Hyaluronan-CD44 interaction promotes c-Jun signaling and miRNA21 expression leading to Bcl-2 expression and chemoresistance in breast cancer cells
© Chen and Bourguignon; licensee BioMed Central Ltd. 2014
Received: 26 September 2013
Accepted: 25 February 2014
Published: 8 March 2014
MicroRNA-21 (miR-21) is associated with the development of solid tumors progression including breast cancer. In this study we investigated matrix hyaluronan (HA)-CD44 (a primary HA receptor) interaction with c-Jun N-Terminal Kinase (JNK)/c-Jun signaling in MDA-MB-468 breast cancer cells [a triple-negative (estrogen receptor-negative/progesterone receptor-negative/HER2-negative) breast cancer cell line]. Our results indicated that HA binding to CD44 promotes c-Jun nuclear translocation and transcriptional activation. Further analyses revealed that miR-21 is regulated by an upstream promoter containing AP1 binding site(s), and chromatin immunoprecipitation (CHIP) assays demonstrated that stimulation of miR-21 expression by HA/CD44 interaction is c-Jun-dependent in these breast cancer cells. This process results in an increase of the anti-apoptosis protein Bcl-2 and upregulation of inhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in MDA-MB-468 cells. Treatment with c-Jun specific small interfering RNAs effectively blocks HA-mediated c-Jun signaling and abrogates miR-21 production as well as causes downregulation of survival proteins (Bcl-2 and IAPs) and enhancement of chemosensitivity. In addition, our results demonstrated that anti-miR-21 inhibitor not only downregulates Bcl-2/IAP expression but also increases chemosensitivity in HA-treated breast cancer cells. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in miR-21 production leading to survival protein (Bcl-2/IAP) upregulation and chemoresistance in triple negative breast cancer cells such as MDA-MB-468 cell line. This novel HA/CD44-mediated c-Jun signaling pathway and miR-21 production provide a new drug target for the future intervention strategies to treat breast cancer.
Matrix Hyaluronan (HA) is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues . As a major component in the extracellular matrix of most mammalian tissues, HA contributes significantly to cell adhesion, proliferation and migration/invasion [2–4]. There is also a great deal of evidence linking high level of HA production in human carcinomas to aggressive phenotypes and metastasis, including the progression of breast cancer [2–7].
CD44 is a family of cell-surface glycoproteins that are expressed in a variety of tissues, including breast cancer tissues [2, 3]. RHAMM whose cell surface form is now designated as CD168, was also found in breast cancer cells [8, 9]. Both CD44 and RHAMM mediate hyaluronan signaling . However, these two HA receptors likely regulate cellular signaling by different mechanisms because they are not homologous proteins, are compartmentalized differently in the cell, and differ in the way by which they bind to HA . Since CD44 was identified as the first integral HA binding “receptor”, HA-mediated CD44 signaling has received a great deal of attention in cancer field. Both CD44 and HA are overexpressed/elevated at sites of tumor attachment [1, 4]. HA binding to CD44 not only affects cell adhesion to extracellular matrix (ECM) components, but also stimulates a variety of tumor cell-specific functions leading to breast cancer progression [2, 3, 11–14]. However, the oncogenic mechanism(s) occurring during HA-activated and CD44-specific breast cancer progression remain(s) to be determined.
Jun N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase family, and are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock . Activation of JNKs by targeting phosphorylation of downstream effector proteins (e.g., c-Jun, ATF2, ELK1, SMAD4, p53 and HSF1) leads to a number of important cellular functions including cell growth, differentiation, survival and apoptosis [15, 16]. Among these JNK-regulated target proteins, c-Jun was initially identified as the c-Fos-binding protein. The association between c-Jun and c-Fos forms the AP-1 early response transcription factor complex which then binds to DNA sequences located in the promoter regions of genes stimulated by externally added agonists . In human cancer, the level of c-Jun and c-fos mRNA and AP-1 expression has been shown to be elevated in drug-resistant tumor cells (such as etoposide resistant human leukemia K562 cells) as compared to the c-Jun/c-fos mRNA/AP-1 levels found in drug-sensitive parental lines . Mitogenic stimulation of breast tumor cells (MCF-7 cell line) by insulin or insulin-like growth factors (IGFs) has been shown to promote c-Jun or c-fos upregulation and AP-1 activity . Previous studies showed that persistent expression of c-Jun protein prevents stromal cells from entering apoptosis during the late secretory phase . CD44 ligation blocks cell cycle progression of myeloid leukemia cells by downregulating c-Jun expression . These observations suggest that c-Jun signaling is involved in regulating tumor cell growth, survival/anti-apoptosis and chemoresisitance.
