J Biol Chem. 2009 Sep 25;284(39):26533-46. Epub 2009 Jul 24.

Hyaluronan-CD44 interaction with protein kinase C(epsilon) promotes oncogenic signaling by the stem cell marker Nanog and the Production of microRNA-21, leading to down-regulation of the tumor suppressor protein PDCD4, anti-apoptosis, and chemotherapy resistance in breast tumor cells.

Bourguignon LY, Spevak CC, Wong G, Xia W, Gilad E.

Source

Endocrine Unit, Department of Medicine, University of California at San Francisco, and Veterans Affairs Medical Center, San Francisco, California 94121, USA. lilly.bourguignon@ucsf.edu

Abstract

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

PMID:: 19633292; [PubMed - indexed for MEDLINE]
PMCID:: PMC2785342

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FIGURE 1.

Analyses of HA-induced PKCϵ-CD44 association and PKCϵ activity in MCF-7 cells. Detection of HA-induced PKCϵ-CD44 complex formation in MCF-7 cells was carried out by solubilizing cells with 1% Nonidet P-40 (Nonidet P-40) buffer, followed by immunoprecipitation (IP) and/or immunoblot by anti-CD44 antibody or anti-PKCϵ antibody, respectively (A). Subsequently, CD44-associated PKCϵ activity using peptide-2 as a substrate was analyzed according to the procedures described under “Materials and Methods.” A, detection of PKCϵ in the CD44 complex by anti-CD44-immunoprecipitation, followed by immunoblotting with anti-PKCϵ antibody (a) or reblotting with anti-CD44 (b) as a loading control using MCF-7 cells treated with no HA (lane 1) or with HA (50 μg/ml) for 10 min (lane 2) or pretreated with anti-CD44 followed by a 10-min HA (50 μg/ml) addition (lane 3). B, peptide-2 phosphorylation by PKCϵ in cell-free system. The PKCϵ kinase reaction was performed in the reaction mixture containing [³²P]ATP, peptide-2, and CD44-associated PKCϵ (isolated from MCF-7 cells treated with no HA (lane 1) or with a 10-min HA (50 μg/ml) addition (lane 2) or pretreated with anti-CD44 antibody followed by a 10-min HA (50 μg/ml) addition (lane 3)). Subsequently, the activity of PKCϵ isolated from these samples was determined by the amount of [³²P]ATP incorporated into a PKCϵ substrate, peptide-2, as described under “Materials and Methods.” (The amount of [³²P]ATP incorporated into peptide-2 by PKCϵ isolated from cells treated with no HA (control) is designated as 100%. The values expressed in this figure represent an average of triplicate determinations of four experiments with an S.D. value less than ±5%.)

Hyaluronan-CD44 Interaction with Protein Kinase Cϵ Promotes Oncogenic Signaling by the Stem Cell Marker Nanog and the Production of MicroRNA-21, Leading to Down-regulation of the Tumor Suppressor Protein PDCD4, Anti-apoptosis, and Chemotherapy Resistance in Breast Tumor Cells

J Biol Chem. J Biol Chem;284(39):26533-26546.

FIGURE 5.

