The NEET (CISD) Proteins - A Novel Target for Prostate Cancer

Introduction: Prostate cancer is the sixth leading cause of cancer-related deaths worldwide in males. The NEET (CISD) protein family, a novel class of 2Fe-2S proteins, with a unique fold and cluster coordination (3Cys-1His). In humans, three proteins comprise the family: MitoNEET(mNT), NAF-1 and MiNT that are encoded by cisd1, cisd2 and cisd3, respectively. Previous work indicated the involvement of these proteins in multiple cancer types, e.g. breast, gastric, pancreas and prostate. This study aims at determining the role the NEET proteins play in prostate cancer.

Materials and Methods: PC3 human prostate cell line was stably transfected to either suppress NEET proteins expression via shRNA (pGFP-RS), or overexpress NEET proteins (with pEGFP-N1) either in normal or mutant form exchanging 3Cys-1His to a 4Cys cluster coordination that stabilize the Fe-S. Stable cell lines were isolated by FACS sorting, and protein expression was determined by western blots. Mitochondrial membrane potential (MMP), iron and ROS levels were determined by Epi-fluorescent microscopy with the TMRE, RPA and DHE/mitoSOX probes, respectively. Animal experiments were performed in compliance with the Hebrew University Authority for biological and biomedical models (NS-17-15262-4). Transfected PC3 cells (2.5 × 10⁵) were injected subcutaneously into male athymic nude (FOXN1NU) 5-wk-old mice. Mouse weight and tumor size were measured twice weekly throughout the experiment.

Results and Discussion: We produced ten different PC3 stably transfected cell lines: PC3-NC (negative control), PC3-mNT-CISD1(+), PC3-mNT-CISD1(H87C), PC3-mNT-CISD1(-), PC3-NAF-1-CISD2(+), PC3-NAF-1-CISD2(H114C), PC3-NAF-1-CISD2(-), PC3-MiNT-CISD3(+), PC3-MiNT-CISD3(-)₁), PC3-MiNT-CISD3(-)₂). Mitochondrial Iron and ROS accumulation studied in the different PC3 lines demonstrated that cells with suppressed expression of any NEET protein showed both reduced MMP and increased mitochondrial iron accumulation. We found a resistance to ROS in all three PC3 lines overexpressing the NEET proteins. Also all lines showed a decrease in ROS accumulation in the presence of an iron chelator. Based on our previous work in breast cancer (Darash-Yahana, PNAS 2016) we performed preliminary in vivo studies on NAF-1 (CISD2) in prostate cancer. We injected the PC3-NC, PC3-NAF-1-CISD2(+) and PC3-NAF-1- CISD2(-) lines to athymic nude mice and found that while PC3-NAF-1-CISD2 (-) developed smaller tumors in mice, PC3-NAF-1-CISD2 (+) developed tumor metastasis.

Conclusion: Our in vitro and in vivo results indicate an important role for the NEET family proteins in controlling mitochondrial function and iron and ROS homeostasis of prostate cancer cells. As such the NEET proteins are novel target for prostate cancer screening and therapy design.