Stem cell marker (Nanog) and Stat-3 signaling promote MicroRNA-21 expression and chemoresistance in hyaluronan/CD44-activated head and neck squamous cell carcinoma cells

Abstract

MicroRNAs are often associated with the pathogenesis of many cancers, including head and neck squamous cell carcinoma (HNSCC). In particular, microRNA-21 (miR-21) appears to have a critical role in tumor cell survival, chemoresistance and HNSCC progression. In this study, we investigated matrix hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with the stem cell markers, Nanog and Stat-3, in HNSCC cells (HSC-3 cells). Our results indicate that HA binding to CD44 promotes Nanog-Stat-3 (also tyrosine phosphorylated Stat-3) complex formation, nuclear translocation and transcriptional activation. Further analyses reveal that miR-21 is controlled by an upstream promoter containing Stat-3 binding site(s), while chromatin immunoprecipitation assays demonstrate that stimulation of miR-21 expression by HA/CD44 signaling is Nanog/Stat-3-dependent in HNSCC cells. This process results in a decrease of a tumor suppressor protein (PDCD4), and an upregulation of i nhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in HSC-3 cells. Treatment of HSC-3 cells with Nanog- and/or Stat-3-specific small interfering RNAs effectively blocks HA-mediated Nanog-Stat-3 signaling events, abrogates miR-21 production and increases PDCD4 expression. Subsequently, this Nanog-Stat-3 signaling inhibition causes downregulation of survival protein (IAP) expression and enhancement of chemosensitivity. To further evaluate the role of miR-21 in tumor cell-specific functions, HSC-3 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and block its target functions. Our results demonstrate that anti-miR-21 inhibitor not only upregulates PDCD4 expression but also decreases IAP expression and enhances chemosensitivity in HA-treated HNSCC cells. Together, these findings indicate that the HA-induced CD44 interaction with Nanog and Stat-3 has a pivotal role in miR-21 production leading to PDCD4 reduction, IAP upregulation and chemoresistance in HNSCC cells. This novel Nanog/Stat-3 signaling pathway-specific mechanism involved in miR-21 production is significant for the formation of future intervention strategies in the treatment of HA/CD44-activated HNSCC.

Fig. 1. Analyses of Nanog and Stat-3 expression in HNSCC patient samples and HSC-3 cells

A: Detection of Nanog and Stat-3 expression in head and neck squamous cell carcinoma (HNSCC) primary tumors. A panel of immune-reagents including rabbit anti-Nanog antibody, rabbit anti-Stat-3 antibody or rabbit anti-phospho-Stat-3 (pY[705]) antibody were used to examine the expression of both Nanog and Stat-3 in HNSCC primary tumors using immuno-peroxidase staining (a and b) or co-localization of Nanog with phosphorylated Stat-3 (p-Stat-3) (c-e) and total stat-3 (f-h) using double immunofluorescence staining as described in the Materials and Methods. B: Time course analyses of HA/CD44-induced Stat-3 tyrosine phosphorylation: Cell lysates were prepared from HSC-3 treated with HA (50μg/ml) for various time intervals (e.g., 0, 5, 10, 15 and 60min) followed by anti-CD44-immunoprecipitation followed by immunoblotting with anti-phospho-Stat-3 [pY⁷⁰⁵] antibody or anti-CD44 antibody (as a loading control). C: Detection of Nanog and Stat-3 (or phosphorylated Stat-3) in the CD44 complex by anti-CD44-immunoprecipitation followed by immunoblotting with anti-Nanog antibody (a) or anti-Stat-3 antibody (b) or anti-phospho-Stat-3 [pY⁷⁰⁵] antibody (c) or reblotting with anti-CD44 (d) as a loading control using HSC-3 cells treated with no HA or with HA (50μg/ml) for 5min or pretreated with anti-CD44 antibody for 1h followed by 5min HA (50μg/ml) addition.

Fig. 2. Immunocytochemical and biochemical analyses of HA/CD44-induced Nanog and Stat-3 nuclear translocation in HSC-3 cells

A: Immunostaining of Nanog [using Texas Red-labeled anti-Nanog (red color) and Stat-3 using FITC-labeled anti-Stat-3 (green color)]: HSC-3 cells (untreated or pretreated with anti-CD44 antibody for 1h) were incubated with HA (50μg/ml) (or without HA) for 15min at 37°C and fixed by 2% paraformaldehyde. Subsequently, these cells were rendered permeable by ethanol treatment and immuno-stained with Texas Red-labeled anti-Nanog (red color) or FITC-labeled anti-Stat-3 (green color) and DAPI (a nuclear marker) (c) as described in the Materials and Methods. B: Analyses of HA/CD44-induced Nanog and Stat-3 complex formation in the nuclear fraction: HSC-3 cells (untreated or pretreated with anti-CD44 antibody for 1h) were incubated with HA (50μg/ml) (or without HA) for 15min at 37°C. Nuclear fractions of these cells were then prepared by anti-Nanog-immunoprecipitation followed by immunoblotting with anti-Stat-3 antibody (a) or anti-phospho-Stat-3 (pY⁷⁰⁵) (b) or reblotting with anti-Nanog (c) as a loading control as described in the Materials and Methods [untreated cells (-HA); cells treated with HA (+HA) for 15min; lane 3: cells pretreated with anti-CD44 antibody for 1h plus 15min HA addition (anti-CD44 +HA)]. [The ratio of Stat-3 or phosphorylated Stat-3 and Nanog (the loading control) (d) was determined by densitometry, and the levels were normalized to untreated (no HA treatment) value (designated as 1.00); the values expressed represent an average of triplicate determination of 3 experiments with an SD of less than 5%].

