Rami Mäkelä

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.

A Comprehensive Panel of Three-Dimensional Models for Studies of Prostate Cancer Growth, Invasion and Drug Responses

Prostate epithelial cells from both normal and cancer tissues, grown in three-dimensional (3D) culture as spheroids, represent promising in vitro models for the study of normal and cancer-relevant patterns of epithelial differentiation. We have developed the most comprehensive panel of miniaturized prostate cell culture models in 3D to date (n = 29), including many non-transformed and most currently available classic prostate cancer (PrCa) cell lines. The purpose of this study was to analyze morphogenetic properties of PrCa models in 3D, to compare phenotypes, gene expression and metabolism between 2D and 3D cultures, and to evaluate their relevance for pre-clinical drug discovery, disease modeling and basic research. Primary and non-transformed prostate epithelial cells, but also several PrCa lines, formed well-differentiated round spheroids. These showed strong cell-cell contacts, epithelial polarization, a hollow lumen and were covered by a complete basal lamina (BL). Most PrCa lines, however, formed large, poorly differentiated spheroids, or aggressively invading structures. In PC-3 and PC-3M cells, well-differentiated spheroids formed, which were then spontaneously transformed into highly invasive cells. These cell lines may have previously undergone an epithelial-to-mesenchymal transition (EMT), which is temporarily suppressed in favor of epithelial maturation by signals from the extracellular matrix (ECM). The induction of lipid and steroid metabolism, epigenetic reprogramming, and ECM remodeling represents a general adaptation to 3D culture, regardless of transformation and phenotype. In contrast, PI3-Kinase, AKT, STAT/interferon and integrin signaling pathways were particularly activated in invasive cells. Specific small molecule inhibitors targeted against PI3-Kinase blocked invasive cell growth more effectively in 3D than in 2D monolayer culture, or the growth of normal cells. Our panel of cell models, spanning a wide spectrum of phenotypic plasticity, supports the investigation of different modes of cell migration and tumor morphologies, and will be useful for predictive testing of anti-cancer and anti-metastatic compounds.

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.

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.

A cell spot microarray method for production of high density siRNA transfection microarrays

BackgroundHigh-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible.ResultsHere, we describe the optimization of a miniaturized cell spot microarray (CSMA) method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells.ConclusionsThe CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.

The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells

About 20% of breast cancers are characterized by amplification and overexpression of the HER2 oncogene. Although significant progress has been achieved for treating such patients with HER2 inhibitor trastuzumab, more than half of the patients respond poorly or become resistant to the treatment. Since the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co‐amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated aCGH data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non‐responding HER2 positive breast cancer cells. Silencing of HER2 caused a greater growth arrest and apoptosis in the responding compared to the non‐responding cell lines, indicating that the resistant cells are inherently less dependent on the HER2 pathway. Several other genes in the amplicon also showed a more pronounced effect when silenced; indicating that expression of HER2 co‐amplified genes may be needed to sustain the growth of breast cancer cells. Importantly, co‐silencing of STARD3, GRB7, PSMD3 and PERLD1 together with HER2 led to an additive inhibition of cell viability as well as induced apoptosis. These studies indicate that breast cancer cells may become addicted to the amplification of several genes that reside in the HER2 amplicon. The simultaneous targeting of these genes may increase the efficacy of the anti‐HER2 therapies and possibly also counteract trastuzumab resistance. The observed additive effects seem to culminate to both apoptosis and cell proliferation pathways indicating that these pathways may be interesting targets for combinatorial treatment of HER2+ breast cancers.

Systematic Identification of MicroRNAs That Impact on Proliferation of Prostate Cancer Cells and Display Changed Expression in Tumor Tissue

BACKGROUND Systematic approaches to functionally identify key players in microRNA (miRNA)-target networks regulating prostate cancer (PCa) proliferation are still missing. OBJECTIVE To comprehensively map miRNA regulation of genes relevant for PCa proliferation through phenotypic screening and tumor expression data. DESIGN, SETTING, AND PARTICIPANTS Gain-of-function screening with 1129 miRNA molecules was performed in five PCa cell lines, measuring proliferation, viability, and apoptosis. These results were integrated with changes in miRNA expression from two cohorts of human PCa (188 tumors in total). For resulting miRNAs, the predicted targets were collected and analyzed for patterns with gene set enrichment analysis, and for their association with biochemical recurrence free survival. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Rank product statistical analysis was used to evaluate miRNA effects in phenotypic screening and for expression differences in the prostate tumor cohorts. Expression data were analyzed using the significance analysis of microarrays (SAM) method and the patient material was subjected to Kaplan-Meier statistics. RESULTS AND LIMITATIONS Functional screening identified 25 miRNAs increasing and 48 miRNAs decreasing cell viability. Data integration resulted in 14 miRNAs, with aberrant expression and effect on proliferation. These miRNAs are predicted to regulate >3700 genes, of which 28 were found up-regulated and 127 down-regulated in PCa compared with benign tissue. Seven genes, FLNC, MSRB3, PARVA, PCDH7, PRNP, RAB34, and SORBS1, showed an inverse association to their predicted miRNA, and were identified to significantly correlate with biochemical recurrence free survival in PCa patients. CONCLUSIONS A systematic in vitro screening approach combined with in vivo expression and gene set enrichment analysis provide unbiased means for revealing novel miRNA-target links, possibly driving the oncogenic processes in PCa. PATIENT SUMMARY This study identified novel regulatory molecules, which impact on PCa proliferation and are aberrantly expressed in clinical tumors. Thus, our study reveals regulatory nodes with potential for therapy.

