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ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth.

Zhang S, Chen L, Cui B, Chuang HY, Yu J, Wang-Rodriguez J, Tang L, Chen G, Basak GW, Kipps TJ - PLoS ONE (2012)

Bottom Line: Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues.We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth.Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth.

View Article: PubMed Central - PubMed

Affiliation: Moores UCSD Cancer Center, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.

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ROR1 interacts with CK1ε to activate PI3K/AKT, leading to activation of CREB.(A) MDA-MB-231tumor cells with or without ROR1 expression were examined for phosphorylated AKT at ser-473 (p-AKT) or total AKT (t-AKT). (B) MDA-MB-231 cells were cultured with (+) or without (−) LY294002 and examined for p-AKT, t-AKT, p-CREB, or t-CREB by immunobot analysis after 16 hours. (C) MDA-MB-231 cells (Ct-shRNA) or cells silenced for ROR1 (ROR1-shRNA) were cultured with different concentrations of LY294002 and monitored for cell growth after 48 hours using the WST-8 assay. The graphs depict the mean numbers of viable cells, ± S.E.M of triplicate samples, which are representative of three independent experiments. *P<0.05 and ***P<0.001). (D–E) MCF7 control cells (Ct-Vector) or cells made to express ROR1 (pCDNA3-ROR1) were examined for protein expression (D) or (E) monitored for growth over 4 days using the WST-8 assay. The graph depicts the mean numbers of viable cells ± S.E.M. over time in each of these cell lines, as indicated in the legend at the top of the figure, which are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 by Student's t test. (F) Protein lysates from MDA-MB-231 cells or cells silenced for ROR1 were immunoprecipitated (IP) with ROR1 antibody. The bound immunoprecipitated products and whole cell lysate (WCL) were probed by the antibodies indicated in the Probe column. (G–H) MDA-MB-231 cells were treated without (−) or with CK1ε siRNA (CK1ε-siRNA) or control siRNA (Ct-siRNA) for 72 hours and examined for protein expression (G) or cell growth (H). The height of each bar in the graph F provides the mean number of viable cells ± S.E.M., which are representative of more than three independent experiments. P indicates the statistical significance.
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pone-0031127-g005: ROR1 interacts with CK1ε to activate PI3K/AKT, leading to activation of CREB.(A) MDA-MB-231tumor cells with or without ROR1 expression were examined for phosphorylated AKT at ser-473 (p-AKT) or total AKT (t-AKT). (B) MDA-MB-231 cells were cultured with (+) or without (−) LY294002 and examined for p-AKT, t-AKT, p-CREB, or t-CREB by immunobot analysis after 16 hours. (C) MDA-MB-231 cells (Ct-shRNA) or cells silenced for ROR1 (ROR1-shRNA) were cultured with different concentrations of LY294002 and monitored for cell growth after 48 hours using the WST-8 assay. The graphs depict the mean numbers of viable cells, ± S.E.M of triplicate samples, which are representative of three independent experiments. *P<0.05 and ***P<0.001). (D–E) MCF7 control cells (Ct-Vector) or cells made to express ROR1 (pCDNA3-ROR1) were examined for protein expression (D) or (E) monitored for growth over 4 days using the WST-8 assay. The graph depicts the mean numbers of viable cells ± S.E.M. over time in each of these cell lines, as indicated in the legend at the top of the figure, which are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 by Student's t test. (F) Protein lysates from MDA-MB-231 cells or cells silenced for ROR1 were immunoprecipitated (IP) with ROR1 antibody. The bound immunoprecipitated products and whole cell lysate (WCL) were probed by the antibodies indicated in the Probe column. (G–H) MDA-MB-231 cells were treated without (−) or with CK1ε siRNA (CK1ε-siRNA) or control siRNA (Ct-siRNA) for 72 hours and examined for protein expression (G) or cell growth (H). The height of each bar in the graph F provides the mean number of viable cells ± S.E.M., which are representative of more than three independent experiments. P indicates the statistical significance.

Mentions: We examined for activation of signaling proteins upstream of p-CREB, such as AKT or phosphoinositol 3′ kinase (PI3K) [26]. We found MDA-MB-231 cells silenced for ROR1 had lower levels of p-AKT relative to AKT than control-treated cells (Fig. 5A). On the other hand, treatment with a PI3K inhibitor (LY294002) reduced the levels of p-AKT and p-CREB in MDA-MB-231 cells that expressed ROR1 (Fig. 5B). Moreover, ROR1-expressing MDA-MB-231 cells were more sensitive to the growth-inhibitory activity of LY294002 than MDA-MB-231 cells silenced for ROR1 (Fig. 5C).


ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth.