Fig. 3. Interaction between Nanog, Stat-3 and the upstream promoter/enhancer region of miR-21 promoter in HSC-3 cells

A: In vivo binding of Nanog and Stat-3 (or phosphorylated Stat-3) to the miR-21 upstream promoter/enhancer region in HSC-3 cells: ChIP assay was performed in HSC-3 cells following protocols described in the Materials and Methods using the Stat-3 binding site-containing miR-21 promoter (upstream promoter/enhancer region)-specific primers by PCR. Identical volumes from the final precipitated materials were used for the PCR reactions [untreated cells (lane 1); cells treated with HA for 30min (lane 2); cells pretreated with anti-CD44 antibody for 1h plus 30min HA addition (lane 3); cells pretreated with scrambled siRNA with no HA (lane 4); cells pretreated with scrambled siRNA plus 30min HA addition (lane 5); cells pretreated with Nanog siRNA plus 30min HA addition (lane 6); cells pretreated with Stat-3 siRNA plus 30min HA addition) (lane 7). (a: anti-Stat-3-mediated immunoprecipitated material; b: anti-phospho-Stat-3 (pY⁷⁰⁵)-mediated immunoprecipitated material; c: anti-Nanog-mediated immunoprecipitated material; d: IgG isotype control-mediated precipitated material; e: total input materials). B: Verification of the specificity of Nanog siRNA or Stat-3 siRNA used in the study. Cell lysates isolated from HSC-3 cells [transfected with Nanog siRNA or Stat-3 siRNA-target or scrambled siRNA] were solubilized by 1% Nonidet P-40 (NP-40) buffer followed by immunoblotting with anti-Stat-3 antibody (a), anti-Nanog antibody (b) or anti-actin (a loading control) (c).

Fig. 4. Detection of HA/CD44-induced miR-21 production in HSC-3 cells

Detection of miR-21 in HSC-3 cells using RNase protection assay as described in the Materials and methods. A: Autoradiogram of miR-21 detected in HSC-3 cells incubated with scrambled sequence siRNA [without HA (lane 1) or with 2h HA treatment (lane 2) or pretreated with anti-CD44 antibody for 1h followed by HA addition for 2h (lane 3) or incubated with Nanog siRNA plus 2h HA treatment (lane 4) or incubated with Stat-3 siRNA plus 2h HA treatment (lane 5) or incubated with miRNA-negative control [without HA (lane 6) or with 2h HA treatment (lane 7)] or incubated with an anti-miR-21 inhibitor plus 2h HA treatment (lane 8). (Autoradiogram of miR-191 in each gel lane was used as a loading control).

Fig. 5. Analyses of HA/CD44-mediated PDCD4 and IAP expression in HSC-3 cells

Detection of HA/CD44-induced PDCD4 and IAP (cIAP-1, cIAP-2 and XIAP) expression in HSC-3 cells was performed by solubilizing cells with 1% Nonidet P-40 (NP-40) buffer followed by immunobloting with anti-PDCD4 antibody or anti-cIAP-1 antibody or anti-cIAP-2 antibody or anti-XIAP antibody, respectively as described in the Materials and Methods. First, cell lysates were prepared from HSC-3 cells treated with scrambled sequence siRNA [without HA (lane 1) or with HA for 24h (lane 2)] or treated with anti-CD44 antibody for 1h followed by 24h HA addition (lane 3) or treated with Nanog siRNA plus HA for 24h (lane 4) or treated with Stat-3 siRNA plus HA for 24h (lane 5) or treated with miRNA-negative control [without HA (lane 6) or with HA for 24h (lane 7)] or treated with anti-miR-21 inhibitor plus HA for 24h (lane 8). These samples were then immunoblotted with anti-PDCD4 antibody (a) or anti-cIAP-1 antibody (b) or anti-cIAP-2 antibody (c) or anti-XIAP antibody (d), respectively. The amount of actin detected by anti-actin-mediated immunoblot (e) in each gel lane was used as a loading control.

Fig. 6. A proposed model for HA/CD44-mediated Nanog and Stat-3 signaling in the regulation of miRNA-21 production, oncogenesis and chemoresistence in HNSCC cells

The binding of HA to CD44 (step 1) promotes a complex formation between Nanog/Stat-3 (also tyrosine phosphorylated Stat-3, p-Stat-3) and CD44 (step 2). Both Nanog and Stat-3 (p-Stat-3) then translocate from the cytosol to the nucleus and interacts with the Stat-3-binding sites located at the upstream promoter/enhancer region of miR-21 (step 4), resulting in miR-21 gene expression and mature miR-21 production (step 5). The resultant miR-21 then functions to downregulate the tumor suppressor protein (PDCD4) (step 6a, indicated by the arrow with solid lines) and promote HNSCC activation leading to IAP (c-IAP-1, c-IAP-2 and XIAP) expression, HNSCC cell anti-apoptosis/survival and chemoresistance (step 7a, indicated by the arrow with solid lines). In direct contrast, treatment of HNSCC cells with an anti-miR-21 inhibitor induces tumor suppressor protein (PDCD4) upregulation (step 6b, indicated by the arrow with dash lines). Subsequently, these changes result in HNSCC cell inactivation including inhibition of IAP (c-IAP-1, c-IAP-2 and XIAP) expression (Fig. 7b, indicated by the arrow with dash lines), stimulation of apoptosis and enhancement of chemosensitivity in HNSCC cells. This newly-discovered Nanog/Stat-3 signaling pathway leading to miR-21 functioning should provide important drug targets for sensitizing tumor cell apoptosis and overcome chemoresistance in HA/CD44-activated head and neck cancer cells.