Nicholas H. Farina

Standardizing Analysis of Circulating MicroRNA: Clinical and Biological Relevance

Circulating microRNAs (c‐miRNAs) provide a new dimension as clinical biomarkers for disease diagnosis, progression, and response to treatment. However, the discovery of individual miRNAs from biofluids that reliably reflect disease states is in its infancy. The highly variable nature of published studies exemplifies a need to standardize the analysis of miRNA in circulation. Here, we show that differential sample handling of serum leads to inconsistent and incomparable results. We present a standardized method of RNA isolation from serum that eliminates multiple freeze/thaw cycles, provides at least three normalization mechanisms, and can be utilized in studies that compare both archived and prospectively collected samples. It is anticipated that serum processed as described here can be profiled, either globally or on a gene by gene basis, for c‐miRNAs and other non‐coding RNA in the circulation to reveal novel, clinically relevant epigenetic signatures for a wide range of diseases. J. Cell. Biochem. 115: 805–811, 2014.

Antagonizing miR-218-5p attenuates Wnt signaling and reduces metastatic bone disease of triple negative breast cancer cells

Wnt signaling is implicated in bone formation and activated in breast cancer cells promoting primary and metastatic tumor growth. A compelling question is whether osteogenic miRNAs that increase Wnt activity for bone formation are aberrantly expressed in breast tumor cells to support metastatic bone disease. Here we report that miR-218-5p is highly expressed in bone metastases from breast cancer patients, but is not detected in normal mammary epithelial cells. Furthermore, inhibition of miR-218-5p impaired the growth of bone metastatic MDA-MB-231 cells in the bone microenvironment in vivo. These findings indicate a positive role for miR-218-5p in bone metastasis. Bioinformatic and biochemical analyses revealed a positive correlation between aberrant miR-218-5p expression and activation of Wnt signaling in breast cancer cells. Mechanistically, miR-218-5p targets the Wnt inhibitors Sclerostin (SOST) and sFRP-2, which highly enhances Wnt signaling. In contrast, delivery of antimiR-218-5p decreased Wnt activity and the expression of metastasis-related genes, including bone sialoprotein (BSP/IBSP), osteopontin (OPN/SPP1) and CXCR-4, implicating a Wnt/miR-218-5p regulatory network in bone metastatic breast cancer. Furthermore, miR-218-5p also mediates the Wnt-dependent up-regulation of PTHrP, a key cytokine promoting cancer-induced osteolysis. Antagonizing miR-218-5p reduced the expression of PTHrP and Rankl, inhibited osteoclast differentiation in vitro and in vivo, and prevented the development of osteolytic lesions in a preclinical metastasis model. We conclude that pathological elevation of miR-218-5p in breast cancer cells activates Wnt signaling to enhance metastatic properties of breast cancer cells and cancer-induced osteolytic disease, suggesting that miR-218-5p could be an attractive therapeutic target for preventing disease progression.

tsRNA signatures in cancer

Significance We found that tRNA-derived small RNAs (tsRNAs) are dysregulated in many cancers and that their expression is modulated during cancer development and staging. Indeed, activation of oncogenes and inactivation of tumor suppressors lead to a dysregulation of specific tsRNAs, and tsRNA-KO cells display a specific change in gene-expression profile. Thus tsRNAs could be key effectors in cancer-related pathways. These results indicate active crosstalk between tsRNAs and oncogenes and suggest that tsRNAs could be useful markers for diagnosis or targets for therapy. Additionally, ts-46 and ts-47 affect cell growth in lung cancer cell lines, further confirming the involvement of tsRNAs in cancer pathogenesis. Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter “tsRNAs”) is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called “ts-101” and “ts-53,” respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.

MicroRNA-378-mediated suppression of Runx1 alleviates the aggressive phenotype of triple-negative MDA-MB-231 human breast cancer cells.

The Runx1 transcription factor, known for its essential role in normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx1 increases concomitantly with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant downregulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mice and in human breast cancer cell lines MCF7 and triple-negative MDA-MB-231 that represent early- and late-stage diseases, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3′ untranslated region (3′UTR) and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion, while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late-stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.