Zhang S, Chen L, Cui B, Chuang HY, Yu J, Wang-Rodriguez J, Tang L, Chen G, Basak GW, Kipps TJ - PLoS ONE (2012)

ROR1 interacts with CK1ε to activate PI3K/AKT, leading to activation of CREB.(A) MDA-MB-231tumor cells with or without ROR1 expression were examined for phosphorylated AKT at ser-473 (p-AKT) or total AKT (t-AKT). (B) MDA-MB-231 cells were cultured with (+) or without (−) LY294002 and examined for p-AKT, t-AKT, p-CREB, or t-CREB by immunobot analysis after 16 hours. (C) MDA-MB-231 cells (Ct-shRNA) or cells silenced for ROR1 (ROR1-shRNA) were cultured with different concentrations of LY294002 and monitored for cell growth after 48 hours using the WST-8 assay. The graphs depict the mean numbers of viable cells, ± S.E.M of triplicate samples, which are representative of three independent experiments. *P<0.05 and ***P<0.001). (D–E) MCF7 control cells (Ct-Vector) or cells made to express ROR1 (pCDNA3-ROR1) were examined for protein expression (D) or (E) monitored for growth over 4 days using the WST-8 assay. The graph depicts the mean numbers of viable cells ± S.E.M. over time in each of these cell lines, as indicated in the legend at the top of the figure, which are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 by Student's t test. (F) Protein lysates from MDA-MB-231 cells or cells silenced for ROR1 were immunoprecipitated (IP) with ROR1 antibody. The bound immunoprecipitated products and whole cell lysate (WCL) were probed by the antibodies indicated in the Probe column. (G–H) MDA-MB-231 cells were treated without (−) or with CK1ε siRNA (CK1ε-siRNA) or control siRNA (Ct-siRNA) for 72 hours and examined for protein expression (G) or cell growth (H). The height of each bar in the graph F provides the mean number of viable cells ± S.E.M., which are representative of more than three independent experiments. P indicates the statistical significance.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293865&req=5

pone-0031127-g005: ROR1 interacts with CK1ε to activate PI3K/AKT, leading to activation of CREB.(A) MDA-MB-231tumor cells with or without ROR1 expression were examined for phosphorylated AKT at ser-473 (p-AKT) or total AKT (t-AKT). (B) MDA-MB-231 cells were cultured with (+) or without (−) LY294002 and examined for p-AKT, t-AKT, p-CREB, or t-CREB by immunobot analysis after 16 hours. (C) MDA-MB-231 cells (Ct-shRNA) or cells silenced for ROR1 (ROR1-shRNA) were cultured with different concentrations of LY294002 and monitored for cell growth after 48 hours using the WST-8 assay. The graphs depict the mean numbers of viable cells, ± S.E.M of triplicate samples, which are representative of three independent experiments. *P<0.05 and ***P<0.001). (D–E) MCF7 control cells (Ct-Vector) or cells made to express ROR1 (pCDNA3-ROR1) were examined for protein expression (D) or (E) monitored for growth over 4 days using the WST-8 assay. The graph depicts the mean numbers of viable cells ± S.E.M. over time in each of these cell lines, as indicated in the legend at the top of the figure, which are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 by Student's t test. (F) Protein lysates from MDA-MB-231 cells or cells silenced for ROR1 were immunoprecipitated (IP) with ROR1 antibody. The bound immunoprecipitated products and whole cell lysate (WCL) were probed by the antibodies indicated in the Probe column. (G–H) MDA-MB-231 cells were treated without (−) or with CK1ε siRNA (CK1ε-siRNA) or control siRNA (Ct-siRNA) for 72 hours and examined for protein expression (G) or cell growth (H). The height of each bar in the graph F provides the mean number of viable cells ± S.E.M., which are representative of more than three independent experiments. P indicates the statistical significance.
Mentions: We examined for activation of signaling proteins upstream of p-CREB, such as AKT or phosphoinositol 3′ kinase (PI3K) [26]. We found MDA-MB-231 cells silenced for ROR1 had lower levels of p-AKT relative to AKT than control-treated cells (Fig. 5A). On the other hand, treatment with a PI3K inhibitor (LY294002) reduced the levels of p-AKT and p-CREB in MDA-MB-231 cells that expressed ROR1 (Fig. 5B). Moreover, ROR1-expressing MDA-MB-231 cells were more sensitive to the growth-inhibitory activity of LY294002 than MDA-MB-231 cells silenced for ROR1 (Fig. 5C).

Bottom Line: Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues.We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth.Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth.

View Article: PubMed Central - PubMed

Affiliation: Moores UCSD Cancer Center, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.

Show MeSH
Related in: MedlinePlus