Zendra E. Zehner

Anibamine, a Natural Product CCR5 Antagonist, as a Novel Lead for the Development of Anti Prostate Cancer Agents

Accumulating evidence indicates that the chemokine receptor CCR5 and the chemokine CCL5 may be involved in the proliferation and metastasis of prostate cancer. Consequently, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents. As the first natural product CCR5 antagonist, anibamine provides a novel chemical structural skeleton compared with other known antagonists identified through high-throughput screening. Our studies demonstrate that anibamine produces significant inhibition of prostate cancer cell proliferation at micromolar to submicromolar concentrations as well as suppressing adhesion and invasion of the highly metastatic M12 prostate cancer cell line. Preliminary in vivo studies indicate that anibamine also inhibits prostate tumor growth in mice. These findings indicate that anibamine may prove to be a novel lead compound for the development of prostate cancer therapeutic agents.

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3’-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression.

microRNA Dysregulation in Prostate Cancer: Network Analysis Reveals Preferential Regulation of Highly Connected Nodes

microRNAs (miRNAs) are small RNAs shown to contribute to a number of cellular processes including cell growth, differentiation, and apoptosis. MiRNAs regulate gene expression of their targets post‐transcriptionally by binding to messenger RNA (mRNA), causing translational inhibition or mRNA degradation. Dysregulation of miRNA expression can promote cancer formation and progression. Research has largely focused on the function and expression of single miRNAs. However, complex physiological processes require the interaction, regulation and coordination of many molecules including miRNAs and proteins. Highly connected molecules often serve important roles in the cell. A proteinprotein interaction network of established miRNA targets confirmed these proteins to be highly connected and essential to the cell, affecting tumorigenesis, cell growth/proliferation, cellular death, cell assembly, and maintenance pathways. This analysis showed that miRNAs contribute to the overall health of the prostate, and their aberrant expression destabilized homeostatic balance. This integrative network approach can reveal important miRNAs and proteins in prostate cancer that will be useful to identify specific disease biomarkers, which may be used as targets for therapeutics or drugs in themselves.

miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis

Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9.

High-throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids.

microRNAs (miRNAs) are small noncoding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRNAs, when fully evaluated, will make excellent candidates for body fluid identification. miRNA analysis of body fluids has been the subject of some recent interest in the forensic community. In this study, small RNAs were isolated from individual donations of eight forensically relevant biological fluids (blood, semen, vaginal fluid, menstrual blood, saliva, urine, feces, and perspiration) and subjected to next generation sequencing using the Illumina Hi‐Seq platform. Sequencing reads were aligned and annotated against miRbase release 21, resulting in a list of miRNAs and their relative expression levels for each sample analyzed. Body fluids with high bacterial loads (vaginal fluid, saliva, and feces) yielded relatively low annotated miRNA counts, likely due to oversaturation of small RNAs from the endogenous bacteria. Both body fluid specific (miRs‐200b, 1246, 320c, 10b‐5p, 26b, and 891a) and potential normalization miRNAs (let‐7g and i) were identified for further analysis as potential body fluid identification tools for each body fluid.

A Panel of MicroRNAs as Diagnostic Biomarkers for the Identification of Prostate Cancer

Prostate cancer is the most common non-cutaneous cancer among men; yet, current diagnostic methods are insufficient, and more reliable diagnostic markers need to be developed. One answer that can bridge this gap may lie in microRNAs. These small RNA molecules impact protein expression at the translational level, regulating important cellular pathways, the dysregulation of which can exert tumorigenic effects contributing to cancer. In this study, high throughput sequencing of small RNAs extracted from blood from 28 prostate cancer patients at initial stages of diagnosis and prior to treatment was used to identify microRNAs that could be utilized as diagnostic biomarkers for prostate cancer compared to 12 healthy controls. In addition, a group of four microRNAs (miR-1468-3p, miR-146a-5p, miR-1538 and miR-197-3p) was identified as normalization standards for subsequent qRT-PCR confirmation. qRT-PCR analysis corroborated microRNA sequencing results for the seven top dysregulated microRNAs. The abundance of four microRNAs (miR-127-3p, miR-204-5p, miR-329-3p and miR-487b-3p) was upregulated in blood, whereas the levels of three microRNAs (miR-32-5p, miR-20a-5p and miR-454-3p) were downregulated. Data analysis of the receiver operating curves for these selected microRNAs exhibited a better correlation with prostate cancer than PSA (prostate-specific antigen), the current gold standard for prostate cancer detection. In summary, a panel of seven microRNAs is proposed, many of which have prostate-specific targets, which may represent a significant improvement over current testing methods.