A microRNA/Runx1/Runx2 network regulates prostate tumor progression from onset to adenocarcinoma in TRAMP mice

While decades of research have identified molecular pathways inducing and promoting stages of prostate cancer malignancy, studies addressing dynamic changes of cancer-related regulatory factors in a prostate tumor progression model are limited. Using the TRAMP mouse model of human prostate cancer, we address mechanisms of deregulation for the cancer-associated transcription factors, Runx1 and Runx2 by identifying microRNAs with reciprocal expression changes at six time points during 33 weeks of tumorigenesis. We molecularly define transition stages from PIN lesions to hyperplasia/neoplasia and progression to adenocarcinoma by temporal changes in expression of human prostate cancer markers, including the androgen receptor and tumor suppressors, Nkx3.1 and PTEN. Concomitant activation of PTEN, AR, and Runx factors occurs at early stages. At late stages, PTEN and AR are downregulated, while Runx1 and Runx2 remain elevated. Loss of Runx-targeting microRNAs, miR-23b-5p, miR-139-5p, miR-205-5p, miR-221-3p, miR-375-3p, miR-382-5p, and miR-384-5p, contribute to aberrant Runx expression in prostate tumors. Our studies reveal a Runx/miRNA interaction axis centered on PTEN-PI3K-AKT signaling. This regulatory network translates to mechanistic understanding of prostate tumorigenesis that can be developed for diagnosis and directed therapy.

Development of a predictive miRNA signature for breast cancer risk among high-risk women

Significant limitations exist in our ability to predict breast cancer risk at the individual level. Circulating microRNAs (C-miRNAs) have emerged as measurable biomarkers (liquid biopsies) for cancer detection. We evaluated the ability of C-miRNAs to identify women most likely to develop breast cancer by profiling miRNA from serum obtained long before diagnosis. 24 breast cancer cases and controls (matched for risk and age) were identified from women enrolled in the High-Risk Breast Program at the UVM Cancer Center. Isolated RNA from serum was profiled for over 2500 human miRNAs. The miRNA expression data were input into a stepwise linear regression model to discover a multivariable miRNA signature that predicts long-term risk of breast cancer. 25 candidate miRNAs were identified that individually classified cases and controls based on statistical methodologies. A refined 6-miRNA risk-signature was discovered following regression modeling that distinguishes cases and controls (AUC0.896, CI 0.804-0.988) in this cohort. A functional relationship between miRNAs that cluster together when cases are contrasted against controls was suggested and confirmed by pathway analyses. The discovered 6 miRNA risk-signature can discriminate high-risk women who ultimately develop breast cancer from those who remain cancer-free, improving current risk assessment models. Future studies will focus on functional analysis of the miRNAs in this signature and testing in larger cohorts. We propose that the combined signature is highly significant for predicting cancer risk, and worthy of further screening in larger, independent clinical cohorts.

Dissection of Individual Prostate Lobes in Mouse Models of Prostate Cancer to Obtain High Quality RNA

Genetically engineered mouse models of prostate cancer allow for study of disease progression from localized tumor formation through distal metastasis. The anatomy of the mouse prostate differs dramatically from the human prostate, being composed of four lobe pairs (anterior, dorsal, lateral, and ventral), making the identification and dissection technically challenging. Although the entire murine prostate and surrounding tissue, including urethra, bladder, seminal vesicles, and associated adipose tissue, can be quickly dissected for en bloc analysis, it is necessary to isolate individual prostate lobes for gene expression studies elucidating the molecular mechanisms of prostate cancer. The procedure as described here includes full color images, allowing the researcher to appreciate the unique prostate morphology and tissue manipulation required to harvest individual prostate lobes. Along with removing all extraneous tissue, the procedure allows for direct comparison of the different prostate lobes by established downstream techniques. Importantly, high quality RNA required for next‐generation gene expression analysis can only consistently be obtained from ventral and lateral lobes. Finally, preclinical studies using prostate targeted therapies can be monitored specifically in individual prostate lobes for histological and gene expression studies. J. Cell. Physiol. 232: 14–18, 2017.

