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CK1epsilon is required for breast cancers dependent on beta-catenin activity.

Kim SY, Dunn IF, Firestein R, Gupta P, Wardwell L, Repich K, Schinzel AC, Wittner B, Silver SJ, Root DE, Boehm JS, Ramaswamy S, Lander ES, Hahn WC - PLoS ONE (2010)

Bottom Line: However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.We also find that expression of CK1epsilon is able to promote oncogenic transformation of human cells in a beta-catenin-dependent manner.More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Methodology: To uncover genetic dependencies in breast cancer cells that harbor active beta-catenin signaling, we performed RNAi-based loss-of-function screens in breast cancer cell lines in which we had characterized beta-catenin activity. Here we identify CSNK1E, the gene encoding casein kinase 1 epsilon (CK1epsilon) as required specifically for the proliferation of breast cancer cells with activated beta-catenin and confirm its role as a positive regulator of beta-catenin-driven transcription. Furthermore, we demonstrate that breast cancer cells that harbor activated beta-catenin activity exhibit enhanced sensitivity to pharmacological blockade of Wnt/beta-catenin signaling. We also find that expression of CK1epsilon is able to promote oncogenic transformation of human cells in a beta-catenin-dependent manner.

Conclusions/significance: These studies identify CK1epsilon as a critical contributor to activated beta-catenin signaling in cancer and suggest it may provide a potential therapeutic target for cancers that harbor active beta-catenin. More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors.

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Characterization of Wnt/β-catenin activity in breast cancer cell lines.Immunoblot analysis of (A) active (upper panel) and total (middle panel) β-catenin levels or (B) cytoplasmic (left) and nuclear (right) β-catenin levels. To verify fractionation, immunoblots for cytoplasmic GAPDH and nuclear lamin are shown. (C) Immunoblot analysis of β-catenin levels after suppression of CTNNB1 with two distinct shRNAs (shBCAT A, B) in β-catenin active (MCF7) and β-catenin inactive (MDA-MB-453) cells. An shRNA against GFP was included as a control (shGFP). Asterisk denotes position of a non-specific cross-reacting band. (D) Effects on proliferation after RNAi-induced suppression of CTNNB1. Graph shows mean ± SD of a representative experiment performed in triplicate.
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pone-0008979-g001: Characterization of Wnt/β-catenin activity in breast cancer cell lines.Immunoblot analysis of (A) active (upper panel) and total (middle panel) β-catenin levels or (B) cytoplasmic (left) and nuclear (right) β-catenin levels. To verify fractionation, immunoblots for cytoplasmic GAPDH and nuclear lamin are shown. (C) Immunoblot analysis of β-catenin levels after suppression of CTNNB1 with two distinct shRNAs (shBCAT A, B) in β-catenin active (MCF7) and β-catenin inactive (MDA-MB-453) cells. An shRNA against GFP was included as a control (shGFP). Asterisk denotes position of a non-specific cross-reacting band. (D) Effects on proliferation after RNAi-induced suppression of CTNNB1. Graph shows mean ± SD of a representative experiment performed in triplicate.

Mentions: We initially characterized the activation status of β-catenin in breast cancer cell lines using an antibody that specifically recognizes the unphosphorylated form of β-catenin, which corresponds to the stable and thus functionally active form [8]. Three of the lines tested, MCF7, MDA-MB-231 and T47D were found to have elevated levels of both active and total β-catenin compared with the MDA-MB-453 cell line, which had much lower levels (Fig. 1A). In addition to determining levels of the active form of β-catenin, we also assessed β-catenin activity status by measuring nuclear β-catenin levels, which corresponds to the transcriptionally active pool of β-catenin, and is physically and functionally separate from the other major cellular pool of β-catenin at adherens junctions. We observed elevated levels of nuclear β-catenin in MCF7, MDA-MB-231 and T47D cells, with barely detectable levels in the MDA-MB-453 cells. (Fig. 1B). Methylation and subsequent downregulation of expression of the Wnt-inhibitory gene SFRP1 has previously been observed in all three β-catenin-expressing lines [12] and may contribute to activation of Wnt/β-catenin signaling in these cells.


