Erythropoietin Signaling In Neuroendocrine-like Transdifferentiation Of LNCaP Cells Under Chronic Hypoxia

BackgroundProstate carcinoma (PCa) is one of the most common malignances among theurological neoplasms that happen in men, with a secondary highest lethality. Its recentincidence, though lower than western countries, is increasing significantly in Chinawhich results from the aging and the changes in life style. In clinic, patients with PCawill eventually become androgen-independent after endocrinotherapy. So far, thereare no effective treatments, and the mechanism of transition to androgen-independentis still unknown. Most of the studies on this field only focus on the important roles ofthe Androgen Receptor (AR) during the transition to androgen-independent; however,they fail to explain any of the related mechanisms, which suggest that the AR is notthe only pathway involved in this transition.It is indicated that the neuroendocrine-like (NE) cells within the prostate cancertissues are closely correlated to tumor progression, poor prognosis and the transitionto androgen-independent. The neuropeptides secreted by these NE cells have beenidentified as potent paracrine and autocrine growth factors, which instead of androgen,can promote cancer cell migration and growth that will lead to theandrogen-independent transition.ObjectiveStudy of neuroendocrine differentiation of the LNCaP cell induced by oxygen-deficient culture, and its related EPO-EPOR signal pathway during thisprogress. Analyze the mechanism of the androgen-independent transition that causedby hypoxia induce.Methods1. Construct a specific culture model of neuroendocrine-like (NE) cell by inducing theLNCaP cell with the10%FBS-phenol red RPMI1640culture medium under anoxygen-deficient culture environment (37℃、5%CO2、1%O2saturation humidity) for2weeks, then test the NE markers expressed by these cells using western blot.2. Establish a human prostate carcinoma xenograft model by orthotopicly injectingLNCaP cells into SCID mice.3. Compare the expression level of EPO and EPOR between the androgen-dependenttissue and the androgen-independent tissue by immunohistochemical analysis.4. Illustrate the cell growth curve of the induced neuroendocrine-like cells thattransformed from hypoxia treated LNCaP cells using CCK-8assay; identify theexpression levels of EPO, EPOR, and HIF-1α of the LNCaP cells both before andafter hypoxic culturing by RT-PCR and western blot; analyze the expression levels ofseveral NE markers of the induced neuroendocrine-like cells both before and afterhypoxic culturing, with the EPOR inhibitory treatment at beginning using SmallInterference RNA (siRNA) transfection.Results1. The LNCaP cells showed a neuroendocrine change after2weeks hypoxic culture.And western blot results revealed some neuron-specific markers, such asneuron-specific enolase (NSE) and chromogranin A (CgA), were increased in thesehypoxic induced LNCaP cells.2. In the LNCaP cells orthotopicly injected group, there were tumors that developed in13mice with the total of15(87％), in which most parts of the prostate wereinvaded by tumor cells. There are confluent atypia epithelial cells, some of whichhave hyperchromatic and polymorphic nuclei, frothy eosinophilic structures in thecytoplasm, and some are abnormally proceeding of cytokinesis. In the LNCaP cellssubcutaneously injected group, only9of10mouse (90%) resulted in tumor growth.In the induced LNCaP cells orthotopicly injected group, all of these four mice(100%) had tumors developed in their prostates, in which the cortices and medullaeof metastatic lymph nodes were destroyed by tumor cells, hyperchromatic andpolymorphic nuclei, frothy eosinophilic structures, and abnormal mitotic figure canbe found in these atypia epithelial cells. There were also visible necrotic tissueswithin the tumors.3. EPO and EPOR were up-regulated in both the androgen-dependent tissue and theandrogen-independent tissue. And statistic significances (p<0.001) of the EPOscores and the EPOR scores can be found between the androgen-dependent tissueand the androgen-independent tissue respectively.4. The induced neuroendocrine-like cells, which transformed from hypoxia treatedLNCaP cells, turned to be androgen-independence that show a continuouslyincreased expression of EPO and EPOR both in the mRNA and the protein level.However, HIF-1α was up-regulated at the beginning and then followed by adown-regulation. NSE and CgA in the induced neuroendocrine-like cells weredecreased when blocking EPOR by siRNA.Conclusions1. The neuroendocrine transformation of the LNCaP cells, after2weeks hypoxicculture, indicates oxygen deficit which may promote theandrogen-independent progress of the prostate carcinoma.2. Compared with androgen-dependent tissue, the up-regulation of EPO andEPOR in androgen-independent tissue suggests that EPO-EPOR autocrineloop plays an important role during tumor development andandrogen-independent progression. 3. The continuously increase of both EPO and EPOR in LNCaP cells underhypoxic culture, together with the sustained decrease of HIF-1α, indicates thatthere is formation of a EPO-EPOR autocrine loop that takes over the functionof the HIF-1α. Besides, the EPO can also regulate cell apoptosis and cellresponse to hypoxic stress by binding to the functional EPOR.