Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Acoustic enrichment of heterogenous circulating tumor cells and clusters from patients with metastatic prostate cancer.

Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Expression of microRNA-379 reduces metastatic spread of prostate cancer.

Introduction: Prostate cancer (PCa) is the most common type of cancer in males, and the metastatic form is a leading cause of death worldwide. There are currently no curative treatments for this subset of patients. To decrease the mortality of this disease, greater focus must be placed on developing therapeutics to reduce metastatic spread. We focus on dissemination to the bone since this is both the most common site of metastatic spread and associated with extreme pain and discomfort for patients. Our strategy is to exploit microRNAs (miRNAs) to disrupt the spread of primary PCa to the bone. Methods: PCa cell lines were transduced to overexpress microRNA-379 (miR-379). These transduced PCa cells were assessed using cell growth, migration, colony formation and adhesion assays. We also performed in vivo intracardiac injections to look at metastatic spread in NSG mice. A cytokine array was also performed to identify targets of miR-379 that may drive metastatic spread. Results: PCa cells with increased levels of miR-379 showed a significant decrease in proliferation, migration, colony formation, and adhesion to bone cells in vitro. In vivo miR-379 overexpression in PC3 cells significantly decreased metastatic spread to bone and reduced levels of miR-379 were seen in patients with metastases. We identified GDF-15 to be secreted from osteoblasts when grown in conditioned media from PCa cells with reduced miR-379 levels. Discussion: Taken together, our in vitro and in vivo functional assays support a role for miR-379 as a tumour suppressor. A potential mechanism is unravelled whereby miR-379 deregulation in PCa cells affects the secretion of GDF-15 from osteoblasts which in turn facilitates the metastatic establishment in bone. Our findings support the potential role of miR-379 as a therapeutic solution for prostate cancer.

Functional consequences of A-to-I editing of miR-379 in prostate cancer cells.

Prostate cancer is the predominant cause of cancer in men, but there is still a lack of biomarkers and treatments for metastatic spread. The initial promise of microRNAs to provide avenues to solve these problems has been dampened by the realisation that microRNAs co-exist in multiple functionally distinct isoforms, for example due to A-to-I editing. We recently found that A-to-I-editing of microRNA-379 (miR-379) was associated with prostate cancer, and that only the unedited isoform was negatively correlated with aggressive disease. Here, we set out to decipher the biological effects of unedited and edited miR-379 in prostate cancer cells. After transfection of four different prostate cancer cell lines with isoform-specific miR-379 mimics, we performed assays for cell growth, colony formation, migration, cell-cell adhesion, and analysed epithelial-mesenchymal transition (EMT) and stemness markers. We found that unedited miR-379 affected cell growth, with a promoting function in androgen receptor (AR)-negative cells and an inhibiting effect in AR-positive cells. This is supported by our in silico analysis that found unedited miR-379 targets are predicted to be predominantly involved in cellular proliferation whereas the targets of edited miR-379 are not. We further found that both miR-379 isoforms could promote colony formation, migration, and cell-cell adhesion. Overall, our data suggests that editing of miR-379 attenuates the growth-suppressive function of unedited miR-379 in androgen-sensitive prostate cancer cells, thereby promoting tumor growth.

Functional In Vivo Screening Identifies microRNAs Regulating Metastatic Dissemination of Prostate Cancer Cells to Bone Marrow.

Distant metastasis is the major cause of cancer-related deaths in men with prostate cancer (PCa). An in vivo functional screen was used to identify microRNAs (miRNAs) regulating metastatic dissemination of PCa cells. PC3 cells transduced with pooled miRZiP™ lentivirus library (anti-miRNAs) were injected intraprostatic to 13 NSG mice followed by targeted barcode/anti-miR sequencing. PCa cells in the primary tumours showed a homogenous pattern of anti-miRNAs, but different anti-miRNAs were enriched in liver, lung, and bone marrow, with anti-miR-379 highly enriched in the latter. The bone metastasis-promoting phenotype induced by decreased miR-379 levels was also confirmed in a less metastatic PCa cell line, 22Rv1, where all mice injected intracardially with anti-miR-379-22Rv1 cells developed bone metastases. The levels of miR-379 were found to be lower in bone metastases compared to primary tumours and non-cancerous prostatic tissue in a patient cohort. In vitro functional studies suggested that the mechanism of action was that reduced levels of miR-379 gave an increased colony formation capacity in conditions mimicking the bone microenvironment. In conclusion, our data suggest that specific miRNAs affect the establishment of primary tumours and metastatic dissemination, with a loss of miR-379 promoting metastases in bone.