The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells - A data description.

A large number of breast cancers are characterized by amplification and overexpression of the chromosome segment surrounding the HER2 (ERBB2) oncogene. As the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated array comparative genomic hybridization (aCGH) data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non-responding HER2 positive breast cancer cells. In this Data in Brief we give a detailed description of the experimental procedures and the data analysis methods used in the study (1).

Abstract 1956: High-throughput screens identify microRNAs essential for HER2-positive breast cancer cell growth.

MicroRNAs (miRNAs) are small non-coding RNAs regulating gene expression post-transcriptionally. Due to their capability to target multiple genes, often in a same pathway, miRNAs would be ideal candidates for therapeutic targeting. Here, our aim was to characterize the role of miRNAs in the regulation of HER2-signaling in breast cancer. HER2 is a receptor tyrosine kinase amplified in over 20% of human breast cancers and it regulates many important cellular processes, including cell growth and survival. HER2 mediates its signals by activating downstream pathways, such as phosphatidyl-inositol-3 kinase (PI3K)/AKT and the mitogen-activated protein kinases. We performed miRNA gain-of-function assays by screening two HER2-positive 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 (cleaved PARP) were detected with specific antibodies using protein lysate microarrays. In order to find the miRNAs most efficiently inhibiting HER2 signaling and proliferation as well as inducing apoptosis, we did rank product analyses through the whole data matrix. These analyses resulted in 38 miRNAs (q≤0.05). Among them were miR-491-5p, miR-342-5p and miR-744*, which were significantly (p≤0.05) down-regulated in HER2-positive breast tumors as compared to HER2-negative tumors. miR-342-5p appeared to specifically inhibit HER2-positive cell growth, as it had no effect on the growth of HER2-negative control cell line. Furthermore, higher expression of miR-342-5p was associated with better survival in breast cancer patients. We characterized also miRNAs directly targeting HER2 and identified eight novel miRNAs (miR-552, miR-541, miR-193a-5p, miR-453, miR-134, miR-498, miR-449b, and miR-449a) as direct regulators of the HER2 3’UTR. Taken together, the miRNAs identified here are potent negative regulators of HER2 function and they may also play a role in vivo during breast cancer progression. These results suggest mechanistic insights in HER2 regulation in breast cancer and open potential new strategies towards prevention and therapeutic inhibition of HER2-positive breast cancer. Citation Format: Suvi-Katri Leivonen, Kristine Kleivi Sahlberg, Rami Makela, Olli Kallioniemi, Anne-Lise Borresen-Dale, Merja Perala. High-throughput screens identify microRNAs essential for HER2-positive breast cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1956. doi:10.1158/1538-7445.AM2013-1956

Abstract 334: Identification of novel regulatory genes controlling epithelial to mesenchymal transition in cultured breast cancer cells

Breast cancer prognosis is tightly correlated with the degree of spread beyond the primary tumor. A prerequisite for breast cancer cell dissemination is activation of a process called “epithelial - mesenchymal transition” (EMT). During EMT, epithelial cells lose epithelial characteristics, such as E-cadherin expression and gain mesenchymal properties, such as vimentin expression. The reversal process to EMT is mesenchymal-epithelial transition (MET), which occurs during colonization of the disseminated cancer cells to distant sites. Thus, EMT should be considered as a transient and reversible process in cancer progression. Despite the clinical importance, EMT and MET have been largely studied from a focused signalling standpoint, which may provide a biased view of the biological processes involved. In this study, RNAi technology followed up by lysate microarray analysis (LMA) using E-cadherin and vimentin as endpoint markers was performed to identify novel EMT and MET regulatory genes in cultured breast cancer cells. The screening conditions were optimized for the estrogen receptor (ER) positive E-cadherin expressing luminal MCF-7 breast cancer cells and for ER negative vimentin expressing mesenchymal bone metastatic MDA-MB 231 cells. The results from the replicate screens with >1012 siRNAs targeting 599 genes (1-3 siRNAs / gene) amplified and /or over-expressed in breast cancer cells were found to correlate (r > 0.9), and the validated siRNAs targeting E-cadherin and vimentin were among the top repressing hits for the corresponding endpoints, indicating reliability of the protocol. Interestingly, several novel putative E-cadherin (E-cadherin reducing siRNAs, genes inducing loss of metastatic potential) and vimentin (vimentin reducing siRNAs, metastasis-promoting candidate genes) regulators were identified and validated using secondary assays, indicating that our knowledge on EMT and MET is still far from comprehensive. Functional characterization of the identified candidate regulators in cultured breast cancer cells is on-going. Since modulation of the differentiation state of breast cancer cells is a promising therapeutic strategy, better understanding of EMT and MET may provide novel therapeutic opportunities to block breast cancer cell dissemination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 334. doi:1538-7445.AM2012-334