MicroRNAs (miRNAs) are single-stranded RNAs of 21–25 nucleotides in length, which have been found to modulate gene expression at the posttranslational level . MicroRNAs (miRNAs) are essential for normal development as modulators of gene expression. An estimated 30%-60% of the genome is regulated by miRNA-mediated silencing , however aberrant expression of miRNAs is associated with many diseases, including cancer. Recent studies indicate that that some microRNAs upregulate the expression of its target gene by binding to the 3′ UTR [23, 24]. Overexpression of miR-21 influences cell proliferation, invasion, metastasis and chemoresistance in different cancer cells including breast cancer cells [25–27]. The identified targets of miR-21 in human cancer cells include a tumor suppressor protein [Program Cell Death 4 (PDCD4)] . A previous study indicated that HA-CD44 interaction promotes miR-21 production, and PDCD4 reduction in both breast cancer cells (MCF-7 cell line) and head and neck cancer cells (HSC-3 cell line) [25, 29]. This event contributes to upregulation of inhibitors of apoptosis proteins (IAPs) and the multidrug resistant protein (MDR1)/P-glycoprotein (P-gp) resulting in anti-apoptosis and chemotherapy resistance in breast tumor cells (MCF-7 cell line) . Thus, miR-21 is currently considered to be an oncogene. A recent report indicates that miR-21 can also stimulate the expression of an anti-apoptosis protein, Bcl-2 by binding directly to the 3′UTR of Bcl-2 mRNA . Upregulation of Bcl-2 expression by miR-21 is associated with anti-apoptosis, chemoresistance and proliferation in pancreatic cancer cells . The question of whether Bcl-2 expression is associated with miR-21 production in HA-treated breast tumor cells has not been addressed.
In this study we investigated a new HA/CD44-mediated c-Jun signaling pathway that regulates miR-21 production and chemoresistance in MDA-MB-468 cell line (a triple negative breast cancer cell line). Our results indicated that HA/CD44 activates c-Jun signaling which, in turn, stimulates miR-21 expression and function. These events lead to the production of an anti-apoptosis protein, Bcl-2 and upregulation of survival proteins (IAPs) and Doxorubicin chemoresistance in MDA-MB-468 cells. cells. Inhibition of c-Jun signaling or silencing miR-21 expression/function not only results in Bcl-2 downregulation, but also causes a reduction of survival protein expression and enhances chemosensitivity to Doxorubicin. Thus, our findings strongly support the contention that HA/CD44-regulated c-Jun and miR-21 form a functional signaling axis that regulates tumor cell survival and Doxorubicin chemoresistance in triple negative breast cancer cells such as MDA-MB-468 cells.
HA-CD44 interaction activates JNK and c-Jun signaling in breast tumor cells
HA-CD44 binding promotes nuclear translocation of c-Jun in MDA-MB-468 cells
Measurement of HA-induced p-c-Jun and c-Jun nuclear accumulation in MDA-MB-468 cells
p-c-Jun nuclear accumulation (% of control)
c-Jun nuclear accumulation (% of control)
No HA (control)
100% ± 5
100% ± 3
275% ± 13a
260% ± 10b
Non-immune IgG (No HA)
102% ± 4a
101% ± 5b
Non-immune IgG (+HA)
268% ± 9a
275% ± 11b
Anti-CD44 antibody + HA
92% ± 3a
87% ± 3b
Vehicle control + HA
262% ± 8a
279% ± 8b
JNK inhibitor + HA
84% ± 4a
75% ± 3b
The reason for showing both phospho-c-Jun and total c-Jun in MDA-MB-468 cells following HA treatment is to determine whether phosphorylated c-Jun represents the majority or a minority species of total c-Jun. The fact that the JNK inhibitor prevents nuclear translocation of both phospho-c-Jun and Jun suggests that majority of c-Jun is phosphorylated by JNK. This explains the effect of JNK inhibitor on blocking both phosphorylated c-Jun and total c-Jun nuclear accumulation in cells treated with HA. These findings strongly suggest that the HA-CD44 interaction promotes c-Jun nuclear translocation in MDA-MB-468 cells in a JNK-dependent manner.