Two-Step Acoustophoresis Separation of Live Tumor Cells from Whole Blood.

There is an unmet clinical need to extract living circulating tumor cells (CTCs) for functional studies and in vitro expansion to enable drug testing and predict responses to therapy in metastatic cancer. Here, we present a novel two-step acoustophoresis (A2) method for isolation of unfixed, viable cancer cells from red blood cell (RBC) lysed whole blood. The A2 method uses an initial acoustofluidic preseparation step to separate cells based on their acoustic mobility. This acoustofluidic step enriches viable cancer cells in a central outlet, but a significant number of white blood cells (WBCs) remain in the central outlet fraction due to overlapping acoustophysical properties of these viable cells. A subsequent purging step was employed to remove contaminating WBCs through negative selection acoustophoresis with anti-CD45-functionalized negative acoustic contrast particles. We processed 1 mL samples of 1:1 diluted RBC lysed whole blood mixed with 10 000 DU145 cells through the A2 method. Additional experiments were performed using 1000 DU145 cells spiked into 1.5 × 106 WBCs in 1 mL of buffer to further elucidate the dynamic range of the method. Using samples with 10 000 DU145 cells, we obtained 459 ± 188-fold depletion of WBC and 42% recovery of viable cancer cells. Based on spiked samples with 1000 DU145 cells, our cancer cell recovery was 28% with 247 ± 156-fold WBC depletion corresponding to a depletion efficacy of ≥99.5%. The novel A2 method provides extensive elimination of WBCs combined with the gentle recovery of viable cancer cells suitable for downstream functional analyses and in vitro culture.

Quantification of microRNA editing using two-tailed RT-qPCR for improved biomarker discovery.

Even though microRNAs have been viewed as promising biomarkers for years, their clinical implementation is still lagging far behind. This is in part due to the lack of RT-qPCR technologies that can differentiate between microRNA isoforms. For example, A-to-I editing of microRNAs through adenosine deaminase acting on RNA (ADAR) enzymes can affect their expression levels and functional roles, but editing isoform-specific assays are not commercially available. Here, we describe RT-qPCR assays that are specific for editing isoforms, using microRNA-379 (miR-379) as a model. The assays are based on two-tailed RT-qPCR, and we show them to be compatible both with SYBR Green and hydrolysis-based chemistries, as well as with both qPCR and digital PCR. The assays could readily detect different miR-379 editing isoforms in various human tissues as well as changes of editing levels in ADAR-overexpressing cell lines. We found that the miR-379 editing frequency was higher in prostate cancer samples compared to benign prostatic hyperplasia samples. Furthermore, decreased expression of unedited miR-379, but not edited miR-379, was associated with treatment resistance, metastasis, and shorter overall survival. Taken together, this study presents the first RT-qPCR assays that were demonstrated to distinguish A-to-I-edited microRNAs, and shows that they can be useful in the identification of biomarkers that previously have been masked by other isoforms.

High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer.

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 µl of urine for small RNA sequencing in a robust and disease discriminatory manner.

Regulation of cell-cell adhesion in prostate cancer cells by microRNA-96 through upregulation of E-Cadherin and EpCAM.

Prostate cancer is one of the most common cancers in men, yet the biology behind lethal disease progression and bone metastasis is poorly understood. In this study, we found elevated levels of microRNA-96 (miR-96) in prostate cancer bone metastasis samples. To determine the molecular mechanisms by which miR-96 deregulation contributes to metastatic progression, we performed an Argonaute2-immunoprecipitation assay, in which mRNAs associated with cell-cell interaction were enriched. The expression of two cell adhesion molecules, E-Cadherin and EpCAM, was upregulated by miR-96, and potential targets sites were identified in the coding sequences of their mRNAs. We further showed that miR-96 enhanced cell-cell adhesion between prostate cancer cells as well as their ability to bind to osteoblasts. Our findings suggest that increased levels of miR-96 give prostate cancer cells an advantage at forming metastases in the bone microenvironment due to increased cell-cell interaction. We propose that miR-96 promotes bone metastasis in prostate cancer patients by facilitating the outgrowth of macroscopic tumours in the bone.

Acoustic Enrichment of Extracellular Vesicles from Biological Fluids.

Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 µL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.

Reducing WBC background in cancer cell separation products by negative acoustic contrast particle immuno-acoustophoresis.

