The role of microRNA-194 in prostate cancer progression

Prostate cancer is a major cause of cancer-related mortality in Australia men. Mortality is primarily due to metastasis and the development of resistance to therapy. While prostate cancer is primarily driven by the androgen receptor signalling, a number of other factors play important roles in its growth and progression. In particular, small non-coding RNA molecules called microRNAs (miRNAs) are known to be key regulators of progression in prostate cancer. Our group previously identified one specific miRNA, miR-194-5p (miR-194), as an important driver of prostate cancer metastasis; however, the molecular mechanisms by miR-194 mediates these effects is not fully understood. My PhD project aimed to identify target genes and pathways that miR-194 regulates in order to better understand its role in prostate cancer. I used cutting-edge genomic techniques and bioinformatics to identify 163 miR-194 target genes in prostate cancer. In Chapter 3, I used this data to identify a new role for miR-194 in prostate cancer. More specifically, I found that miR-194 activity was inversely correlated with androgen receptor (AR) activity in clinical samples, an observation explained mechanistically by AR-mediated repression of miR-194 expression. In concordance with these findings, miR-194 activity was significantly elevated in treatment-induced neuroendocrine prostate cancer (NEPC), an aggressive AR-independent subtype of prostate cancer. Furthermore, miR-194 can enhance transdifferentiation of epithelial LNCaP cells to neuroendocrine-like cells, a function mediated at least in part by its ability to target the FOXA1 transcription factor. Importantly, targeting miR-194 effectively inhibited the growth of aggressive models of NEPC, including patient-derived organoids. By integrating the miR-194 “targetome” with transcriptomic data, my work has provided important insights into miRNA function in cancer cells (Chapter 4). Specifically, I have found that miR-194 functions potently through canonical interactions and can mediate co-operative repression through targeting multiple sites in the same mRNA transcript. Further, I have demonstrated that miR-194 is associated with widespread non-canonical interactions that can regulate gene expression, albeit to a lesser extent than canonical sites. Finally, in Chapter 5 I have demonstrated that miR-194 has dichotomous effects on proliferation and invasion in breast and prostate cancer despite both cancers having several underlying biological similarities. Furthermore, in breast cancer I have found that miR-194 inhibits estrogen receptor expression, potentially by targeting FOXA1. Overall, my work has provided unique insights into the pathobiology of miR-194, demonstrated its role as a potential therapeutic target in aggressive AR-independent prostate cancer subtypes, and identified novel functions for miR-194 in breast cancer.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 202

Detection of YAP1 and AR-V7 mRNA for Prostate Cancer prognosis using an ISFET Lab-On-Chip platform

AbstractProstate cancer (PCa) is the second most common cause of male cancer-related death worldwide. The gold standard of treatment for advanced PCa is androgen deprivation therapy (ADT). However, eventual failure of ADT is common and leads to lethal metastatic castration resistant PCa (mCRPC). As such, the detection of relevant biomarkers in the blood for drug resistance in mCRPC patients could lead to personalized treatment options. mRNA detection is often limited by the low specificity of qPCR assays which are restricted to specialised laboratories. Here, we present a novel reversetranscription loop-mediated isothermal amplification (RT-LAMP) assay and have demonstrated its capability for sensitive detection of AR-V7 and YAP1 RNA (3×101 RNA copies per reaction). This work presents a foundation for the detection of circulating mRNA in PCa on a non-invasive Lab-on-chip (LoC) device for use at point-of-care. This technique was implemented onto a Lab-on-Chip platform integrating an array of chemical sensors (ion-sensitive field-effect transistors - ISFETs) for real-time detection of RNA. Detection of RNA presence was achieved through the translation of chemical signals into electrical readouts. Validation of this technique was conducted with rapid detection (&lt;15 min) of extracted RNA from prostate cancer cell lines 22Rv1s and DU145s.</jats:p

Novel Androgen Receptor Coregulator GRHL2 Exerts Both Oncogenic and Antimetastatic Functions in Prostate Cancer.

Alteration to the expression and activity of androgen receptor (AR) coregulators in prostate cancer is an important mechanism driving disease progression and therapy resistance. Using a novel proteomic technique, we identified a new AR coregulator, the transcription factor Grainyhead-like 2 (GRHL2), and demonstrated its essential role in the oncogenic AR signaling axis. GRHL2 colocalized with AR in prostate tumors and was frequently amplified and upregulated in prostate cancer. Importantly, GRHL2 maintained AR expression in multiple prostate cancer model systems, was required for cell proliferation, enhanced AR's transcriptional activity, and colocated with AR at specific sites on chromatin to regulate genes relevant to disease progression. GRHL2 is itself an AR-regulated gene, creating a positive feedback loop between the two factors. The link between GRHL2 and AR also applied to constitutively active truncated AR variants (ARV), as GRHL2 interacted with and regulated ARVs and vice versa. These oncogenic functions of GRHL2 were counterbalanced by its ability to suppress epithelial-mesenchymal transition and cell invasion. Mechanistic evidence suggested that AR assisted GRHL2 in maintaining the epithelial phenotype. In summary, this study has identified a new AR coregulator with a multifaceted role in prostate cancer, functioning as an enhancer of the oncogenic AR signaling pathway but also as a suppressor of metastasis-related phenotypes. Cancer Res; 77(13); 3417-30. ©2017 AACR

MicroRNA-194 promotes prostate cancer metastasis by inhibiting SOCS2

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer