High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer

Suvi-Katri Leivonen, Rami Mäkelä, Päivi Östling, Pekka Kohonen, Saija Haapa-Paananen, Anna Aakula, Kirsi Hellström, Niko Sahlberg, Kristine Kleivi, Espen Enerly, Vessela N. Kristensen, Anne-Lise Borresen-Dale, Petri Saviranta, Merja Perälä, Olli Kallioniemi

Research output: Contribution to conferenceConference articleScientific

Abstract

MicroRNAs (miRNAs) regulate protein expression by targeting messenger RNAs (mRNAs) for cleavage or by inducing translational repression. In breast cancer, estrogen receptor-a (ERa) levels are critical for cancer cell growth, but our knowledge of miRNA-dependent ERa regulation is limited. In order to identify miRNAs impacting ERa expression in breast cancer, we transiently transfected 319 pre-miRs into MCF-7 and BT-474 cell lines and monitored their effects on ERa expression by protein lysate microarrays. There was a significant enrichment of the in silico predicted ERa targeting miRNAs among the miRNAs that experimentally reduced ERa protein expression. The ERa inhibition by 21 pre-miRs was validated with western blotting and qRT-PCR. Proliferation assays demonstrated that the impact of ERa-downregulating miRNAs on the inhibition of estrogen-stimulated growth of MCF-7 cells was as strong as that of an siRNA for ERa. Overexpression of the top pre-miR hits (miR-18a/b, miR-193b, miR-206, and miR-302c) followed by gene microarray profiling revealed downregulation of ERa responsive genes, and ERa 3'UTR-luciferase reporter assays confirmed that these miRNAs directly targeted ERa. Finally, miR-18a and miR-18b showed increased expression in ERa-negative as compared to ERa-positive clinical tumors. Taken together, these results suggest that miRNAs may be useful in the therapeutic inhibition of ERa expression, and that upregulation of certain miRNAs may be involved in the silencing of ERa in the progression towards estrogen-independent breast cancers.
Original languageEnglish
Publication statusPublished - 2009
MoE publication typeNot Eligible
Event100th Annual Meeting of the American Association for Cancer Research - Denver, United States
Duration: 18 Apr 200922 Apr 2009

Conference

Conference100th Annual Meeting of the American Association for Cancer Research
CountryUnited States
CityDenver
Period18/04/0922/04/09

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Estrogen Receptor alpha
MicroRNAs
Estrogen Receptors
Breast Neoplasms
Estrogens
Down-Regulation
RNA Cleavage
Protein Array Analysis
MCF-7 Cells
3' Untranslated Regions
Protein Transport
Growth
Luciferases
Computer Simulation
Small Interfering RNA
Genes

Cite this

Leivonen, S-K., Mäkelä, R., Östling, P., Kohonen, P., Haapa-Paananen, S., Aakula, A., ... Kallioniemi, O. (2009). High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer. Paper presented at 100th Annual Meeting of the American Association for Cancer Research, Denver, United States.
Leivonen, Suvi-Katri ; Mäkelä, Rami ; Östling, Päivi ; Kohonen, Pekka ; Haapa-Paananen, Saija ; Aakula, Anna ; Hellström, Kirsi ; Sahlberg, Niko ; Kleivi, Kristine ; Enerly, Espen ; Kristensen, Vessela N. ; Borresen-Dale, Anne-Lise ; Saviranta, Petri ; Perälä, Merja ; Kallioniemi, Olli. / High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer. Paper presented at 100th Annual Meeting of the American Association for Cancer Research, Denver, United States.
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title = "High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer",
abstract = "MicroRNAs (miRNAs) regulate protein expression by targeting messenger RNAs (mRNAs) for cleavage or by inducing translational repression. In breast cancer, estrogen receptor-a (ERa) levels are critical for cancer cell growth, but our knowledge of miRNA-dependent ERa regulation is limited. In order to identify miRNAs impacting ERa expression in breast cancer, we transiently transfected 319 pre-miRs into MCF-7 and BT-474 cell lines and monitored their effects on ERa expression by protein lysate microarrays. There was a significant enrichment of the in silico predicted ERa targeting miRNAs among the miRNAs that experimentally reduced ERa protein expression. The ERa inhibition by 21 pre-miRs was validated with western blotting and qRT-PCR. Proliferation assays demonstrated that the impact of ERa-downregulating miRNAs on the inhibition of estrogen-stimulated growth of MCF-7 cells was as strong as that of an siRNA for ERa. Overexpression of the top pre-miR hits (miR-18a/b, miR-193b, miR-206, and miR-302c) followed by gene microarray profiling revealed downregulation of ERa responsive genes, and ERa 3'UTR-luciferase reporter assays confirmed that these miRNAs directly targeted ERa. Finally, miR-18a and miR-18b showed increased expression in ERa-negative as compared to ERa-positive clinical tumors. Taken together, these results suggest that miRNAs may be useful in the therapeutic inhibition of ERa expression, and that upregulation of certain miRNAs may be involved in the silencing of ERa in the progression towards estrogen-independent breast cancers.",
author = "Suvi-Katri Leivonen and Rami M{\"a}kel{\"a} and P{\"a}ivi {\"O}stling and Pekka Kohonen and Saija Haapa-Paananen and Anna Aakula and Kirsi Hellstr{\"o}m and Niko Sahlberg and Kristine Kleivi and Espen Enerly and Kristensen, {Vessela N.} and Anne-Lise Borresen-Dale and Petri Saviranta and Merja Per{\"a}l{\"a} and Olli Kallioniemi",
year = "2009",
language = "English",
note = "100th Annual Meeting of the American Association for Cancer Research ; Conference date: 18-04-2009 Through 22-04-2009",

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Leivonen, S-K, Mäkelä, R, Östling, P, Kohonen, P, Haapa-Paananen, S, Aakula, A, Hellström, K, Sahlberg, N, Kleivi, K, Enerly, E, Kristensen, VN, Borresen-Dale, A-L, Saviranta, P, Perälä, M & Kallioniemi, O 2009, 'High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer' Paper presented at 100th Annual Meeting of the American Association for Cancer Research, Denver, United States, 18/04/09 - 22/04/09, .