Detection of HA-CD44-induced miR-21 production in MCF-7 cells. A and B, detection of both pre-miRNA and mature miR-21 in MCF-7 cells using Northern blot analysis, as described under “Materials and Methods.” A, autoradiogram of both pre-miRNA and mature miR-21 detected in MCF-7 cells treated with no HA (lane 1), treated with HA for 2 h (lane 2), or pretreated with anti-CD44, followed by HA addition for 2 h (lane 3). B, autoradiogram of both pre-miRNA and mature miR-21 detected in MCF cells incubated with scrambled sequence siRNA (without HA (lane 1) or with a 2-h HA treatment (lane 2)), incubated with PKCϵ siRNA plus a 2-h HA treatment (lane 3), incubated with Nanog siRNA plus a 2-h HA treatment (lane 4), incubated with miRNA-negative control (without HA (lane 5) or with a 2-h HA treatment (lane 6)), or incubated with anti-miR-21 inhibitor plus a 2-h HA treatment (lane 7). nt, nucleotide; 10 and 75 nucleotides were used as miRNA size markers. Ethidium bromide-stained 5 S rRNA in each gel lane was used as a loading control. C and D, detection of miR-21 in MCF-7 cells using RNase protection assay as described under “Materials and Methods.” C, autoradiogram of miR-21 detected in MCF-7 cells treated with no HA (lane 1), treated with HA for 2 h (lane 2), or pretreated with anti-CD44 followed by the addition of HA for 2 h (lane 3). D, autoradiogram of miR-21 detected in MCF cells incubated with scrambled sequence siRNA (without HA (lane 1) or with a 2-h HA treatment (lane 2)), incubated with PKCϵ siRNA plus a 2-h HA treatment (lane 3), incubated with Nanog siRNA plus a 2-h HA treatment (lane 4), incubated with miRNA-negative control (without HA (lane 5) or with a 2-h HA treatment (lane 6)), or incubated with anti-miR-21 inhibitor plus a 2-h HA treatment (lane 7). (An autoradiogram of miR-191 in each gel lane was used as a loading control.)

J Biol Chem. J Biol Chem;284(39):26533-26546.

FIGURE 2.

Detection of HA-CD44-mediated Nanog phosphorylation by PKCϵ in cell-free systems and in whole cells. A, Nanog phosphorylation by PKCϵ in a cell-free system. The PKCϵ kinase reaction was carried out in the reaction mixture containing [³²P]ATP, purified Nanog, and CD44-associated PKCϵ isolated from MCF-7 cells treated with no HA (lane 1), treated with HA for 10 min (lane 2), pretreated with anti-CD44 antibody followed by a 10-min HA addition (lane 3), treated with scrambled sequence siRNA (without HA (lane 4) or with a 10-min HA addition (lane 5)), or treated with PKCϵ siRNA (without HA (lane 6) or with a 10-min HA addition (lane 7)). Subsequently, the activity of PKCϵ isolated from these samples was determined by the amount of [³²P]ATP incorporated into a PKCϵ substrate, Nanog, as described under “Materials and Methods.” (The amount of [³²P]ATP-incorporated into Nanog by PKCϵ isolated from cells treated with no HA (control) is designated as 100%. The values expressed in this figure represent an average of triplicate determinations of three experiments with an S.D. less than ±5%.) B, Nanog phosphorylation in whole cells. Phosphorylation of Nanog was analyzed by solubilizing MCF-7 cells with 1% Nonidet P-40 (Nonidet P-40) buffer, followed by immunoprecipitation (IP) with anti-Nanog antibody followed by immunoblotting with anti-phosphoserine antibody (a) or anti-Nanog antibody (as a loading control) (b), using cell lysates obtained from MCF-7 cells treated with no HA (lane 1), treated with HA for 15 min (lane 2), pretreated with anti-CD44 followed by a 15-min HA addition (lane 3), treated with scrambled sequence siRNA (without HA (lane 4) or with HA for 15 min (lane 5)), or treated with PKCϵ siRNA (without HA (lane 6) or with HA for 15 min (lane 7)).

J Biol Chem. J Biol Chem;284(39):26533-26546.

FIGURE 6.