Nanoparticle‐based targeted cancer strategies for non‐invasive prostate cancer intervention

Prostate cancer is screened by testing circulating levels of the prostate‐specific antigen (PSA) biomarker, monitoring changes over time, or a digital rectal exam. Abnormal results often lead to prostate biopsy. Prostate cancer positive patients are stratified into very low‐risk, low‐risk, intermediate‐risk, and high‐risk, based on clinical classification parameters, to assess therapy options. However, there remains a gap in our knowledge and a compelling need for improved risk stratification to inform clinical decisions and reduce both over‐diagnosis and over‐treatment. Further, current strategies for clinical intervention do not distinguish clinically aggressive prostate cancer from indolent disease. This mini‐review takes advantage of a large number of functionally characterized microRNAs (miRNA), epigenetic regulators of prostate cancer, that define prostate cancer cell activity, tumor stage, and circulate as biomarkers to monitor disease progression. Nanoparticles provide an effective platform for targeted delivery of miRNA inhibitors or mimics specifically to prostate tumor cells to inhibit cancer progression. Several prostate–specific transmembrane proteins expressed at elevated levels in prostate tumors are under investigation for targeting therapeutic agents to prostate cancer cells. Given that prostate cancer progresses slowly, circulating miRNAs can be monitored to identify tumor progression in indolent disease, allowing identification of miRNAs for nanoparticle intervention before the crucial point of transition to aggressive disease. Here, we describe clinically significant and non‐invasive intervention nanoparticle strategies being used in clinical trials for drug and nucleic acid delivery. The advantages of mesoporous silica‐based nanoparticles and a number of candidate miRNAs for inhibition of prostate cancer are discussed.

Abstract LB-251: Development of a circulating microRNA risk signature predictive of breast cancer diagnosis among high-risk women

Introduction: Current models predicting short and long term risk of breast cancer based on clinical factors, such as family history, have significant limitations. Circulating microRNAs (c-miRNAs) have emerged as measurable ‘liquid biopsy’ biomarkers for cancer detection. However, the value of c-miRNAs, an epigenetic regulator of protein levels, as a novel prediction tool of breast cancer development years before detection has not been explored. Methods: Twenty-four breast cancer cases and matched were selected from a source population cohort of 584 unaffected women enrolled in the High Risk Breast Program at the UVM Cancer Center. Women with a mutation in BRCA1 or BRCA2 were excluded. Cases were women diagnosed with stage I-III breast cancer. One control was matched to each case on age (± 3.5 yrs) and reason for high-risk status (family history or benign breast disease). Clinical data and serum samples were collected at study enrollment and at subsequent cancer-free 4-year intervals. RNA was isolated from serum and over 2500 mature human miRNAs were profiled (Affymetrix microRNA v4.0 microarray). Two sets of candidate miRNAs were identified that distinguished cases from controls and a multivariate risk score for each set calculated using Cox regression models. Set 1: 20 miRNAs with greatest area under the receiver operating characteristic curve (AUC) after univariate regression. Set 2: 19 miRNAs with significantly different expression (ANOVA p Results: We identified 2 panels of c-miRNAs that distinguish high-risk unaffected women who later develop breast cancer from those remaining cancer-free; the AUC values of 0.896 and 0.870, greatly exceed modeled risk based on clinical parameters (Gail model AUC = 0.497; Claus model AUC = 0.507; IBIS model AUC = 0.503). Panel 1: 6 model-selected c-miRNAs from set 1. Panel 2: 5 model-selected c-miRNAs from set 2. Panels 1 and 2 included 9 c-miRNAs; 2 shared, 4 unique to panel 1, and 3 unique to panel 2. Of these 9 c-miRNAs, 6 are detected at lower levels in serum from women going onto develop breast cancer while 3 are elevated in cases as compared to cancer-free women. Pathway analysis for modeled miRNAs revealed roles in many cancer-related pathways and biological functions including Regulation of EMT, Molecular Mechanisms of Cancer, and malignant solid tumor. Conclusions: In contrast to clinical breast cancer risk models, the identified miRNA panels perform well at distinguishing cases from controls, with AUCs approaching 0.9. While these data require validation, this non-invasive ‘risk signature’ of c-miRNAs will help identify high-risk women who will most benefit from enhanced screening and prevention strategies. Pathways targeted by ‘risk signature’ c-miRNAs may provide insight into mechanisms promoting breast cancer development. Citation Format: Nicholas H. Farina, Jon E. Ramsey, Melissa E. Sands, Tiffany J. Rounds, David J. Shirley, Thomas P. Ahern, Janet L. Stein, Gary S. Stein, Jane B. Lian, Marie E. Wood. Development of a circulating microRNA risk signature predictive of breast cancer diagnosis among high-risk women [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 LB-251. doi:10.1158/1538-7445.AM2017-LB-251

