Notch signaling modulates hypoxia-induced neuroendocrine differentiation of human prostate cancer cells

Abstract

Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro.

Results: Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur.

Conclusions: Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription.

Figure 1

A. Western blotting for the detection of HIF-1α in LNCaP cells exposed to normoxia (N) or hypoxia (H) for 24 hours. Detection of GAPDH was used for normalization of protein loading; B–C. LNCaP cells exposed to hypoxia (H) for 7 days, compared to cells exposed to normoxia (N), assessed by phase contrast microscopy (B) and by immunoperoxidase staining (brown) for the detection of NSE (C); D–E. Representative immunoblot for the expression of NSE (D) and β3-tubulin (E) in LNCaP cells exposed to normoxia (N) and hypoxia (H) for 7 days. Detection of GAPDH was used for normalization of protein loading. F–G. Densitometric analysis of three independent experiments for the detection of NSE (F) and β3-tubulin (G); values are expressed as mean ± SE. H. Neurogenin (Ngn)3 mRNA expression in LNCaP cells exposed to normoxia (N) and hypoxia (H), at different time points, assessed by quantitative real time PCR.

Figure 2

A–B. Notch 1 (N1), N2, Jagged (J)1, J2, Delta-like (Dll)-1, Dll-4 mRNA expression in LNCaP cells exposed to normoxia and hypoxia for 7 days, assessed by quantitative real time PCR. Data are expressed as fold expression of mRNA levels under hypoxia, normalized to normoxia level (equal 1). C. Western blotting for the detection of transmembrane N1 and N2 proteins in LNCaP cells exposed to normoxia (1), hypoxia (2) and hypoxia + DAPT [20 μM] (3), for 7 days. D. Expression of Notch target genes, Hes1 and Hey1, mRNA in LNCaP cells exposed to normoxia and hypoxia for 7 days, assessed by quantitative real time PCR. Cells treated with the γ-secretase inhibitor DAPT [20 μM] were used as control for inhibition of Notch signal.

Figure 3

A–C. LNCaP cells virally transduced with LacZ or with a dominant negative form of Hes1 (dnHes1), in frame with a HA-tag, were analyzed for Ngn3 and CGA mRNA expression 4 days after transduction, by quantitative real time PCR (A–B) and for NSE and β3-tubulin protein expression, by Western blotting (C). Detection of HA-tag was used to determine the expression of the transduced dnHes1 construct. Detection of GAPDH was used for normalization of protein loading. D–E. LNCaP cells virally transduced with LacZ or with a constitutively active form of N1 (caN1), in frame with a V5-tag, were analyzed for CGA mRNA expression 3 days after transduction, by quantitative real time PCR (D), and for NSE and β3-tubulin protein expression, by Western blotting (E). Detection of V5-tag was used to determine the expression of the transduced caN1 construct. Detection of GAPDH was used for normalization of protein loading.

Figure 4

A. Exponential growth curve of LNCaP cells grown under normoxic and hypoxic conditions; B. AR mRNA expression in LNCaP cells exposed to normoxia (N) and hypoxia (H) for 7 days, assessed by quantitative real time PCR; C. Growth of LNCaP cells under normoxic (N) and hypoxic (H) conditions, in the absence or presence of DHT [10 nM]. Cells were counted at day 7. Data are shown as mean ± standard deviation; D. Expression of AR mRNA in LNCaP cells virally transduced with LacZ, caN1 and dnHES1, 4 days after transduction, assessed by quantitative real time PCR.