Analyses of HA-CD44-mediated PDCD4-eIF4A expression and PDCD4-eIF4A interaction in MCF-7 cells. Detection of HA-CD44-induced PDCD4-eIF4A expression in MCF-7 cells was carried out by solubilizing cells with 1% Nonidet P-40 (Nonidet P-40) buffer, followed by immunoblotting with anti-PDCD4 antibody or anti-eIF4A antibody, respectively (A). The formation of PDCD4-eIF4A complex was also analyzed by immunoprecipitating the cell lysate with anti-PDCD4 antibody, followed by immunoblotting with anti-eIF4A antibody (B), as described under “Materials and Methods.” A, detection of the expression of PDCD4 or eIF4A by anti-PDCD4-mediated (a) or anti-eIF4A-mediated (b) immunoblotting using cell lysate isolated from MCF-7 cells treated with no HA (lane 1), treated with HA for 24 h (lane 2), pretreated with anti-CD44 followed by a 24-h HA addition (lane 3), treated with scrambled sequence siRNA (without HA (lane 3) or with HA for 24 h (lane 4)), treated with PKCϵ siRNA plus HA for 24 h (lane 6), treated with Nanog siRNA plus HA for 24 h (lane 7), treated with miRNA-negative control (without HA (lane 8) or with HA for 24 h (lane 9)), or treated with anti-miR-21 inhibitor plus HA for 24 h (lane 10). The amount of actin detected by anti-actin-mediated immunoblot in each gel lane was used as a loading control. B, detection of the PDCD4-eIF4A complex by anti-PDCD4-mediated immunoprecipitation (IP) (a), followed by anti-eIF4A-mediated immunoblotting (b) (as a loading control) using cell lysate isolated from MCF-7 cells treated with no HA (lane 1), treated with HA for 24 h (lane 2), pretreated with anti-CD44 followed by a 24-h HA addition (lane 3), treated with scrambled sequence siRNA (without HA (lane 3) or with HA for 24 h (lane 4)), treated with PKCϵ siRNA plus HA for 24 h (lane 6), treated with Nanog siRNA plus HA for 24 h (lane 7), treated with miRNA-negative control (without HA (lane 8) or with HA for 24 h (lane 9), or treated with anti-miR-21 inhibitor plus HA for 24 h (lane 10).

J Biol Chem. J Biol Chem;284(39):26533-26546.

FIGURE 8.

A proposed model for HA-CD44-mediated PKCϵ activation and Nanog signaling in the regulation of miRNA-21 production, oncogenesis, and chemoresistance in breast tumor cells. The binding of HA to CD44 (step 1) promotes PKCϵ activity (step 2), which, in turn, causes phosphorylation of Nanog (step 3). Phosphorylated Nanog then translocates from the cytosol to the nucleus and interacts with the microprocessor complex containing the RNase III (DROSHA) and the RNA helicase (p68) (step 4), resulting in miR-21 production (step 5). The resultant miR-21 then functions to down-regulate the tumor suppressor protein (PDCD4) (step 6a; indicated by the arrow with solid lines) and promotes oncogenesis (step 7a; indicated by the arrow with solid lines), leading to IAP (survivin and XIAP)/MDR1 (P-gp) expression, breast tumor cell anti-apoptosis/survival, and chemoresistance (step 8a; indicated by the arrow with solid lines). In direct contrast, treatment of breast tumor cells with an anti-miR-21 inhibitor (indicated by the arrow with dashed lines) induces tumor suppressor protein (PDCD4) up-regulation and PDCD4-eIF4A complex formation, which then inhibits translational machinery (step 6b; indicated by the arrow with dashed lines). Subsequently, these changes result in the inhibition of IAP (survivin and XIAP) and MDR1 expression (step 7b; indicated by the arrow with dashed lines), stimulation of apoptosis/cell death, and enhancement of chemosensitivity (step 8b; indicated by the arrow with dashed lines) in breast tumor cells. This newly discovered PKCϵ-Nanog signaling pathway leading to miR-21 functioning should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemoresistance in HA-CD44-activated breast cancer cells.

J Biol Chem. J Biol Chem;284(39):26533-26546.

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Hyaluronan-CD44 interaction with protein kinase C(epsilon) promotes oncogenic signaling by the stem cell marker Nanog and the Production of microRNA-21, leading to down-regulation of the tumor suppressor protein PDCD4, anti-apoptosis, and chemotherapy resistance in breast tumor cells.

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