Role of c-Jun in regulating miR-21 gene expression in HA/CD44
To confirm the direct involvement of JNK-mediated c-Jun signaling in miR-21 gene upregulation, JNK activity was blocked by a JNK Inhibitor, 420116, (Figure 3B) and c-Jun was downregulated by c-Jun small interfering RNA (siRNA), (Figure 3C) followed by the miR-21 promoter-specific ChIP assay as described above. Our results indicate that (i) inhibition of c-JNK (but not vehicle control samples) (Figure 3B-a, b-lane 2 vs. lane 1) or (ii) transfection of MDA-MB-468 cells with c-Jun siRNAs (but not scrambled sequenced siRNA) (Figure 3C-a, b-lane 2 vs. lane 1) effectively blocked HA-mediated c-Jun/phospho-c-Jun binding to the miR-21 upstream/enhancer promoter region with AP1 binding sites in MDA-MB-468 cells. Identical amplification products were detected in the positive controls from total input chromatin (Figure 3A, B, C-d-all lines). Moreover, no amplification was seen in samples that were processed by IgG isotype control-mediated precipitation (Figure 3A, B, C-c-all lanes). Therefore, we concluded that downregulation of JNK activity or c-Jun/phospho-c-Jun expression by either JNK inhibitor (420116) or c-Jun siRNA is specific.
HA-CD44-activated JNK/c-Jun signaling stimulates miRNA-21 production in MDA-MB-468 Cells
The impact of HA/CD44-mediated miR-21 (induced by c-Jun signaling) on Bcl2/IAP expression, anti-apoptosis and chemoresistance in MDA-MB-468 cells
Measurement of doxorubicin-induced MDA-MB-468 cell apoptosis and growth inhibition
Apoptotic cells (Annexin V-positive cell/total cells × 100%)*
Doxorubicin-induced tumor cell growth inhibition IC50(μM)**
(A) Effects of various signaling perturbation agents on doxorubicin--induced apoptosis and cell growth inhibition in MDA-MB-468 cells.
(a) Effects of Doxorubicin-induced apoptosis and cell growth inhibition in MDA-MB-468 cells following HA treatment:
No HA (Untreated cells)
4.1 ± 0.5
30.2 ± 3.1a
73.5 ± 5.1
2.4 ± 0.3a
12.6 ± 2.3a
188.0 ± 10.5b
Non-immune rat IgG-treated cells (No HA)
3.8 ± 0.5a
29.2 ± 2.2a
70.3 ± 4.1b
Non-immune rat IgG-treated cells (+ HA)
2.2 ± 0.2a
10.5 ± 2.0a
176.0 ± 10.3b
Anti-CD44-treated cells (+ HA)
3.8 ± 0.2a
29.2 ± 2.2a
28.4 ± 3.6b
(b) Effects of Doxorubicin-induced apoptosis and cell growth inhibition in MDA-MB-468 cells treated with c-Jun siRNA plus HA:
Scrambled siRNA-treated cells (No HA)
5.2 ± 0.6
32.4 ± 3.6c
85.0 ± 2.4
Scrambled siRNA-treated cells (+ HA)
4.5 ± 0.5c
11.9 ± 3.1c
190.0 ± 6.00d
c-Jun siRNA-treated cells (No HA)
5.2 ± 0.6c
33.6 ± 2.8c
40.5 ± 0.30d
c-Jun siRNA-treated cells (+ HA)
4.5 ± 0.5c
31.9 ± 3.2c
39.2 ± 0.80d
(c) Effects of Doxorubicin-induced apoptosis and cell growth inhibition in MDA-MB-468 cells treated with anti-miR-21 inhibitor plus HA:
miRNA negative control-treated cells (No HA)
3.7 ± 0.4
31.6 ± 2.9e
62.8 ± 0.5
miRNA negative control-treated cells (+ HA)
2.5 ± 0.2e
12.3 ± 0.4e
196.0 ± 4.2f
Anti-miR-21 inhibitor-treated cells (No HA)
3.2 ± 0.6e
38 ± 2.5e
65.0 ± 3.0f
Anti-miR-21 inhibitor-treated cells (+ HA)
3.1 ± 0.4e
34 ± 2.1e
70.0 ± 0.2f
(B) Effects of JNK inhibitor (420116) on Doxorubicin-induced apoptosis and cell growth inhibition in MDA-MB-468 cells.
