  • Male
  • Microscopy, Confocal
  • Oligonucleotide Array Sequence Analysis
  • Prostatic Neoplasms / metabolism*
  • Prostatic Neoplasms / physiopathology
  • Real-Time Polymerase Chain Reaction
  • Transcriptome
  • Up-Regulation

Substances

  • LAMP2 protein, human
  • Lysosomal-Associated Membrane Protein 2

Grants and funding

This work was supported by the University of Alcalá (Grant n° PI2005/063) to ART; the Spanish Ministry of Economy and Competitiveness (Grant n° BFU2012-31444); Junta de Comunidades Castilla-LaMancha and European Commission FEDER Funds (Grant n° POII11-0159-0054); Comunidad de Madrid (Grant S2010-BMD2308); and the Fundación Tatiana Pérez de Guzmán el Bueno (Grant n° Patrocinio 2013-001) to AB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.