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The discovery that androgens play an important role in the progression of prostate cancer led to the development of androgen deprivation therapy (ADT) as a first line of treatment. However, paradoxical growth inhibition has been observed in a subset of prostate cancer upon administration of supraphysiologic levels of testosterone (SupraT), both experimentally and clinically. Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive prostate cancer cells. This was initiated by the induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activated nucleic acid sensors converge on NFκB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT resulted in increased migration of cytotoxic immune cells to tumor beds in xenograft models and patient tumors. Collectively, these findings indicate that SupraT may inhibit a subset of prostate cancer by activating nucleic acid sensors and downstream immune signaling.Significance:This study demonstrates that supraphysiologic testosterone induces two parallel autophagy-mediated processes, ferritinophagy and nucleophagy, which then activate nucleic acid sensors to drive immune signaling pathways in prostate cancer.

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Obesity, a major healthcare problem worldwide, induces metabolic endotoxemia through the gut translocation of lipopolysaccharides (LPS), a major cell wall component of Gram-negative bacteria, causing a chronic inflammatory state. A combination of several probiotics including Lactobacillus acidophilus 5 (LA5), a potent lactic acid-producing bacterium, has previously been shown to attenuate obesity. However, data on the correlation between a single administration of LA5 versus microbiota alteration might be helpful for the probiotic adjustment. LA5 was administered daily together with a high-fat diet (HFD) for 8 weeks in mice. Furthermore, the condition media of LA5 was also tested in a hepatocyte cell-line (HepG2 cells). Accordingly, LA5 attenuated obesity in mice as demonstrated by weight reduction, regional fat accumulation, lipidemia, liver injury (liver weight, lipid compositions, and liver enzyme), gut permeability defect, endotoxemia, and serum cytokines. Unsurprisingly, LA5 improved these parameters and acidified fecal pH leads to the attenuation of fecal dysbiosis. The fecal microbiome analysis in obese mice with or without LA5 indicated; (i) decreased Bacteroidetes (Gram-negative anaerobes that predominate in non-healthy conditions), (ii) reduced total fecal Gram-negative bacterial burdens (the sources of gut LPS), (iii) enhanced Firmicutes (Gram-positive bacteria with potential benefits) and (iv) increased Verrucomycobia, especially Akkermansia muciniphila, a bacterium with the anti-obesity property. With LA5 administration, A. muciniphila in the colon were more than 2,000 folds higher than the regular diet mice as determined by 16S rRNA. Besides, LA5 produced anti-inflammatory molecules with a similar molecular weight to LPS that reduced cytokine production in LPS-activated HepG2 cells. In conclusion, LA5 attenuated obesity through (i) gut dysbiosis attenuation, partly through the promotion of A. muciniphila (probiotics with the difficulty in preparation processes), (ii) reduced endotoxemia, and (iii) possibly decreased liver injury by producing the anti-inflammatory molecules.

Fat reduction and anti-inflammation are commonly claimed properties of probiotics. Lactobacillus plantarum and Enterococcus faecium were tested in high fat-induced obesity mice and in vitro experiments. After 16 weeks of probiotics, L. plantarum outperforms E. faecium on the anti-obesity property as indicated by body weight, regional fat accumulation, serum cholesterol, inflammatory cytokines (in blood and colon tissue), and gut barrier defect (FITC-dextran assay). With fecal microbiome analysis, L. plantarum but not E. faecium reduced fecal abundance of pathogenic Proteobacteria without an alteration in total Gram-negative bacteria when compared with non-probiotics obese mice. With palmitic acid induction, the condition media from both probiotics similarly attenuated supernatant IL-8, improved enterocyte integrity and down-regulated cholesterol absorption-associated genes in Caco-2 cell (an enterocyte cell line) and reduced supernatant cytokines (TNF-α and IL-6) with normalization of cell energy status (extracellular flux analysis) in bone-marrow-derived macrophages. Because the anti-inflammatory effect of the condition media of both probiotics on palmitic acid-activated enterocytes was neutralized by amylase, the active anti-inflammatory molecules might, partly, be exopolysaccharides. As L. plantarum out-performed E. faecium in anti-obesity property, possibly through the reduced fecal Proteobacteria, with a similar anti-inflammatory exopolysaccharide; L. plantarum is a potentially better option for anti-obesity than E. faecium.

