Suvi-Katri Leivonen

Transforming growth factor-β signaling in cancer invasion and metastasis

Transforming growth factor‐β (TGF‐β) family members are polypeptides with dual tumor suppressive and oncogenic effects. They signal through serine/threonine kinase receptor complexes, which phosphorylate cytoplasmic mediators, the Smads. Upon phosphorylation, Smads translocate to the nucleus and associate with transcriptional coactivators or corepressors, and regulate the transcriptional activation of various TGF‐β responsive genes. In addition, TGF‐β activates cellular mitogen‐activated protein kinase signaling pathways, which crosstalk with Smad signaling and regulate growth, survival and motility of cells. During tumorigenesis, malignantly transformed cells often lose the response to the tumor suppressive effects of TGF‐β, which, in turn, starts to act as an autocrine tumor promoting factor by enhancing cancer invasion and metastasis. In this review, we summarize current view on the role of TGF‐β signaling in tumorigenesis, with emphasis on cancer invasion and metastasis. On the basis of these recent observations, we discuss new therapeutic strategies targeting TGF‐β signaling at distinct levels as a basis for inhibiting tumor growth, angiogenesis, invasion and metastasis.

EGF-R regulates MMP function in fibroblasts through MAPK and AP-1 pathways

EGF‐R regulates cell proliferation, migration, and invasion in fibroblasts. However, the connection of EGF‐R with downstream signaling pathways mediating these responses has remained elusive. Here we provide genetic and biochemical evidence that EGF‐R‐ and AP‐1‐mediated signals are required for MMP expression and collagen contraction in fibroblasts. In EGF‐R (−/−) mouse embryonal fibroblasts, basal and inducible expression of several MMPs, including MMP‐2, ‐3, and ‐14 is impaired in comparison to wild‐type counterparts. The loss of MMP expression is associated with a suppression of EGF‐induced Erk and Jnk activities, and AP‐1 DNA‐binding and transactivation capacities. While inhibition of Jnk mainly prevents EGF‐induced phosphorylation of c‐Jun, inhibition of Erk pathway suppresses both the expression and phosphorylation of c‐Jun and c‐Fos proteins. Moreover, the expression of MMP‐3 and ‐14, and collagen contraction is partially prevented by Mek/Erk and Jnk inhibitors. However, Jnk inhibitor also suppresses cell growth independently of EGF‐R activity. The central role of AP‐1 as a mediator of EGF‐R signaling in fibroblasts is emphasized by the finding that expression of a dominant negative c‐Jun downregulates the expression of MMP‐3. Conversely, expression of a constitutively active Mek1 can induce MMP‐3 expression independently of upstream signals. The results indicate that ERK pathway and AP‐1 are downstream effectors of the EGF‐R‐mediated MMP‐3 expression and collagen contraction in fibroblasts. J. Cell. Physiol. 212: 489–497, 2007.

Human papillomavirus 16 E5 modulates the expression of host microRNAs.

Human papillomavirus (HPV) infection is a prerequisite of developing cervical cancer, approximately half of which are associated with HPV type 16. HPV 16 encodes three oncogenes, E5, E6, and E7, of which E5 is the least studied so far. Its roles in regulating replication and pathogenesis of HPV are not fully understood. Here we utilize high-throughput screening to coordinately investigate the effect of E5 on the expression of host protein-coding and microRNA genes. MicroRNAs form a class of 22nt long noncoding RNAs with regulatory activity. Among the altered cellular microRNAs we focus on the alteration in the expression of miR-146a, miR-203 and miR-324-5p and their target genes in a time interval of 96 hours of E5 induction. Our results indicate that HPV infection and subsequent transformation take place through complex regulatory patterns of gene expression in the host cells, part of which are regulated by the E5 protein.

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.

Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

BackgroundThe global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.ResultsWe identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.ConclusionsOur data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.

Identification of miR-193b Targets in Breast Cancer Cells and Systems Biological Analysis of Their Functional Impact

Identification of protein targets for microRNAs (miRNAs) is a significant challenge due to the complexity of miRNA-mediated regulation. We have previously demonstrated that miR-193b targets estrogen receptor-α (ERα) and inhibits estrogen-induced growth of breast cancer cells. Here, we applied a high-throughput strategy using quantitative iTRAQ (isobaric tag for relative and absolute quantitation) reagents to identify other target proteins regulated by miR-193b in breast cancer cells. iTRAQ analysis of pre-miR-193b transfected MCF-7 cells resulted in identification of 743 unique proteins, of which 39 were down-regulated and 44 up-regulated as compared with negative control transfected cells. Computationally predicted targets of miR-193b were highly enriched (sevenfold) among the proteins whose level of expression decreased after miR-193b transfection. Only a minority of these (13%) showed similar effect at the mRNA level illustrating the importance of post-transcriptional regulation. The most significantly repressed proteins were selected for validation experiments. These data confirmed 14–3-3ζ (YWHAZ), serine hydroxyl transferase (SHMT2), and aldo-keto reductase family 1, member C2 (AKR1C2) as direct, previously uncharacterized, targets of miR-193b. Functional RNAi assays demonstrated that specific combinations of knockdowns of these target genes by siRNAs inhibited growth of MCF-7 cells, mimicking the effects of the miR-193b overexpression. Interestingly, the data imply that besides targeting ERα, the miR-193b effects include suppression of the local production of estrogens and other steroid hormones mediated by the AKR1C2 gene, thus provoking two separate molecular mechanisms inhibiting steroid-dependent growth of breast cancer cells. In conclusion, we present here a proteomic screen to identify targets of miR-193b, and a systems biological approach to mimic its effects at the level of cellular phenotypes. This led to the identification of multiple genes whose combinatorial knock-down likely mediates the strong anti-cancer effects observed for miR-193b in breast cancer cells.

Hypoxia-activated Smad3-specific dephosphorylation by PP2A.

The transforming growth factor-β (TGF-β) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-β promotes malignancy. TGF-β activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-β-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-β receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-β-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-β on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses.

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.

Integrative clustering reveals a novel split in the luminal A subtype of breast cancer with impact on outcome

BackgroundBreast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes.MethodsTumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a “cluster-of-clusters” approach with consensus clustering.ResultsBased on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed.ConclusionsThe six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.

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