Cancer cells display acoustic properties enabling acoustophoretic separation from white blood cells (WBCs) with 2-3 log suppression of the WBC background. However, a subset of WBCs has overlapping acoustic properties with cancer cells, which is why label-free acoustophoretic cancer cell isolation needs additional purification prior to analysis. This paper reports for the first time a proof of concept for continuous flow acoustophoretic negative selection of WBCs from cancer cells using negative acoustic contrast elastomeric particles (EPs) activated with CD45-antibodies that specifically bind to WBCs. The EP/WBC complexes align at the acoustic pressure anti-nodes along the channel walls while unbound cancer cells focus to the pressure node in the channel center, enabling continuous flow based depletion of WBC background in a cancer cell product. The method does not provide a single process solution for the CTC separation challenge, but provides an elegant part to a multi-step process by further reducing the WBC background in cancer cell separation products derived from an initial step of label-free acoustophoresis. We report the recorded performance of the negative selection immuno-acoustophoretic WBC depletion and cancer cell recovery. To eliminate the negative impact of the separation due to the known problems of aggregation of negative acoustic contrast particles along the sidewalls of the acoustophoresis channel and to enable continuous separation of EP/WBC complexes from cancer cells, a new acoustic actuation method has been implemented where the ultrasound frequency is scanned (1.991MHz ± 100 kHz, scan rate 200 kHz ms-1). Using this frequency scanning strategy EP/WBC complexes were acoustophoretically separated from mixtures of WBCs spiked with breast and prostate cancer cells (DU145 and MCF-7). An 86-fold (MCF-7) and 52-fold (DU145) reduction of WBCs in the cancer cell fractions were recorded with separation efficiencies of 98.6% (MCF-7) and 99.7% (DU145) and cancer cell recoveries of 89.8% (MCF-7) and 85.0% (DU145).

Clinical-Scale Cell-Surface-Marker Independent Acoustic Microfluidic Enrichment of Tumor Cells from Blood.

Enumeration of circulating tumor cells (CTCs) predicts overall survival and treatment response in metastatic cancer, but as many commercialized assays isolate CTCs positive for epithelial cell markers alone, CTCs with little or no epithelial cell adhesion molecule (EpCAM) expression stay undetected. Therefore, CTC enrichment and isolation by label-free methods based on biophysical rather than biochemical properties could provide a more representative spectrum of CTCs. Here, we report on a clinical-scale automated acoustic microfluidic platform processing 5 mL of erythrocyte-depleted paraformaldehyde (PFA)-fixed blood (diluted 1:2) at a flow rate of 75 µL/min, recovering 43/50 (86 ± 2.3%) breast cancer cell line cells (MCF7), with 0.11% cancer cell purity and 162-fold enrichment in close to 2 h based on intrinsic biophysical cell properties. Adjustments of the voltage settings aimed at higher cancer cell purity in the central outlet provided 0.72% cancer cell purity and 1445-fold enrichment that resulted in 62 ± 8.7% cancer cell recovery. Similar rates of cancer-cell recovery, cancer-cell purity, and fold-enrichment were seen with both prostate cancer (DU145, PC3) and breast cancer (MCF7) cell line cells. We identified eosinophil granulocytes as the predominant white blood cell (WBC) contaminant (85%) in the enriched cancer-cell fraction. Processing of viable cancer cells in erythrocyte-depleted blood provided slightly reduced results as to fixed cells (77% cancer cells in the enriched cancer cell fraction, with 0.2% WBC contamination). We demonstrate feasibility of enriching either PFA-fixed or viable cancer cells with a clinical-scale acoustic microfluidic platform that can be adjusted to meet requirements for either high cancer-cell recovery or higher purity and can process 5 mL blood samples in close to 2 h.

microRNAs as cancer therapeutics: A step closer to clinical application.

During the last decades, basic and translational research has enabled great improvements in the clinical management of cancer. However, scarcity of complete remission and many drug-induced toxicities are still a major problem in the clinics. Recently, microRNAs (miRNAs) have emerged as promising therapeutic targets due to their involvement in cancer development and progression. Their extraordinary regulatory potential, which enables regulation of entire signalling networks within the cells, makes them an interesting tool for the development of cancer therapeutics. In this review we will focus on miRNAs with experimentally proven therapeutic potential, and discuss recent advances in the technical development and clinical evaluation of miRNA-based therapeutic agents.

The Potential of MicroRNAs as Prostate Cancer Biomarkers.

