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.
Highlights
Prostate cancer is the second most common cancer in men, with an estimated 1.1 million cases diagnosed worldwide in 2012 (GLOBOCAN 2012) [1]
LNCaP cells underwent neuroendocrine (NE) differentiation by serum deprivation of culture medium for 6 days. βIII Tubulin and neuron-specific enolase (NSE) levels were assessed as NE markers
We assessed the levels of NSE mRNA in NE and control cells by qRT-PCR (Fig 1B) and verified that increased protein levels of NSE correlated with enhanced levels of NSE mRNA in NE cells
Summary
Prostate cancer is the second most common cancer in men, with an estimated 1.1 million cases diagnosed worldwide in 2012 (GLOBOCAN 2012) [1]. Prostate cancer represents an important public health problem throughout the world and for developed countries in particular, since almost 70% of the cases (759,000) occur in more developed regions. Androgen deprivation therapy (ADT) is used to treat advanced prostate and yields transient efficacy. This therapy consists in administrating LHRH agonists or antagonist which prevent the secretion of the pituitary hormone LH which, in turn, reduces the production of androgens by the testicles [2]. ADT has limited and transient efficacy and most patients receiving it progress to a more aggressive form of the disease termed castration-resistant prostate cancer (CRPC) [5, 6]. There is evidence of androgen receptor (AR) reactivation despite decreased serum levels of androgens as an adaptive survival response [4]
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