Merja Perälä

miRNA-mRNA Integrated Analysis Reveals Roles for miRNAs in Primary Breast Tumors

Introduction Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information. Methods We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods. Results We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process. Conclusion This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.

Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines.

Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-α (ERα), as validated by western blotting and quantitative real time–PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERα-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERα in 3′-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERα, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERα-negative as compared with ERα-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERα signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.

A Mutation in COL9A1 Causes Multiple Epiphyseal Dysplasia: Further Evidence for Locus Heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.

High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram as an Inhibitor of Prostate Cancer Cell Growth

Purpose: To identify novel therapeutic opportunities for patients with prostate cancer, we applied high-throughput screening to systematically explore most currently marketed drugs and drug-like molecules for their efficacy against a panel of prostate cancer cells. Experimental Design: We carried out a high-throughput cell-based screening with proliferation as a primary end-point using a library of 4,910 drug-like small molecule compounds in four prostate cancer (VCaP, LNCaP, DU 145, and PC-3) and two nonmalignant prostate epithelial cell lines (RWPE-1 and EP156T). The EC50 values were determined for each cell type to identify cancer selective compounds. The in vivo effect of disulfiram (DSF) was studied in VCaP cell xenografts, and gene microarray and combinatorial studies with copper or zinc were done in vitro for mechanistic exploration. Results: Most of the effective compounds, including antineoplastic agents, were nonselective and found to inhibit both cancer and control cells in equal amounts. In contrast, histone deacetylase inhibitor trichostatin A, thiram, DSF, and monensin were identified as selective antineoplastic agents that inhibited VCaP and LNCaP cell proliferation at nanomolar concentrations. DSF reduced tumor growth in vivo, induced metallothionein expression, and reduced DNA replication by downregulating MCM mRNA expression. The effect of DSF was potentiated by copper in vitro. Conclusions: We identified three novel cancer-selective growth inhibitory compounds for human prostate cancer cells among marketed drugs. We then validated DSF as a potential prostate cancer therapeutic agent. These kinds of pharmacologically well-known molecules can be readily translated to in vivo preclinical studies and clinical trials. (Clin Cancer Res 2009;15(19):6070–8)

High-throughput screens identify microRNAs essential for HER2 positive breast cancer cell growth

MicroRNAs (miRNAs) are non‐coding RNAs regulating gene expression post‐transcriptionally. We have characterized the role of miRNAs in regulating the human epidermal growth factor receptor 2 (HER2)‐pathway in breast cancer. We performed miRNA gain‐of‐function assays by screening two HER2 amplified cell lines (KPL‐4 and JIMT‐1) with a miRNA mimic library consisting of 810 human miRNAs. The levels of HER2, phospho‐AKT, phospho‐ERK1/2, cell proliferation (Ki67) and apoptosis (cPARP) were analyzed with reverse‐phase protein arrays. Rank product analyses identified 38 miRNAs (q < 0.05) as inhibitors of HER2 signaling and cell growth, the most effective being miR‐491‐5p, miR‐634, miR‐637 and miR‐342‐5p. We also characterized miRNAs directly targeting HER2 and identified seven novel miRNAs (miR‐552, miR‐541, miR‐193a‐5p, miR‐453, miR‐134, miR‐498, and miR‐331‐3p) as direct regulators of the HER2 3′UTR. We demonstrated the clinical relevance of the miRNAs and identified miR‐342‐5p and miR‐744* as significantly down‐regulated in HER2‐positive breast tumors as compared to HER2‐negative tumors from two cohorts of breast cancer patients (101 and 1302 cases). miR‐342‐5p specifically inhibited HER2‐positive cell growth, as it had no effect on the growth of HER2‐negative control cells in vitro. Furthermore, higher expression of miR‐342‐5p was associated with better survival in both breast cancer patient cohorts. In conclusion, we have identified miRNAs which are efficient negative regulators of the HER2 pathway that may play a role in vivo during breast cancer progression. These results give mechanistic insights in HER2 regulation which may open potential new strategies towards prevention and therapeutic inhibition of HER2‐positive breast cancer.

High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells.