CONTEXT: Short noncoding RNAs known as microRNAs (miRNAs) control protein expression through the degradation of RNA or the inhibition of protein translation. The miRNAs influence a wide range of biologic processes and are often deregulated in cancer. This family of small RNAs constitutes potentially valuable markers for the diagnosis, prognosis, and therapeutic choices in prostate cancer (PCa) patients, as well as potential drugs (miRNA mimics) or drug targets (anti-miRNAs) in PCa management. OBJECTIVE: To review the currently available data on miRNAs as biomarkers in PCa and as possible tools for early detection and prognosis. EVIDENCE ACQUISITION: A systematic review was performed searching the PubMed database for articles in English using a combination of the following terms: microRNA, miRNA, cancer, prostate cancer, miRNA profiling, diagnosis, prognosis, therapy response, and predictive marker. EVIDENCE SYNTHESIS: We summarize the existing literature regarding the profiling of miRNA in PCa detection, prognosis, and response to therapy. The articles were reviewed with the main goal of finding a common recommendation that could be translated from bench to bedside in future clinical practice. CONCLUSIONS: The miRNAs are important regulators of biologic processes in PCa progression. A common expression profile characterizing each tumor subtype and stage has still not been identified for PCa, probably due to molecular heterogeneity as well as differences in study design and patient selection. Large-scale studies that should provide additional important information are still missing. Further studies, based on common clinical parameters and guidelines, are necessary to validate the translational potential of miRNAs in PCa clinical management. Such common signatures are promising in the field and emerge as potential biomarkers. PATIENT SUMMARY: The literature shows that microRNAs hold potential as novel biomarkers that could aid prostate cancer management, but additional studies with larger patient cohorts and common guidelines are necessary before clinical implementation.

Non-coding RNAs in Prostate Cancer: From Discovery to Clinical Applications.

Prostate cancer is a heterogeneous disease for which the molecular mechanisms are still not fully elucidated. Prostate cancer research has traditionally focused on genomic and epigenetic alterations affecting the proteome, but over the last decade non-coding RNAs, especially microRNAs, have been recognized to play a key role in prostate cancer progression. A considerable number of individual microRNAs have been found to be deregulated in prostate cancer and their biological significance elucidated in functional studies. This review will delineate the current advances regarding the involvement of microRNAs and their targets in prostate cancer biology as well as their potential usage in the clinical management of the disease. The main focus will be on microRNAs contributing to initiation and progression of prostate cancer, including androgen signalling, cellular plasticity, stem cells biology and metastatic processes. To conclude, implications on potential future microRNA-based therapeutics based on the recent advances regarding the interplay between microRNAs and their targets are discussed.

The Molecular Evolution of Castration-resistant Prostate Cancer.

CONTEXT: Androgen deprivation therapy (ADT) is the backbone of treatment for advanced prostate cancer. However, castration-resistant prostate cancer (CRPC) nearly invariably develops through a range of different molecular mechanisms accompanied by progression to a more aggressive phenotype. OBJECTIVE: To understand the key molecular mechanisms leading to CRPC and the functional implications of this progression. Understanding molecular evolutionary mechanisms in CRPC is essential for the development of novel curative therapeutic approaches. EVIDENCE ACQUISITION: A systematic literature search to identify relevant original articles was conducted using PubMed. Findings verified in independent studies and supported by in vivo data were prioritised. From the eligible collection, 50 papers were selected. EVIDENCE SYNTHESIS: The majority of CRPC tumours harbour alterations in the androgen receptor (AR) at the DNA, RNA, and/or protein level, and/or other alterations involving the AR signalling pathway, so this central molecule is the focus of this review. To survive and resume growth despite low levels of circulating androgens, prostate cancer cells can also adapt androgen synthesis or induce alternative pathways. CONCLUSIONS: Despite more efficient ADT strategies, most evidence points to persistent AR signalling as a major mechanism of progression to CRPC. Resistance due to transdifferentiation or AR independence is also emerging as a mechanism of resistance. The diversity of potential resistance mechanisms supports the need for combination treatment and serial monitoring for adaptive treatment strategies. PATIENT SUMMARY: In this review, we summarise how prostate cancer cells evade androgen deprivation therapy and become more aggressive. Defining the molecular mechanisms will be critical for the development of new treatment approaches and hence improved survival.

The Axl-Regulating Tumor Suppressor miR-34a Is Increased in ccRCC but Does Not Correlate with Axl mRNA or Axl Protein Levels.

