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Background: Cancer cells notoriously escape radiation therapy, chemotherapy, hormonal therapy, and other anti-cancer therapies. Among various breast cancer subtypes, triple negative breast cancers (TNBCs) are known for their poor clinical outcome and dearth of effective targeted therapies due to the absence of druggable receptors. Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment agent that selectively kills basal-like/TN breast cancer cells. Method: In this study, we used plasma-activated media (PAM) to capture and store the multi-modal chemical species of CAP, which significantly expands the treatment scope and flexibility of plasma medicine as a clinical therapeutic approach. The PAM was diluted into different doses and cells were treated for 12 or 24 hours. The effects of PAM on cell viability and ROS levels in 9 different cell lines; normal cells (MCF-10A and HEK-293T), luminal breast cancer cell lines (MCF-7, T47D, HCC70), and TNBC cell lines (MDA-MB-468, SUM-159PT, SUM-149PT, MDA-MB-231), were assessed. Results: The results showed that TNBC cells were more highly sensitive, in a dose-dependent manner, to PAM treatment. PAM selectively caused apoptosis of TNBC cell lines compared to luminal lines and had little effect on the normal cell lines. It has been hypothesised that the cancer-selective CAP and PAM cytotoxicity is due in part to reactive oxygen (ROS) and nitrogen species (RNS). Constitutive RONS levels, combined with those present in and/or induced by PAM, can combine to push the cells over a cytotoxic threshold. In support of this, we found a tight correlation (R2=0.7502) between the constitutive ROS level of different cell lines used and their response (cell viability) to 100% PAM treatment, and found that ROS levels selectively increased further in the TNBC>luminal>normal cells in response to PAM. We have also identified a strong synergistic molecular partner that further promotes sensitivity and selectivity of PAM for TNBC cells. Conclusion: We demonstrated that treatment with PAM promotes apoptosis n cell lines representing the most aggressive TNBC subtype. This discovery opens new TNBC-selective opportunities for simultaneous inhibition of diverse (parallel) cancer development factors through synergistic interactions enabled through plasma activated biochemical media. Citation Format: Peiyu Wang, Renwu Zhou, Rusen Zhou, Jennifer Gunter, Fiona Simpson, Kostya (Ken) Ostrikov, Derek Richard, Xiaofeng Dai, Erik (Rik) Walter Thompson. Cold atmospheric plasma therapy selectively targets triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 996.

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis. Cancer Res; 71(17); 5754–64. ©2011 AACR.

Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated with metastasis and poor prognosis. Finally, we developed two distinct antisense oligonucleotides (ASOs) targeting GHSROS, achieving >60% knockdown, and their function was assessed in vitro. ASO inhibition of GHSROS expression reciprocally regulated cell growth and migration and the expression of a range of genes. These ASOs are currently being assessed in preclinical animal models. Our findings suggest that the long noncoding RNA GHSROS reprograms prostate cancer cells toward a more aggressive phenotype and that the lncRNA represents a promising therapeutic target. Citation Format: Patrick B. Thomas, Penny L. Jeffery, Gahete D. Manuel, Eliza J. Whiteside, Michelle Maugham, Carina Walpole, Jennifer H. Gunter, Elizabeth D. Williams, Colleen C. Nelson, Adrian C. Herington, Raul M. Luque, Rakesh N. Veedu, Lisa K. Chopin, Inge Seim. Dysregulated expression of the human long noncoding RNA GHSROS may influence prostate cancer progression and resistance to docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2452.