CK1epsilon is required for breast cancers dependent on beta-catenin activity.

Kim SY, Dunn IF, Firestein R, Gupta P, Wardwell L, Repich K, Schinzel AC, Wittner B, Silver SJ, Root DE, Boehm JS, Ramaswamy S, Lander ES, Hahn WC - PLoS ONE (2010)

Characterization of Wnt/β-catenin activity in breast cancer cell lines.Immunoblot analysis of (A) active (upper panel) and total (middle panel) β-catenin levels or (B) cytoplasmic (left) and nuclear (right) β-catenin levels. To verify fractionation, immunoblots for cytoplasmic GAPDH and nuclear lamin are shown. (C) Immunoblot analysis of β-catenin levels after suppression of CTNNB1 with two distinct shRNAs (shBCAT A, B) in β-catenin active (MCF7) and β-catenin inactive (MDA-MB-453) cells. An shRNA against GFP was included as a control (shGFP). Asterisk denotes position of a non-specific cross-reacting band. (D) Effects on proliferation after RNAi-induced suppression of CTNNB1. Graph shows mean ± SD of a representative experiment performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2813871&req=5

pone-0008979-g001: Characterization of Wnt/β-catenin activity in breast cancer cell lines.Immunoblot analysis of (A) active (upper panel) and total (middle panel) β-catenin levels or (B) cytoplasmic (left) and nuclear (right) β-catenin levels. To verify fractionation, immunoblots for cytoplasmic GAPDH and nuclear lamin are shown. (C) Immunoblot analysis of β-catenin levels after suppression of CTNNB1 with two distinct shRNAs (shBCAT A, B) in β-catenin active (MCF7) and β-catenin inactive (MDA-MB-453) cells. An shRNA against GFP was included as a control (shGFP). Asterisk denotes position of a non-specific cross-reacting band. (D) Effects on proliferation after RNAi-induced suppression of CTNNB1. Graph shows mean ± SD of a representative experiment performed in triplicate.
Mentions: We initially characterized the activation status of β-catenin in breast cancer cell lines using an antibody that specifically recognizes the unphosphorylated form of β-catenin, which corresponds to the stable and thus functionally active form [8]. Three of the lines tested, MCF7, MDA-MB-231 and T47D were found to have elevated levels of both active and total β-catenin compared with the MDA-MB-453 cell line, which had much lower levels (Fig. 1A). In addition to determining levels of the active form of β-catenin, we also assessed β-catenin activity status by measuring nuclear β-catenin levels, which corresponds to the transcriptionally active pool of β-catenin, and is physically and functionally separate from the other major cellular pool of β-catenin at adherens junctions. We observed elevated levels of nuclear β-catenin in MCF7, MDA-MB-231 and T47D cells, with barely detectable levels in the MDA-MB-453 cells. (Fig. 1B). Methylation and subsequent downregulation of expression of the Wnt-inhibitory gene SFRP1 has previously been observed in all three β-catenin-expressing lines [12] and may contribute to activation of Wnt/β-catenin signaling in these cells.

Bottom Line: However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.We also find that expression of CK1epsilon is able to promote oncogenic transformation of human cells in a beta-catenin-dependent manner.More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.

Methodology: To uncover genetic dependencies in breast cancer cells that harbor active beta-catenin signaling, we performed RNAi-based loss-of-function screens in breast cancer cell lines in which we had characterized beta-catenin activity. Here we identify CSNK1E, the gene encoding casein kinase 1 epsilon (CK1epsilon) as required specifically for the proliferation of breast cancer cells with activated beta-catenin and confirm its role as a positive regulator of beta-catenin-driven transcription. Furthermore, we demonstrate that breast cancer cells that harbor activated beta-catenin activity exhibit enhanced sensitivity to pharmacological blockade of Wnt/beta-catenin signaling. We also find that expression of CK1epsilon is able to promote oncogenic transformation of human cells in a beta-catenin-dependent manner.

Conclusions/significance: These studies identify CK1epsilon as a critical contributor to activated beta-catenin signaling in cancer and suggest it may provide a potential therapeutic target for cancers that harbor active beta-catenin. More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors.

Show MeSH
Related in: MedlinePlus