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2-Methoxyestradiol (2-ME2) possesses anti-tumorigenic activities in multiple tumor models with acceptable tolerability profile in humans. Incomplete understanding of the mechanism has hindered its development as an anti-tumorigenic compound. We have identified for the first-time macrophage stimulatory protein 1 receptor (MST1R) as a potential target of 2-ME2 in prostate cancer cells. Human tissue validation studies show that MST1R (a.k.a RON) protein levels are significantly elevated in prostate cancer tissues compared to adjacent normal/benign glands. Serum levels of macrophage stimulatory protein (MSP), a ligand for RON, is not only associated with the risk of disease recurrence, but also significantly elevated in samples from African American patients. 2-ME2 treatment inhibited mechanical properties such as adhesion and elasticity that are associated with epithelial mesenchymal transition by downregulating mRNA expression and protein levels of MST1R in prostate cancer cell lines. Intervention with 2-ME2 significantly reduced tumor burden in mice. Notably, global metabolomic profiling studies identified significantly higher circulating levels of bile acids in castrated animals that were decreased with 2-ME2 intervention. In summary, findings presented in this manuscript identified MSP as a potential marker for predicting biochemical recurrence and suggest repurposing 2-ME2 to target RON signaling may be a potential therapeutic modality for prostate cancer.

There is an urgent need for innovative strategies such as the discovery of adjuvants that can prevent relapse and improve quality of life for patients treated with radiotherapy. Previously we demonstrated the utility of Nexrutine (Nex) as a neo-adjuvant with radiation. Nex was safe and well tolerated in PCa patients and potentiated radiation response in part through downregulation of ribosomal protein S6K (encoded byRPS6KB1). We now show that RPS6KB1 depleted prostate cancer cells with higher basal levels of γ-H2AX, a marker for DNA double strand breaks (i) are more sensitive to radiation and (ii) form smaller tumors with reduced levels of prostate specific antigen (PSA). Depletion of RPS6KB1 hindered DNA double-strand break repair predominantly through the alternate end-joining pathway, induction of G2/M checkpoint and NFκB pathway activation. Collectively these events led to improved radiation sensitivity. We further identified Berberine (Ber), one of the active constituents of Nex as a potential pharmacological inhibitor of RPS6KB1. In an orthotopic implantation model of C4-2B, treatment with Ber alone or Ber plus radiation decreased PSA levels that was sustained during the course of the experiment. On the other hand animals treated with radiation alone developed recurrent cancer as evidenced by a resurgence of PSA. Animals administered Ber followed by XRT intervention had increased levels of RANTES while there was no change in animals that received XRT followed by Ber. The observed reversal of the Bereffect with the sequence of intervention is statistically significant (p=0.0298). Among animals not subject to XRT, the mean PSA increased in those that did not receive Ber relative to those that did; mean difference=-1.93, 95% CI -3.75 to -0.105, p=0.04 with no significant changes in body weight. Notably,RPS6KB1 mRNA levels increased in tumor samples in patients experiencing biochemical recurrence(BCR). Given that rising PSA following conventional therapeutic approaches such as radiation remain a major clinical challenge, targeting RPS6KB1 signaling with radiation therapy is an attractive strategy to prevent BCR. Supported in part by CPRIT RP190012 (APK). Citation Format: Addanki Pratap Kumar, Alison Clark, Michelle Villarreal, Sridharan Jayamohan, Shih-Bo Huang, Suleman S. Hussain, Xiaoyu Yang, Paul Rivas, Darpan Patel, Bethany L. Pierce, Shreya Tripathy, Pawel Osmulski, Maria Gaczynska, Lai Zhao, Li-Ju Wang, Yidong Chen, Caroline Xavier Paul Ezhilan, Mohan Natarajan, Joel E. Michalek, Robert L. Reddick, Rita Ghosh. Intercepting ribosomal protein S6KB1 signaling: Prevention of prostate cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4.

Radiation therapy (RT) is a standard treatment for organ confined prostate cancer (PCa). Given that both intrinsic and acquired resistance to radiation causes progression to advanced metastatic PCa, there is an unmet need for adjuvant therapies that synergize with radiation to improve therapeutic efficacy. Previously, we demonstrated strong synergistic tumor growth inhibitory activities for Phellodendron amurense bark extract in combination with radiation in part through downregulation of ribosomal protein S6K (encoded by RPS6KB1). Subsequently we identified berberine (Ber), one of the active constituents of Nexrutine as a potential pharmacological inhibitor of RPS6KB1. Given these findings, we hypothesized that targeting RPS6KB1 could be an approach to enhance sensitivity to radiation. We tested the efficacy of Ber alone and in combination with radiation using an orthotopic implantation model and the mechanism associated with radio-sensitization using prostate cancer cells stably silenced and deleted for RPS6KB1. We observed that (i) RPS6KB1 deletion in androgen responsive early stage LNCaP cells and (ii) stable silencing in castrate resistant C4-2B cells increased sensitization to radiation relative to respective parental or non-targeted cells. Furthermore, pharmacological inhibition of RPS6KB1 with Ber sensitized cells to radiation. Mechanistically, RPS6KB1 silencing increased autophagic activity as revealed by changes in proteins identified through reverse phase protein array (RPPA) and decreased expression and levels of SQSTM1 via downregulation of NFkB. Pretreatment with chloroquine (CQ) rescued the observed radio-sensitization effects. Treatment with Ber alone or Ber plus radiation decreased levels of PSA that was sustained during the course of the experiment in mice orthotopically implanted with C4-2B cells. On the other hand animals treated with radiation alone developed recurrent cancer as evidenced by a resurgence of PSA. Notably, RPS6KB1 mRNA levels increased in tumor samples in patients experiencing biochemical recurrence (BCR). Taken together, these findings demonstrate that RPS6KB1 is a clinically relevant target and that Ber sensitizes prostate cancer cells to radiation in vitro and in vivo. Therefore, targeting RPS6KB1 signaling is an attractive strategy to prevent progression to BCR; given that rising PSA following conventional therapeutic approaches such as radiation remain a major clinical challenge. This work is supported in part by CPRIT RP190012 (APK). Citation Format: Addanki P. Kumar, Alison Clark, Michelle R. Villarreal, Shih-Bo Huang, Suleman S. Hussain, Xiaoyu Yang, Roble G. Bedolla, Paul Rivas, Sridharan Jayamohan, Li-Ju Wang, Yidong Chen, Caroline Xavier Paul Ezhilan, Mohan Natarajan, Joel E. Michalek, Robert Reddick, Hiroshi Miyamoto, Rita Ghosh. Therapeutic targeting of RPS6KB1/SQSTM1 axis to prevent biochemical recurrence [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-085.