A networks method for ranking microRNA dysregulation in cancer

BackgroundDespite the lack of agreement on their exact roles, it is known that miRNAs contribute to cancer progression. Many studies utilize methods to detect differential regulation of miRNA expression. It is prohibitively expensive to examine all potentially dysregulated miRNAs and traditionally, researchers have focused their efforts on the most extremely dysregulated miRNAs. These methods may overlook the contribution of less differentially expressed but more functionally relevant miRNAs. The purpose of this study was to outline a method that not only utilizes differential expression but ranks miRNAs based on the functional relevance of their targets. This work uses a networks based approach to determine the sum node degree for all experimentally verified miRNA targets to identify potential regulators of prostate cancer initiation, progression and metastasis.ResultsHere, we present a method for identifying functionally relevant miRNAs that contribute to prostate cancer development. This paper shows that miRNAs preferentially regulate highly connected, central proteins within a protein-protein interaction network. Known targets of miRNAs differentially regulated during prostate cancer progression are enriched in pathways with known involvement in tumorigenesis. To demonstrate the applicability of our method, we utilized a unique model of prostate cancer progression to identify five miRNAs that may contribute to the oncogenic state of the cell. Three of these miRNAs have been shown by other studies to have a role in cancer but their exact role in prostate cancer remains undefined.ConclusionDeveloping methods to determine which miRNAs to carry forward into biological and biochemical analyses is important as traditional approaches often overlook miRNAs that contribute to oncogenesis. Our method applied to a model of prostate cancer progression was able to identify miRNAs with roles in prostate cancer development.

FindSUMO: A PSSM-Based Method for Sumoylation Site Prediction

Post-translation modification by sumoylation is an important step in the regulation of many cellular processes. Existing programs predict sumoylation sites based on a consensus motif ( φ-K-X-E/D. However, ~23% of real SUMO sites do not match this motif, which makes prediction of sumoylation sites complicated. Here, we present a new method, FindSUMO, which predicts sumoylation sites using a position-specific scoring matrix. The comparison of FindSUMO with other two programs (SUMOsp and SUMOplot) indicates that FindSUMO accurately predicts sumoylation sites out of fewer total candidates and, in many cases, real sites with the highest scores. Overall, FindSUMO has at least the same performance as the other methods. While many improvements are expected, FindSUMO can also be applied to predict sumoylation sites that do not follow the consensus motif.

Abstract B54: miR-125b and miR-17-3p: Newly identified tumor suppressors for prostate cancer

