Valosin-Containing Protein (VCP/p97) Mediates Neuroendocrine Differentiation in Prostate Cancer Cells Through Pim1 Signaling Inducing Autophagy

Background: Neuroendocrine Prostate Cancer (NEPC) is an aggressive type of androgen-independent prostate Cancer (AIPC) associated with resistance to treatment. Valosin-containing protein (VCP/p97) has been found to be overexpressed in prostate Cancer (PCa) cells undergoing neuroendocrine differentiation (NED) in response to interleukin-6 (IL-6). This study explores the molecular mechanisms through which VCP/p97 contributes to the progression of NEPC.

Methods: To investigate the role of VCP/p97 in the NED of PCa, we overexpressed the VCP/p97 in PCa cells. The molecular mechanisms underlying VCP/p97 induced NED were assessed by using western blot analysis and RT-PCR. Morphological changes were analyzed by using both bright field and confocal microscope. Lysotracker staining was performed to identify Autophagy in VCP positive PCa cells.

Results: In the present study, we found that VCP/p97 expression was notably higher in neuroendocrine (NE) cells NCI-H660 and PC3 than in Other PCa cells. IL-6 treatment led to significant VCP/p97 overexpression in LNCaP and VCaP cells, with a marked increase in NE markers NSE and CHR-A. Inhibition of VCP/p97 using NMS-873 attenuated NED features, suggesting that VCP/p97 is required for NED progression. Moreover, VCP's role in NED is linked to its regulation via PIM1 in differentiating cells. Exogenous expression of VCP/p97 enhanced PIM1 and c-Myc expression, which were diminished upon VCP/p97 inhibition which is corroborated by reduced NED markers. PIM1 inhibition using AZD1208 and c-Myc knockdown further supported Pim1's involvement in VCP mediated NED. To promote NED, VCP/p97 regulated Autophagy, as evidenced by increased LC3B and decreased SQSTM1/p62 levels upon VCP overexpression. Inhibition of VCP/p97 or Autophagy disrupted NED and autophagic flux, arresting NED of LNCaP cells. Lysotracker staining and autophagic flux assays confirmed VCP's role in enhancing lysosomal-mediated Autophagy and autophagolysosome formation. Furthermore, we show that AMPK activation, via LKB1 is essential for VCP/p97 mediated NED and Autophagy.

Conclusion: VCP drives NED in PCa cells through a complex interplay involving the PIM1 axis and Autophagy pathways. These findings highlight the potential of targeting VCP/p97 and its associated mechanisms as therapeutic strategies to inhibit NED progression.

IL‐6; LC3A/B; LNCaP cells; NE markers; Pim‐1; VCaP cells; autophagy.