TY - JOUR
T1 - Dynamics of antioxidant heme oxygenase-1 and pro-oxidant p66Shc in promoting advanced prostate cancer progression
AU - Miller, Dannah R.
AU - Ingersoll, Matthew A.
AU - Chou, Yu Wei
AU - Kosmacek, Elizabeth A.
AU - Oberley-Deegan, Rebecca E.
AU - Lin, Ming Fong
N1 - Funding Information:
This study was supported in part by awards of the National Institutes of Health, United States [ CA88184 , CA230950 , CA233664 ], the Nebraska Redox Biology Center, Newbraska, United States, the University of Nebraska Food for Health Grant, the University of Nebraska Advanced Microscopy Core Facility, the Fred and Pamela Buffet Cancer Center Support Grant, United States [ P30CA036727 ], the UNMC Bridge Fund, the UNMC Cancer Biology Training Grant, United States [ T32CA009476 ], the UNMC Fellowship, and the Purdue Pharma Scholars Award, United States, at UNMC . We thank Dr. Melissa Teoh-Fitzgerald for the NBT and Keap1 antibody. We also thank Ms. Fen-Fen Lin for help establish LNCaP-AI and MDA PCa 2b-AI cells as well as p66Shc stable subclones in LNCaP-AS cells.
Funding Information:
p66Shc is a member of the Shc (Src homology-collagen homolog) adaptor protein family [ 18–20]. p66Shc can enhance oxidant species including superoxide production via two pathways: within the cytosol, p66Shc through Rac1 can interact with NOX enzymes to generate O2−; p66Shc can also localize to mitochondria and interact with cytochrome C to promote the leakage of electrons from the electron transport chain to form O2−. The function of p66Shc in enhancing ROS production thus lends its role to a variety of cellular processes, e.g., longevity, senescence, cell proliferation and apoptosis, resulting in its role in aging and diseases [ 20–22]. In PCa, p66Shc protein levels are elevated in clinical adenocarcinomas compared to that of benign prostate tissues [11,23]. In androgen-sensitive (AS) PCa cells, androgen treatments increase p66Shc protein that leads to increased oxidant species production and cell proliferation, and p66Shc protein level is also higher in androgen-independent (AI) cells than in the corresponding AS cells [ 10–13, 23,24]. Furthermore, elevated p66Shc protein levels by cDNA transfection supports AS cell proliferation and migration under steroid-reduced (SR) conditions in culture and tumor development in xenograft animals with low circulating androgens, a CR PCa phenotype [ 11–13]. Together, elevated p66Shc enhances PCa cell tumorigenecity and metastasis as well as obtains the CR phenotype in xenograft animals [11,13].In this study, we discovered a pair of redox proteins: one prooxidant protein and its corresponding antioxidant protein; together they maintain a high level of ROS for supporting CR PCa progression without allowing the ROS balance to reach toxic levels. We found that in PCa cells, an increase or decrease of p66Shc protein correlates with respective increase or reduction of HO-1 protein and its potent inducer heme levels. Conversely, knockdown HO-1 leads to increased cellular ROS levels and nucleotide and protein oxidation as well as the induction of cell death particularly in SR conditions. Further, treatment of p66Shc-elevated PCa cells with HO-1 competitive inhibitor zinc protoporphyrin IX (ZnPPIX) in combination with radiation led to enhanced anti-tumor activity. Hence, elevated HO-1 protects PCa cells from aberrantly excessive p66Shc-induced ROS and preventing them from oxidative damage, concurrently, those cells maintain a high level of ROS resulting in promoting advanced CR PCa progression. p66Shc and HO-1 together can serve as functional targets for developing an effective treatment for CR PCa.We first analyzed the effects of ROS treatment on AS and AI PCa cell lines, including LNCaP, MDA PCa2b and VCaP cells. Interestingly, there is an increase in cell growth upon treatment of AS PCa cells with 10 μM H2O2, while AI PCa cells having 2-3-fold higher basal levels of ROS experienced reduced cell proliferation and cell death with same concentration of H2O2 (Fig. 1) [11]. Analyses on the redox signaling profile of those PCa cell lines as well as vector-alone transfected V1 cells compared to p66Shc cDNA-transfected subclones, demonstrated that p66Shc was increased in all AI PCa cells examined as well as p66Shc subclones, higher than the corresponding AS cells. Nrf2 and HO-1 were also consistently upregulated in p66Shc-elevated AI PCa cells and p66Shc subclones, while SOD protein levels and activities were not significantly altered (Fig. 2). Hence, we propose that HO-1 is the vital antioxidant counterpart to p66Shc, which is also indicated by observations in other cell types including hepatic cells and renal cells that the elevation of HO-1 in response to increased p66Shc can reduce oxidative damage in those cells [42,43]. The importance of HO-1 in p66Shc-elevated cells is further supported by metabolomic analyses, which revealed that heme, an inducer of HO-1 protein and activity, was also increased in LNCaP-AI and MDA PCa2b-AI cells and p66Shc subclones compared to their AS counterparts, all of which have