The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional monolayers on plastic. However, many cellular features are impaired in these artificial conditions, and large changes in gene expression compared to tumors have been reported. Three-dimensional cell culture models have become increasingly popular and are suggested to be better models than two-dimensional monolayers due to improved cell-to-cell contact and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput three-dimensional drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in two dimensions, in poly(2-hydroxyethyl methacrylate) induced anchorage-independent three-dimensional models, and in Matrigel three-dimensional cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating, or they were allowed to grow in three-dimensional cultures for 4 days before the drug treatment. Large variations in drug responses were observed between the models indicating that comparisons of culture model-influenced drug sensitivities cannot be made based on the effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in two-dimensional cultures, while the responses of cells grown in poly(2-hydroxyethyl methacrylate) resembled those of the two-dimensional cultures. Furthermore, comparing the gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study, we also suggest that three-dimensional cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode and be used as alternatives to traditional two-dimensional screens for better comparability to the in vivo state.

Deregulation of cancer-related miRNAs is a common event in both benign and malignant human breast tumors

MicroRNAs (miRNAs) are endogenous non-coding RNAs, which play an essential role in the regulation of gene expression during carcinogenesis. The role of miRNAs in breast cancer has been thoroughly investigated, and although many miRNAs are identified as cancer related, little is known about their involvement in benign tumors. In this study, we investigated miRNA expression profiles in the two most common types of human benign tumors (fibroadenoma/fibroadenomatosis) and in malignant breast tumors and explored their role as oncomirs and tumor suppressor miRNAs. Here, we identified 33 miRNAs with similar deregulated expression in both benign and malignant tumors compared with the expression levels of those in normal tissue, including breast cancer-related miRNAs such as let-7, miR-21 and miR-155. Additionally, messenger RNA (mRNA) expression profiles were obtained for some of the same samples. Using integrated mRNA/miRNA expression analysis, we observed that overexpression of certain miRNAs co-occurred with a significant downregulation of their candidate target mRNAs in both benign and malignant tumors. In support of these findings, in vitro functional screening of the downregulated miRNAs in non-malignant and breast cancer cell lines identified several possible tumor suppressor miRNAs, including miR-193b, miR-193a-3p, miR-126, miR-134, miR-132, miR-486-5p, miR-886-3p, miR-195 and miR-497, showing reduced growth when re-expressed in cancer cells. The finding of deregulated expression of oncomirs and tumor suppressor miRNAs in benign breast tumors is intriguing, indicating that they may play a role in proliferation. A role of cancer-related miRNAs in the early phases of carcinogenesis and malignant transformation can, therefore, not be ruled out.

Arachidonic Acid Pathway Members PLA2G7, HPGD, EPHX2, and CYP4F8 Identified as Putative Novel Therapeutic Targets in Prostate Cancer

The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.

Up-regulation of cartilage oligomeric matrix protein at the onset of articular cartilage degeneration in a transgenic mouse model of osteoarthritis

OBJECTIVE To investigate the suitability of cartilage oligomeric matrix protein (COMP) as a marker for articular cartilage degeneration in a transgenic mouse model of osteoarthritis (OA). METHODS Northern blot analysis of total RNA extracted from the knee joints of transgenic Del1 mice, which harbor a short deletion in a type II collagen transgene, and of their nontransgenic littermates was used to monitor changes in COMP messenger RNA (mRNA) levels during cartilage degeneration. Immunohistochemistry was used to determine the distribution of COMP in articular cartilage, and serum levels of COMP were measured by immunoassay. RESULTS Transient up-regulation of COMP mRNA was seen in articular cartilage of transgenic Del1 mice at the onset of OA lesions at the age of 3 months. Compared with nontransgenic controls, COMP immunostaining of articular cartilage in 3-9-month-old transgenic mice was increased, especially at the border of uncalcified and calcified cartilage. There was also a change from predominantly interterritorial to pericellular/territorial deposition of COMP. This difference persisted until the age of 15 months, when the nontransgenic controls also demonstrated articular cartilage degeneration and increased COMP immunostaining. Increased serum levels of COMP were seen in Del1 mice at the age of 4 months, correlating temporally with the onset of cartilage degeneration. CONCLUSION These findings suggest that upregulation of COMP mRNA and redistribution of the protein are characteristic of the early stages of articular cartilage degeneration in the transgenic mouse model in which OA results from a dominant-negative mutation in the type II collagen gene. The data provide additional support for the notion that COMP is a useful marker for altered cartilage metabolism in developing OA.

Identification of MicroRNAs Inhibiting TGF-β-Induced IL-11 Production in Bone Metastatic Breast Cancer Cells

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor β (TGF-β) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-β induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-β-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3′ UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-β signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.