Ionizing radiation induces neuroendocrine differentiation of prostate cancer cells in vitro, in vivo and in prostate cancer patients

Abstract

Prostate cancer remains the most common noncutaneous cancer among American men. Although most patients can be cured by surgery and radiotherapy, 32,050 patients still died of the disease in 2010. Many patients receive radiotherapy either as a primary therapy, salvage therapy, or in combination with surgery or hormonal therapy. Despite initial treatment, several studies suggest that approximately 10% of low-risk prostate cancer patients and up to 30-60% with more advanced cancer patients experience biochemical recurrence within five years after radiotherapy. Thus, elucidating the molecular mechanisms underlying radioresistance and tumor recurrence has the potential to significantly reduce prostate cancer mortality. We previously demonstrated that fractionated ionizing radiation (IR) can induce the prostate cancer cell line LNCaP to undergo neuroendocrine differentiation (NED) by activation of cAMP response element binding protein (CREB) and cytoplasmic sequestration of ATF2, two CRE-binding transcription factors that oppose each other to regulate NED. Importantly, IR-induced NED is reversible and de-differentiated cells are cross-resistant to IR, androgen depletion and docetaxel treatments. These findings suggest that radiation-induced NED may allow prostate cancer cells to survive treatment and contribute to tumor recurrence. In the present study, we further demonstrated that IR also induces NED in a subset of DU-145 and PC-3 cells. In addition, we confirmed that IR induces NED in LNCaP xenograft tumors in nude mice, and observed that the plasma chro-mogranin A (CgA) level, a biomarker for NED, is increased by 2- to 5-fold in tumor-bearing mice after fractionated radiation doses of 20 and 40 Gy, respectively. Consistent with these in vivo findings, a pilot study in prostate cancer patients showed that the serum CgA level is elevated in 4 out of 9 patients after radiotherapy. Taken together, these findings provide evidence that radiation-induced NED is a general therapeutic response in a subset of prostate cancer patients. Thus, a large scale analysis of radiotherapy-induced NED in prostate cancer patients and its correlation to clinical outcomes will likely provide new insight into the role of NED in prostate cancer radiotherapy and prognosis.

Keywords: ATF2; CREB; Ionizing radiation; neuroendocrine differentiation; prostate cancer; radiotherapy.

Figure 1

IR induces NED in prostate cancer cells. A). Representative images acquired from the indicated prostate cancer cells that were treated with 40 Gy of fractionated IR (IR+) or without IR treatment (IR-) (microscopy at 200x for LNCaP and 100x for PC-3 and DU-145). Note that enlarged and flat cell bodies were observed for irradiated PC-3 when compared with non-irradiated PC-3. B). Approximately 40 μg of total lysate from non-irradiated (IR-) and irradiated (40 Gy, IR+) cells was used for immunoblot analysis of CgA, NSE and β-actin. Similar results were reproduced from at least three independent experiments.

Figure 2

IR induces CREB activation and cytoplasmic sequestration of ATF2 in prostate cancer cells. A). A representative im-munoblot analysis of phosphorylated CREB (pCREB) from non-irradiated cells (IR-) or from cells that received 10 Gy of fractionated IR (IR+). B). Shown are DIC and fluorescent images for ATF2 and DNA (DAPI) acquired from the indicated non-irradiated prostate cancer cells (IR-) or from cells that received 10 Gy of fractionated IR (IR+) (microscopy at 600x). These experiments were reproduced at least three times and similar results were obtained.

Figure 3

Activated CREB induces neurite outgrowth and the expression of CgA and NSE in PC-3 and DU-145 cells. A). Prostate cancer cells PC-3 and DU-145 were transfected with a pHA-CMV plasmid encoding a constitutively activated CREB, VP16-bCREB (bCREB), or the pHA-CMV empty vector (Vec). Shown are phase contrast images acquired five days after the transfection (microscopy at 200x). The number indicates the percentage of cells showing neurite outgrowth. B) and C). Expression of HA-VP16-bCREB (HA), CgA, NSE and β-actin in PC-3 cells (B) or DU-145 cells (C) from the experiments in A. Note that CgA was not detectable in PC-3 cells transfected with the vector control pHA-CMV or pHA-VP16-bCREB.

Figure 4

ATF2 knockdown induces neurite outgrowth and the expression of CgA and NSE in prostate cancer cells. A). Prostate cancer cells PC-3 and DU-145 were transfected with the ATF2 shRNA plasmid (ATF2 KD) or the scrambled control (SC). Shown are phase contrast images acquired five days after the transfection (Microscopy at 200x). The number indicates the percentage of cells showing neurite outgrowth. B) and C). Expression of ATF2, CgA, NSE and β-actin in PC-3 cells (B) or DU-145 cells (C) from the experiments in A. Note that CgA was not detectable in PC-3 cells transfected with either SC or ATF2 shRNA plasmids.

Figure 5

Ionizing radiation induces CgA expression in LNCaP xenograft tumors and an increase of plasma CgA levels in nude mice. A). IHC analysis of CgA expression in irradiated LNCaP xenograft tumors after 40 Gy (IR+) or in non-irradiated xenograft tumors (IR-) (microscopy at 400x). Scale bar represents 10 μm. B). Average fold change of plasma CgA levels normalized to plasma PSA at the end of week 2 (20 Gy) and week 4 (40 Gy) when compared with pre-irradiation (0 Gy). Similar time points were followed for blood collection from non-irradiated tumor-bearing mice. The average fold change presented is from all 10 mice for each group.

Figure 6

Radiotherapy increases serum CgA levels in prostate cancer patients. All 9 prostate cancer patients were diagnosed with localized tumors and treated at the Indiana University Hospital or the Midwest Proton Radiotherapy Institute with 70.2-79.2 Gy (2 Gy/fraction). Blood samples were collected for pre-treatment, mid-treatment, and post-treatment. The serum CgA levels were normalized to the serum PSA levels, and the fold change at mid- and post-treatment time points is presented for each patient.