MicroRNAs (miRs) are small molecules that post-transcriptionally regulate expression of target mRNAs and are thought to play a major role in prostate cancer (CaP) tumorigenesis. We compared miR expression in a progressive prostate cancer cell model consisting of the immortalized p69 human prostate cell line compared to M12, a highly tumorigenic, metastatic subline, and F6, a weakly tumorigenic variant using Exiqons miRCURY LNA™ miR human panels. Most notable was a decrease in expression of miR-125b and miR-17-3p, which was confirmed by single miR analysis via RT-qPCR. Previously, miR array screens have been done on RNA extracted from a variety of whole tumors. However, due to the heterogeneous nature of prostate tumors, it is impossible to determine which cell-type is contributing to differential miR expression. To address this concern, we have developed laser capture microdissection (LCM) to validate miR expression in RNA extracted from the individual cell-types (normal stroma, normal epithelial, PIN, and tumor cell of increasing Gleason score) that comprise human prostate tumors. This analysis validated that the expression of miR-17-3p and miR-125b is reduced in the prostate tumor cell compared to normal epithelium. Previously, we have shown that miR-17-3p targets vimentin and IGF1R. Restoration of miR-17-3p expression in the M12 cell line resulted in decreased cell motility and invasion in transwell (Boyden) chamber assays as well as decreased growth of subcutaneous and orthotopic tumors in male, athymic nude mice confirming miR-17-3p is behaving as a tumor suppressor. Previously, data from miRNA array screens coupled to gene arrays have been used to predict mRNA targets. This approach assumes that all miRNAs regulate gene expression by degrading their respective targets, which is not always the case. Since the end result of miRNA targeting is reduced protein synthesis, we propose that proteomics using RPMA, Reverse Phase Protein MicroArrays, is a better approach for deducing targets of miR regulation. Proteomic analysis of these same cell lines confirmed a correlative increase in pErB2/ErB3 and vimentin protein levels in cell lines where miR-125b and miR-17-3p expression are low. MiR-125b is proposed to target ErbB2/ErbB3. Interestingly, a network analysis of oncogenic miRNA targets in the prostate revealed that the EGFR family of which ErB2/ErB3 is thought to be a major driver of CaP was one of the most highly connected proteins. Currently, we are using a variety of in vitro and in vivo experiments to prove that miR-125b can function as a tumor suppressor as we have shown for miR-17-3p. Altogether this multi-factorial approach of miRNA array analysis coupled to proteomics, validated by LCM analysis of human tumor samples and additional experiments has identified two new tumor suppressors, miR-17-3p and miR-125b, which could be relevant biomarkers for CaP. Restoration of miR-17-3p and/or miR-125 expression could provide new targets/treatments to block prostate cancer progression. Citation Format: William T. Budd, Danielle E. Weaver, Valerie Calvert, Emanuel F. Petricoin, III, Joy L. Ware, Zendra E. Zehner. miR-125b and miR-17-3p: Newly identified tumor suppressors for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B54.

Abstract 261: Epidermal growth factor receptor (EGFR)-mediated upregulation of matrix metalloproteinase-1(MMP-1) in glioblastoma cell lines involves multiple signaling pathways

Glioblastoma Multiforme (GBM) is a devastating disease with an extremely poor prognosis primarily due to its invasive and infiltrative nature. Matrix metalloproteinases (MMPs) are important enzymes that regulate tumor microenvironment, alter cellular behavior and increase invasion and metastasis in wide variety of cancers. Previous studies from our laboratory have shown that MMP-1 is over expressed in GBM tissue and can influence glioma cell invasion in vitro. Since the epidermal growth factor receptor (EGFR) is amplified in human GBM, we wanted to determine the effect of EGFR activation by epidermal growth factor (EGF) on MMP-1 expression and invasion in human glioma cell lines and if so, determine the signaling mechanisms responsible for this induction. We stimulated glioma cell lines (T98 and U87) with EGF in the presence and absence of the EGFR inhibitor, AG1478. Inhibitors to multiple signaling pathways activated by EGFR induction were also used. All inhibitors tested were added to cells for 2 hrs prior to addition of EGF. In addition, transfections using AKT and STAT3 siRNA were conducted. Western Blotting was performed to examine the levels of MMP-1, EGFR, AKT, STAT3 and ERK. We observed that with EGF treatment, in addition to EGFR activation, AKT was phosphorylated, MMP-1 was induced and that in the presence of AG1478, AKT and EGFR phosphorylation was inhibited in a dose dependent manner. Importantly, MMP-1 up-regulation by EGF was abrogated in cells pretreated with AG1478. EGFR has been shown to up-regulate MAPK, PI3K and STAT3 pathways in glioma and other malignancies. Involvement of the MAPK pathway in the EGFR- mediated induction of MMP-1 was examined using the MAPK inhibitor, U0126. Only a modest decrease in MMP-1 levels was observed following treatment with EGF and U0126, suggesting potential inputs from other signaling pathways. Although there was a modest inhibition of AKT protein levels in cells treated with AKT siRNA, the levels of MMP-1 in these cells were significantly reduced. Current experiments focus on the role of AKT/PI3K and STAT3 signaling pathways in MMP-1 mediated glioma cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 261.