High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer. / Leivonen, Suvi-Katri; Mäkelä, Rami; Östling, Päivi; Kohonen, Pekka; Haapa-Paananen, Saija; Aakula, Anna; Hellström, Kirsi; Sahlberg, Niko; Kleivi, Kristine; Enerly, Espen; Kristensen, Vessela N.; Borresen-Dale, Anne-Lise; Saviranta, Petri; Perälä, Merja; Kallioniemi, Olli.

2009. Paper presented at 100th Annual Meeting of the American Association for Cancer Research, Denver, United States.

Research output: Contribution to conferenceConference articleScientific

TY - CONF

T1 - High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer

AU - Leivonen, Suvi-Katri

AU - Mäkelä, Rami

AU - Östling, Päivi

AU - Kohonen, Pekka

AU - Haapa-Paananen, Saija

AU - Aakula, Anna

AU - Hellström, Kirsi

AU - Sahlberg, Niko

AU - Kleivi, Kristine

AU - Enerly, Espen

AU - Kristensen, Vessela N.

AU - Borresen-Dale, Anne-Lise

AU - Saviranta, Petri

AU - Perälä, Merja

AU - Kallioniemi, Olli

PY - 2009

Y1 - 2009

N2 - MicroRNAs (miRNAs) regulate protein expression by targeting messenger RNAs (mRNAs) for cleavage or by inducing translational repression. In breast cancer, estrogen receptor-a (ERa) levels are critical for cancer cell growth, but our knowledge of miRNA-dependent ERa regulation is limited. In order to identify miRNAs impacting ERa expression in breast cancer, we transiently transfected 319 pre-miRs into MCF-7 and BT-474 cell lines and monitored their effects on ERa expression by protein lysate microarrays. There was a significant enrichment of the in silico predicted ERa targeting miRNAs among the miRNAs that experimentally reduced ERa protein expression. The ERa inhibition by 21 pre-miRs was validated with western blotting and qRT-PCR. Proliferation assays demonstrated that the impact of ERa-downregulating miRNAs on the inhibition of estrogen-stimulated growth of MCF-7 cells was as strong as that of an siRNA for ERa. Overexpression of the top pre-miR hits (miR-18a/b, miR-193b, miR-206, and miR-302c) followed by gene microarray profiling revealed downregulation of ERa responsive genes, and ERa 3'UTR-luciferase reporter assays confirmed that these miRNAs directly targeted ERa. Finally, miR-18a and miR-18b showed increased expression in ERa-negative as compared to ERa-positive clinical tumors. Taken together, these results suggest that miRNAs may be useful in the therapeutic inhibition of ERa expression, and that upregulation of certain miRNAs may be involved in the silencing of ERa in the progression towards estrogen-independent breast cancers.

AB - MicroRNAs (miRNAs) regulate protein expression by targeting messenger RNAs (mRNAs) for cleavage or by inducing translational repression. In breast cancer, estrogen receptor-a (ERa) levels are critical for cancer cell growth, but our knowledge of miRNA-dependent ERa regulation is limited. In order to identify miRNAs impacting ERa expression in breast cancer, we transiently transfected 319 pre-miRs into MCF-7 and BT-474 cell lines and monitored their effects on ERa expression by protein lysate microarrays. There was a significant enrichment of the in silico predicted ERa targeting miRNAs among the miRNAs that experimentally reduced ERa protein expression. The ERa inhibition by 21 pre-miRs was validated with western blotting and qRT-PCR. Proliferation assays demonstrated that the impact of ERa-downregulating miRNAs on the inhibition of estrogen-stimulated growth of MCF-7 cells was as strong as that of an siRNA for ERa. Overexpression of the top pre-miR hits (miR-18a/b, miR-193b, miR-206, and miR-302c) followed by gene microarray profiling revealed downregulation of ERa responsive genes, and ERa 3'UTR-luciferase reporter assays confirmed that these miRNAs directly targeted ERa. Finally, miR-18a and miR-18b showed increased expression in ERa-negative as compared to ERa-positive clinical tumors. Taken together, these results suggest that miRNAs may be useful in the therapeutic inhibition of ERa expression, and that upregulation of certain miRNAs may be involved in the silencing of ERa in the progression towards estrogen-independent breast cancers.

M3 - Conference article

ER -

Leivonen S-K, Mäkelä R, Östling P, Kohonen P, Haapa-Paananen S, Aakula A et al. High-throughput functional screens and clinical profiling identify microRNAs regulating estrogen receptor (ER)-alfa signaling in breast cancer. 2009. Paper presented at 100th Annual Meeting of the American Association for Cancer Research, Denver, United States.