Vehicle control-treated cells (No HA)
4.3 ± 0.2
32 ± 2.4g
64.00 ± 6.2
Vehicle control-treated cells (+ HA)
2.6 ± 0.4g
14 ± 0.5g
180.0 ± 10.0h
JNK inhibitor (420116)-treated cells (No HA)
12.0 ± 0.5g
35 ± 2.6g
26.0 ± 3.5h
JNK inhibitor (420116)-treated cells (+ HA)
3.2 ± 0.3g
38 ± 3.3g
25.0 ± 0.2h
Hyaluronan (HA) is an important structural component of the extracellular matrix (ECM). In cancer patients, the level of HA is usually higher in malignant tumors than in corresponding benign or normal tissues, and in some tumor types the level of HA is predictive of malignancy [2–7, 42]. In particular, HA level is elevated in the serum of breast cancer patients [4, 42]. The aberrant HA production by HA synthases [43–45] and HMW-HA degradation into LMW-HA by hyaluronidases  are thought to be closely associated with breast tumor cell progression .
HA binds specifically to CD44, a family of multifunctional transmembrane glycoproteins expressed in numerous cells and tissues, including breast tumor cells and various carcinoma tissues [2–4]. The crystal structure of the HA-CD44 complex was reported previously and a single HA binding site was identified . CD44 is generally expressed in a variety of isoforms that are products of a single gene generated by alternative splicing of variant exons inserted into an extracellular membrane-proximal site [48, 49]. CD44 is also expressed in tumor stem cells that have the unique ability to initiate tumor cell-specific properties [38, 50]. In fact, CD44 is considered to be one of the important surface markers on cancer stem cells [38, 50]. HA binding to CD44 is involved in the stimulation of both receptor kinases (e.g., ErbB2, EGFR and TGFβ receptors) and non-receptor kinases (e.g., c-Src and ROK)  required for a variety of tumor cell-specific functions leading to tumor progression.
Abnormal JNK/c-Jun signaling also appears to play a critical role in oncogenesis [34, 35]. JNK-activated c-Jun is a signal-transducing transcription factor of the AP-1 family that is implicated in cell cycle progression, differentiation and cell transformation . It has a direct role in regulating the transcription of p53 and cyclinD1 [52, 53]. It has also been shown that c-Jun accelerates leukemogenesis and regulates the activation of genes required for cell cycle progression in tumor cells . The AP-1 factor c-Jun is thought to act as a “bodyguard”, preventing methylation of a distinct set of genes after oncogenic transformation . Recently, c-Jun is found to trigger miR-21 transcription through AP-1 binding sites present in the miR-21 promotor region . In this study we observed that HA-CD44 binding results in c-Jun (also causes phosphorylation of c-Jun) nuclear localization in MDA-MB-468 cells (Figure 2). Thus, identifying specific genes that are transcriptionally controlled by the JNK/c-Jun signaling during HA-CD44 interaction in the nucleus may be essential for understanding the disease mechanism occurring during breast cancer progression.