increased p66Shc protein levels (Fig. 2B).Our results of HO-1 counter-balancing ROS by p66Shc in supporting of AI cell proliferation are consistent with those reports on the influence of HO-1 on CR PCa growth and metastasis in vivo xenograft animals. Elevated p66Shc supports AI PCa cell growth and metastasis in xenograft animals [13]. PC-3 cells express high levels of p66Shc [25] and HO-1 [46]. HO-1 shRNA transfection of PC-3 cells resulted in reduced tumor growth as well as lymph node and tumor metastasis in vivo. Similarly, CR PCa tumor growth and metastasis were also reduced upon treatment with HO-1 inhibitor OB-24, which had an additive anti-proliferative effect when combined with paclitaxel [31]. Importantly, ZnPPIX treatment has effective anti-tumor activity in a variety of cancers [ 47–50] and daily administration of this molecule does not demonstrate any apparent side effects in mice [51], thereby providing a potential therapeutic option for CR PCa.This study was supported in part by awards of the National Institutes of Health, United States [CA88184, CA230950, CA233664], the Nebraska Redox Biology Center, Newbraska, United States, the University of Nebraska Food for Health Grant, the University of Nebraska Advanced Microscopy Core Facility, the Fred and Pamela Buffet Cancer Center Support Grant, United States [P30CA036727], the UNMC Bridge Fund, the UNMC Cancer Biology Training Grant, United States [T32CA009476], the UNMC Fellowship, and the Purdue Pharma Scholars Award, United States, at UNMC. We thank Dr. Melissa Teoh-Fitzgerald for the NBT and Keap1 antibody. We also thank Ms. Fen-Fen Lin for help establish LNCaP-AI and MDA PCa 2b-AI cells as well as p66Shc stable subclones in LNCaP-AS cells.
Publisher Copyright:
© 2022 Elsevier Inc.
PY - 2022/11/20
Y1 - 2022/11/20
N2 - The castration-resistant (CR) prostate cancer (PCa) is lethal and is the second leading cause of cancer-related deaths in U.S. males. To develop effective treatments toward CR PCa, we investigated reactive oxygen species (ROS) signaling pathway for its role involving in CR PCa progression. ROS can regulate both cell growth and apoptosis: a moderate increase of ROS promotes proliferation; its substantial rise results in cell death. p66Shc protein can increase oxidant species production and its elevated level is associated with the androgen-independent (AI) phenotype of CR PCa cells; while heme oxygenase-1 (HO-1) is an antioxidant enzyme and elevated in a sub-group of metastatic PCa cells. In this study, our data revealed that HO-1 and p66Shc protein levels are co-elevated in various AI PCa cell lines as well as p66Shc cDNA-transfected cells. Knockdown and/or inhibition of either p66Shc or HO-1 protein leads to reduced tumorigenicity as well as a reduction of counterpart protein. Knockdown of HO-1 alone results in increased ROS levels, nucleotide and protein oxidation and induction of cell death. Together, our data indicate that elevated HO-1 protein levels protect PCa cells from otherwise apoptotic conditions induced by aberrant p66Shc/ROS production, which thereby promotes PCa progression to the CR phenotype. p66Shc and HO-1 can serve as functional targets for treating CR PCa.
AB - The castration-resistant (CR) prostate cancer (PCa) is lethal and is the second leading cause of cancer-related deaths in U.S. males. To develop effective treatments toward CR PCa, we investigated reactive oxygen species (ROS) signaling pathway for its role involving in CR PCa progression. ROS can regulate both cell growth and apoptosis: a moderate increase of ROS promotes proliferation; its substantial rise results in cell death. p66Shc protein can increase oxidant species production and its elevated level is associated with the androgen-independent (AI) phenotype of CR PCa cells; while heme oxygenase-1 (HO-1) is an antioxidant enzyme and elevated in a sub-group of metastatic PCa cells. In this study, our data revealed that HO-1 and p66Shc protein levels are co-elevated in various AI PCa cell lines as well as p66Shc cDNA-transfected cells. Knockdown and/or inhibition of either p66Shc or HO-1 protein leads to reduced tumorigenicity as well as a reduction of counterpart protein. Knockdown of HO-1 alone results in increased ROS levels, nucleotide and protein oxidation and induction of cell death. Together, our data indicate that elevated HO-1 protein levels protect PCa cells from otherwise apoptotic conditions induced by aberrant p66Shc/ROS production, which thereby promotes PCa progression to the CR phenotype. p66Shc and HO-1 can serve as functional targets for treating CR PCa.
KW - Castration-resistant prostate cancer
KW - Dynamic redox balance
KW - HO-1
KW - Prostate cancer
KW - p66Shc
UR - http://www.scopus.com/inward/record.url?scp=85140339024&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85140339024&partnerID=8YFLogxK
U2 - 10.1016/j.freeradbiomed.2022.10.269
DO - 10.1016/j.freeradbiomed.2022.10.269
M3 - Article
C2 - 36265795
AN - SCOPUS:85140339024
VL - 193
SP - 274
EP - 291
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
SN - 0891-5849
ER -