Reactivation of the embryonic developmental pathway, epithelial-to-mesenchymal transition (EMT) is associated with prostate cancer (PCa) metastasis and therapy resistance. Recent evidence has demonstrated EMT is stimulated following androgen targeted therapy (ATT). In the present study we investigated the role of EMT transcription factor ZEB1 in ATT-driven EMT, PCa metastasis and drug resistance. Immunohistochemistry (IHC) staining of ZEB1 on tissue microarrays of primary tumors from clinical samples demonstrated ZEB1 expression correlated with increased tumor aggressiveness and Gleason score. Upon stratification of patient data (n=198), high ZEB1 protein expression correlated with a shorter time to biochemical recurrence (BCR). Furthermore, IHC staining of primary tumours from 148 treatment-naïve patients with and without metastasis demonstrated high ZEB1 levels and correlated to a reduced time to metastasis. To delineate the role of ZEB1 as a molecular driver of late-stage PCa, microarray analysis of an in vivo LNCaP progression model demonstrated ZEB1 levels increased following castration. Significantly, high ZEB1 levels were associated with patients who had undergone neoadjuvant hormone therapy with or without docetaxel and high ZEB1 levels were associated with a shorter time to BCR and metastasis. Inhibition of the androgen/androgen receptor (AR) axis in LNCaP cells using antiandrogen treatment or shRNA targeting AR resulted in upregulation of ZEB1 in LNCaP cells suggesting enhanced levels of ZEB1 may drive the early adaptive response of PCa after hormone therapy. Interestingly, in models where ZEB1 expression was elevated this was accompanied with repression of classical androgen-regulated genes suggesting ZEB1 is able to regulate the AR transcriptional pathway. We also investigated the role of ZEB1 in cancer invasion and chemoresistance. An inducible model of ZEB1 overexpression in LNCaP cells produced a robust EMT upon ZEB1 expression and was accompanied by an invasive phenotype in 3D cultures. ZEB1 overexpression also conferred resistance to docetaxel (IC50 of 8.17±2.45nM in ZEB1-expressing cells vs. 3.35±0.23nM in control cells) potentially as a result of a reduced apoptotic cell death response mediated by ZEB1. At suboptimal doses of docetaxel, the percentage of apoptotic cells (annexin-V+/propidium iodide-) decreased 4-fold when ZEB1 was expressed compared with control cells and was accompanied by a reduction in PARP cleavage and cleaved caspase-7 expression. In summary, we provide evidence that ZEB1 expression is increased in response to ATTs and correlates with disease progression, metastasis and therapy resistance. We also show ZEB1 is a transcriptional regulator of AR signalling in PCa, Together this provides the rationale to target ZEB1 for the development of novel therapies for the treatment of CRPC. Note: This abstract was not presented at the meeting. Citation Format: Katrina G. Sweeney, Nataly Stylianou, Gregor Tevz, Atefeh Taherianfard, Katrina Pirlo, Akanksha Upadhyaya, Ellca Ratther, Melanie Lehman, Martin Gleave, Jennifer Gunter, Elizabeth D. Williams, Colleen C. Nelson, Brett Hollier. Androgen targeted therapy induces ZEB1 expression and is associated with suppression of androgen signalling and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4909. doi:10.1158/1538-7445.AM2017-4909

Long non-coding RNAs (lncRNAs) play key regulatory roles in cancer progression and are emerging therapeutic targets. The objective of this study was to investigate the expression and function of the lncRNA GHSROS in prostate cancer. Quantitative RT-PCR revealed that GHSROS is highly expressed in a subset of prostate cancers (Gleason score 8-10; z-score >1; Mann-Whitney-Wilcoxon test P=0.0021). Forced overexpression of the lncRNA stimulated cell migration in vitro in the PC3 (1.82 ± 0.35, P=0.006; Student’s t-test), DU145 (1.94 ± 0.34, P=0.017), and LNCaP (1.27 ± 0.02, P=0.0002) prostate cancer cell lines. Cell proliferation was increased in GHSROS overexpressing PC3 (3.36 ± 1.91, P=0.029), DU145 (1.749 ± 0.59, P=0.026), and LNCaP (1.39 ± 0.26, P=0.040) prostate cancer cell lines. These results were recapitulated in NOD/SCID mice, with increased tumor growth and Ki67 immunohistochemical staining in PC3 (P=0.0040) and DU145 (P = 0.036) xenografts overexpressing the lncRNA.High-throughput transcriptome sequencing (RNA-seq) identified 400 differentially expressed genes in GHSROS overexpressing PC3 cells, with enrichment of genes associated with motility, migration and regulation of cell growth. Further interrogation of the 400 gene set using Oncomine concept mapping, and interrogation of publicly-available clinical prostate cancer data sets, revealed a 34-gene signature associated with poorer disease outcome and metastatic progression. Preliminary analysis of The Cancer Genome Atlas (TCGA) data, suggest that the signature has potential as a prognostic indicator for disease free- or overall survival in numerous cancers. Finally, locked antisense oligonucleotide (LNA-ASO) inhibition of endogenous GHSROS reciprocally regulated cell growth (Student’s t-test; RNV124: -1.14 ± 0.06, P=0.049 and RNV104L: -1.18 ± 0.05, P=0.030, migration (RNV124: -1.96 ± 0.11, P=0.004) and gene expression changes, supporting the observations from forced GHSROS overexpression experiments.In summary, we provide evidence that GHSROS is a prostate cancer associated lncRNA that promotes a gene expression signature which enhances the propensity for metastasis and adverse disease outcomes. We also demonstrate that GHSROS can be targeted using antisense oligonucleotides. Further studies on this lncRNA may provide new prognostic and therapeutic opportunities. Citation Format: Patrick B. Thomas, Penny L. Jeffery, Eliza Whiteside, Carina Walpole, Michelle Maugham, Lidija Jovanovic, Jennifer H. Gunter, Colleen C. Nelson, Adrian C. Herington, Rakesh Veedu, Lisa K. Chopin, Inge Seim. The long non-coding RNA GHSROS mediates expression of genes associated with tumor growth, metastasis and adverse disease outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3442. doi:10.1158/1538-7445.AM2017-3442