Background: Prostate cancer continues to show challenges in disease management and ultimately leads to advanced disease with poor survival. These unmet needs, despite recent advances, are leading researchers to identify novel targets to develop next-generation therapeutics. Considerable evidence from the published reports and TCGA database analysis indicates that CRM1, which functions as a nuclear export protein, amplifies prostate cancer, resulting in dysregulated nuclear export machinery. This dysregulation is also seen to correlate with mislocalization of p53, FOXO-1, and 3a, cyclinD1, p16, p21, and p27 in prostate cancer. Besides these mislocalized proteins, key DNA repair pathway members such as BRCA1 and Rad51 exhibit a nuclear export signal (NES) and are putative CRM1 cargoes. Nuclear-cytoplasmic shuttling of these repair proteins may play an essential role in repairing damaged DNA. We hypothesize that inhibition of CRM1 would result in defective DNA repair through disruption of nuclear-cytoplasmic shuttling of important repair protein cargoes. Methods: PCa cells (both AR-positive and negative lines) were treated with Selinexor to estimate the subtoxic doses. Cell lysates and total mRNA were collected to evaluate the relative levels of BRCA-1, RAD51, p53, ATM, ATR transcripts, and proteins. Activation of the DNA damage pathway was measured through a phosphorylated form of these proteins, and functional assessment of homologous recombination(HR) was measured through an HR sensor plasmid construct. Results: Treatment of PCa cells with Selinexor affected expression, localization, and kinetics of DNA repair leading to unrepaired DNA and induced apoptosis. Citation Format: Rajendra Kumar, Janet Mendonca, Kavya Boyapati, Yuhan Yang, Deven Topiwala, Suthicha Kanacharoen, Keerti Soundappan, Lillian Wilson, Eleni Panagopoulos, Michael Carducci, Sushant K. Kachhap. Exploring the role of CRM1 in DNA repair pathway in prostate cancer cells [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 1505.

The discovery that androgens play an important role in the progression of prostate cancer (PCa) has led to the development of androgen deprivation therapy as a first line of treatment against PCa. However, paradoxical growth inhibition has been observed, both experimentally and clinically, in a subset of PCa upon administration of supraphysiological levels of testosterone (SupraT). Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive PCa cells. This is initiated by induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activates nucleic acid sensors that converge on NF-kappaB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT results in increased migration of cytotoxic immune cells to tumor beds of animal xenografts and patient tumors. Collectively, our findings indicate that SupraT may inhibit a subset of PCa by activating nucleic acid sensors and downstream immune signaling.

Import of ligand-bound androgen receptor (AR) into the nucleus occurs with the help of nuclear importin. Past studies have identified a nuclear export signal (NES) in AR. CRM1 (also called XPO1 or Exportin1) is a well-studied exportin that exports proteins and RNA from the nucleus to the cytoplasm. GTP-bound active Ran, CRM1, and the cargo protein form a ternary complex that is exported through the nuclear pore complex. Previous experimental data indicate that RanGTP plays a vital role in Prostate cancer (PCa) progression. Our analyses further suggest that patients who have overexpression of CRM1 and/or Ran have significantly decreased survival. These results indicate that nucleocytoplasmic transport is essential for prostate cancer progression and inhibiting CRM1 could selectively inhibit cancer cell growth. We demonstrate that CRM1 does not affect the nuclear export of AR protein. However, quite surprisingly, we provide novel evidence that CRM1 binds to AR mRNA and is also required for AR protein stability. Inhibiting CRM1 using CRM1 inhibitor results in a dose-dependent rapid loss of AR and AR-V7 splice variants in PCa cells. This observation opens up an entirely new avenue to therapeutically regulate AR signaling using CRM1 inhibitors. Citation Format: Naiju Thomas, Rajendra Kumar, Deven Topiwala, Kavya Boyapati, Suthicha Kanacharoen, Michael Carducci, Samuel Denmeade, Sushant K. Kachhap. Regulation of androgen receptor stability by nuclear export protein CRM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5953.

Castration resistant prostate cancer (CRPC) first manifests as a sustained rise in the androgen-responsive gene, PSA, consistent with reactivation of a functioning androgen receptor (AR) axis. This observation led to the development of “second-line” therapies aimed at further blocking androgen/AR signaling. Unfortunately, resistance to these agents can also develop quickly. Paradoxically, several studies have suggested that the growth of AR-positive human CRPC cell lines may be inhibited by supraphysiologic levels of testosterone (SupT). These studies suggested that the adaptive reliance on AR signaling by CRPC cells becomes a therapeutic liability that can be exploited through the administration of SupT, which we termed as bipolar androgen therapy (BAT). Understanding how BAT works at the molecular and cellular levels might help in rationally combining BAT with other agents to achieve increased efficacy and tumor responses. Our data indicates that SupT induces autophagy mediated degradation of ferritin in prostate cancer (PCa) cells. Degradation of ferritin results in increase in labile iron pool increasing lipid peroxidation. Our data further indicates that SupT distinctly induces ferroptosis, a nonapoptotic regulated form of cell death induced by the accumulation of labile iron. Ferroptosis is thought to have tumor suppressing capabilities by clearing tumor cells via immune system activation. BAT has been discussed as a potential therapy for prostate cancer, but further research is needed to understand its full potential. Future combination of BAT with existing immunotherapeutics including immune checkpoint blockade may prove beneficial for treatment of CRPC. Citation Format: Rajendra Kumar, Kavya Boyapati, Naiju Thomas, Deven Topiwala, Suthicha Kanacharoen, Olutosin Owoyemi, Max Coffey, Michael Carducci, Mark C. Markowski, Emmanuel S. Antonarakis, Drew Pardoll, Samuel Denmeade, Sushant K. Kachhap. Supraphysiological androgens induce ferroptotic cell death in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2410.