BACKGROUND: High expression of the receptor tyrosine kinase Axl is associated with poor prognosis in patients with Renal Cell Carcinoma (RCC), the most common malignancy of the kidney. The miR-34a has been shown to directly regulate Axl in cancer cells. The miR-34a is a mediator of p53-dependent tumor suppression, and low expression of miR-34a has been associated with worse prognosis in several cancers. Our aim was to elucidate whether miR-34a or the other members of the miR-34 family (miR-34b/c) regulate Axl in RCC. METHODOLOGY AND RESULTS: Using western blot, flow cytometry, and RT-qPCR, we showed that Axl mRNA and protein are downregulated in 786-O cells by miR-34a and miR-34c but not by miR-34b. A luciferase reporter assay demonstrated direct interaction between the Axl 3' UTR and miR-34a and miR-34c. The levels of miR-34a/b/c were measured in tumor tissue in a cohort of 198 RCC patients, and the levels of miR-34a were found to be upregulated in clear cell RCC (ccRCC) tumors, but not associated with patient outcome. Neither of the miR-34 family members correlated with Axl mRNA, soluble Axl protein in serum, nor with immunohistochemistry of Axl in tumor tissue. In addition, we measured mRNA levels of a known miR-34a target, HNF4A, and found the HNF4A levels to be decreased in ccRCC tumors, but unexpectedly correlated positively rather than negatively with miR-34a. CONCLUSIONS: Although miR-34a and miR-34c can regulate Axl expression in vitro, our data indicates that the miR-34 family members are not the primary regulators of Axl expression in RCC.

miR-145 suppress the androgen receptor in prostate cancer cells and correlates to prostate cancer prognosis.

Androgen signalling through the androgen receptor (AR) is essential for prostate cancer initiation, progression and transformation to the lethal castration-resistant state. The aim of this study was to characterize the mechanisms by which miR-145 deregulation contribute to prostate cancer progression. The miR-145 levels, measured by quantitative reverse transcription-polymerase chain reaction, were found to inversely correlate with occurrence of metastases, survival and androgen deprivation therapy response in a well-characterized prostate cancer cohort. Introduction of ectopic miR-145 in prostate cancer cells generated an inhibitory effect on the AR at both transcript and protein levels as well as its activity and downstream targets prostate-specific antigen (PSA), kallikrein-related peptidase 2 and TMPRSS2. The regulation was shown to be mediated by direct binding using Ago2-specific immunoprecipitation, but there was also indication of synergetic AR activation. These findings were verified in clinical prostate specimens by demonstrating inverse correlations between miR-145 and AR expression as well as serum PSA levels. In addition, miR-145 was found to regulate androgen-dependent cell growth in vitro. Our findings put forward novel possibilities of therapeutic intervention, as miR-145 potentially could decrease both the stem cells and the AR expressing bulk of the tumour and hence reduce the transformation to the deadly castration-resistant form of prostate cancer.

MicroRNA-135b regulates ERα, AR and HIF1AN and affects breast and prostate cancer cell growth.

MicroRNAs (miRNAs) regulate a wide range of cellular signaling pathways and biological processes in both physiological and pathological states such as cancer. We have previously identified miR-135b as a direct regulator of androgen receptor (AR) protein level in prostate cancer (PCa). We wanted to further explore the relationship of miR-135b to hormonal receptors, particularly estrogen receptor α (ERα). Here we show that miR-135b expression is lower in ERα-positive breast tumors as compared to ERα-negative samples in two independent breast cancer (BCa) patient cohorts (101 and 1302 samples). Additionally, the miR-135b expression is higher in AR-low PCa patient samples (47 samples). We identify ERα as a novel miR-135b target by demonstrating miR-135b binding to the 3'UTR of the ERα and decreased ERα protein and mRNA level upon miR-135b overexpression in BCa cells. MiR-135b reduces proliferation of ERα-positive BCa cells MCF-7 and BT-474 as well as AR-positive PCa cells LNCaP and 22Rv1 when grown in 2D. To identify other genes regulated by miR-135b we performed gene expression studies and found a link to the hypoxia inducible factor 1α (HIF1α) pathway. We show that miR-135b influences the protein level of the inhibitor for hypoxia inducible factor 1α (HIF1AN) and is able to bind to HIF1AN 3'UTR. Our study demonstrates that miR-135b regulates ERα, AR and HIF1AN protein levels through interaction with their 3'UTR regions, and proliferation in ERα-positive BCa and AR-positive PCa cells.

Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer.

The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.