Abstract 3142: Circulating microRNAs can identify cancer-free women at risk for breast cancer

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: MicroRNAs (miRNAs) are well documented to regulate cancer cell activity by modulating signaling pathways to promote disease onset and progression. Recent evidence correlates miRNAs measured in the serum of breast cancer (BCa) patients to expression in breast tumors. Thus, miRNAs are important biomarkers for stages of tumor development. However, no studies have evaluated the potential of circulating miRNAs (c-miRNAs) as risk biomarkers that predict BCa development years before tumor identification. Objective: Use global miRNA analysis of serum from at-risk but pre-cancerous women who have subsequently been diagnosed with BCa or remain cancer-free to 1) identify expression levels of miRNAs with known BCa association, 2) define cohorts of c-miRNAs differentiating women later developing cancer from cancer-free women with similar clinical risk, and 3) provide insight into c-miRNA mechanisms that promote BCa development. Methods: Subjects were identified from a cohort of 571 women enrolled in the High Risk Breast Program at the UVM Cancer Center; 35 diagnosed with BCa since enrollment. Enrollment criteria included no prior cancer and increased BCa risk due to family history, benign breast disease, prior irradiation for Hodgkins disease, known pathogenic abnormality, and/or > 20% lifetime modeled BCa risk score. Clinical data and serum are collected at study enrollment and subsequent cancer-free 4-year interval visits. 28 BCa cases were matched to controls for risk factor and age (+/- 5 yrs) at serum sampling (DCIS, Stage IV BCa, BRCA1/2 excluded). RNA was isolated from 68 serum samples representing 52 individual women following a standardized protocol (Farina et al J Cell Biochem 2014) and over 2500 mature human miRNAs profiled on Affymetrix microRNA v4.0 microarrays. Principal component analyses (PCA), hierarchical clustering, differential expression testing, gene ontology, and biological pathway analyses were performed. Results: Our analyses show that women who developed BCa (largely hormone receptor positive, HER2 and lymph node negative) share similar patterns of miRNA expression that differ from women who remain cancer-free. PCA, based on a subset of dynamic miRNAs, segregates serum samples into 3 groups: cancer, cancer-free, and overlapped. Several miRNAs consistently cluster together when cases are contrasted against controls suggesting a functional relationship in providing an environment ideal for BCa development. Pathway analyses of miRNA gene targets reveal enriched PDGF, Wnt, Ras, and MAPK signaling pathways. Conclusion: These data suggest that a cohort of dynamic c-miRNAs discriminates at-risk women who will develop BCa from those who will remain cancer-free. While we consider these findings preliminary until a larger sample set is screened, we conclude that circulating miRNAs will become valuable biomarkers of BCa risk. Citation Format: Nicholas H. Farina, Jon E. Ramsey, Melissa E. Sands, Tiffany J. Rounds, Janet L. Stein, Gary S. Stein, Jane B. Lian, Marie E. Wood. Circulating microRNAs can identify cancer-free women at risk for breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3142.