Overexpression of miR-21 is detected in various breast cancer cell lines and patient specimens [25, 26]. Accumulating evidence indicates that miR-21 is closely associated with both cancer development and chemotherapy resistance . The stem cell marker, Nanog, has been found to be involved in the regulation of pri-miRNA expression during cancer development . Our previous work indicated that HA/CD44-activated PKCϵ promotes Nanog interaction with p68 and DROSHA leading to biosynthetic processing and production of miR-21 in breast tumor cells . These findings suggest that HA/CD44-mediated Nanog signaling is closely linked to miR-21 production and function during oncogenesis.
In this study, we provided new evidence that miR-21 expression is controlled by an upstream promoter/enhancer containing AP-1 binding sites in MDA-MB-468 cells while chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-21 production by HA is JNK and c-Jun-dependent in breast tumor cells (Figure 4). Most importantly, downregulation of JNK/c-Jun signaling (by treating cells with JNK inhibitor or c-Jun siRNA) or miR-21 (by treating cells with anti-miR-21 inhibitor) reduces the expression of the target protein, Bcl2, and anti-apoptotic proteins [e.g., IAPs (cIAP1/cIAP2/XIAP)] (Figure 5) in breast tumor cells. Determining the cellular and molecular mechanisms involved in the regulation of these causal links between JNK/c-Jun signaling and miR-21 function, including Bcl2 and IAP upregulation, awaits further investigation.
Chemotherapy resistance is one of the primary causes of morbidity in patients diagnosed with solid tumors including breast cancer [54–56]. Chemotherapeutic agents, such as doxorubicin, are commonly used to inhibit DNA synthesis in the treatment of breast cancer patients . In particular, the ability of doxorubicin to bind to DNA and/or produce free radicals is thought to be the mechanism for the induction of cytotoxic effects on tumor cells . However, this drug often displays limited cytotoxic killing and anti-tumor effects due to chemoresistance which occurs in de novo tumor cells .
It is now certain that a number of oncogenic signaling pathways are closely involved with chemotherapeutic drug resistant phenotypes [25, 29–31, 38, 58, 59]. In particular, matrix HA interaction with CD44 in cancer cells have been strongly implicated in the development of chemoresistance [25, 29–31, 38, 58, 59]. Specifically, HA is capable of stimulating MDR1 (P-gp) expression and drug resistance in breast tumor cells [30, 58, 59]. CD44 also interacts with MDR1 (P-gp) to promote cell migration and invasion of breast tumor cells [30, 58, 59]. Previously we reported that activation of HA-CD44-mediated oncogenic signaling events [e.g., mir-302/miR-21, intracellular Ca2+ mobilization, epidermal growth factor receptor (EGFR)-mediated ERK signaling, topoisomerase activation, and ankyrin-associated cytoskeleton function] leads to multidrug resistance in a variety of tumor cells [3, 10, 33]. These observations strongly suggest a functional link between HA-mediated CD44 signaling and drug resistance.
In this study we demonstrated that HA/CD44-activated JNK/c-Jun signaling and miR-21 increases survival protein, Bcl2, resulting in oncogenesis by enhancing the expression of inhibitors of anti-apoptosis proteins (IAPs) (Figure 5). Furthermore, downregulation of HA/CD44-activated JNK/c-Jun signaling (by JNK I nhibitor/c-Jun siRNA) and miR-21 production (by anti-miR-21 inhibitor) not only reduces Bcl2 upregulation (Figure 5), but also inhibits the expression of survival proteins (e.g., c-IAP1, c-IAP2 and XIAP) (Figure 5). Consequently, these signaling perturbation events contribute to apoptosis and chemosensitivity (Table 1). Furthermore, this newly-discovered HA/CD44-activated JNK/c-Jun signaling pathway and miR-21 production/function should provide important new drug targets to cause tumor cell apoptosis and overcome chemotherapy resistance in breast tumor cells.
Materials and methods
The cell line, MDA-MB-468 cells from ATCC, was isolated in 1977 by R. Cailleau, et al., from a pleural effusion of a 51-year-old Black female patient with metastatic adenocarcinoma of the breast. This cell line was cultured in ATCC-formulated Leibovitz’s L-15 Medium, Catalog No. 30-2008, with 10% fetal bovine serum.
Antibodies and reagents
Monoclonal rat anti-CD44 antibody (Clone: 020; Isotype: IgG2b; obtained from CMB-TECH Inc., San Francisco, CA, USA) recognizes a determinant of the HA-binding region common to CD44 and its principal variant isoforms. This rat anti-CD44 was routinely used for HA-related blocking experiments. Immunoreagents such as rabbit anti-C-JUN antibody, mouse anti-Bcl-2 antibody and goat anti-actin antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-c-IAP-1 antibody, mouse anti-c-IAP-2 and mouse anti-XIAP antibody were from BD (Franklin Lakes, NJ, USA). Rabbit anti-phospho-c-Jun [pS63] antibody, rabbit anti-c-Jun antibody, rabbit anti-JNK[pS63] antibody and rabbit anti-JNK antibody were from Cell Signaling Technology (Beverly, MA, USA). JNK Inhibitor I, 420116 was purchased from EMD Millipore (Billerica, MA, USA). Doxorubicin hydrochloride was from Sigma Chemicals (St. Louis, MO). Healon HA polymers (~500,000-dalton polymers), purchased from Pharmacia & Upjohn Co. (Kalamazoo, MI), were prepared as described previously [29, 31, 38].
Anti-miR-21 inhibitor preparation and transfection
MDA-MB-468 cells were transfected with anti-miR-21 inhibitor (Ambion, catalog number, 17000) (30 nmol/l) and its corresponding miRNA negative control (Ambion, catalog number, 17010) (30 nmol/l) using Lipofectamine 2000 reagent (Invitrogen) for 24 hours. Cells were then treated with HA or no HA in various experiments as described below.
The NP-40 solubilized cell lysate materials from MDA-MB-468 cells [untreated or pretreated with anti-CD44 antibody or JNK Inhibitor I, 420116 (20 μM) or vehicle control] plus 50 μg/ml HA (or no HA) for various time intervals (e.g. 0, 5 min, 15 min or 60 min or 24 h) at 37°C] were immunoblotted with rabbit anti-c-Jun antibody (2 μg/ml) or rabbit anti-phospho-c-Jun [pS63] antibody (2 μg/ml) or rabbit anti-c-JNK[pS183] antibody (2 μg/ml), respectively. In some cases, cell lysate of MDA-MB-468 cells (transfected with c-Jun siRNA or siRNA with scrambled sequences; or anti-miR-21 inhibitor or miRNA-negative control; or without any treatment) followed by HA (50 μg/ml) addition (or no HA or anti-CD44 antibody pretreatment plus HA) at 37°C were also immunoblotted using various immuno-reagents (e.g., mouse anti-Bcl-2 (2 μg/ml) or mouse anti-c-IAP-1 or mouse anti-c-IAP-2 antibody and anti-XIAP (2 μg/ml) or goat anti-actin (2 μg/ml) (as a loading control), respectively.
Chromatin immunoprecipitation assay
To examine whether c-Jun or phospho-c-Jun directly interacts with the upstream promoter/enhancer region (containing AP-1 binding site) of miR-21, chromatin immunoprecipitation (ChIP) assays was performed in MDA-MB-468 cells [pretreated with anti-CD44 antibody or JNK Inhibitor I, 420116 (20 μM)/vehicle control or transfected with c-Jun siRNA or siRNA with scrambled sequences] treated with HA (50 μg/ml) or without HA using a kit (EZ ChIP) from Millipore Corp according to the manufacturer’s instructions. Crosslinked chromatin lysates were sonicated and diluted with ChIP sonication buffer plus protease inhibitors, divided and incubated with normal rabbit IgG or rabbit anti-c-Jun antibody or rabbit phospho-c-Jun[pS63] antibody at 4°C overnight, then precipitated with protein G agarose. Crosslinking was reversed by overnight at 65°C incubation; DNA fragments were then extracted with PCR purification kit, analyzed by PCR and quantitated by PCR using primer pairs specific for the miR-21 upstream promoter/enhancer region containing the c-Jun binding sites: forward primer: 5′-TGGATAAGGATGACGCACAG-3′ and reverse primer: 5′-TGGTTTGAACCAATTAATAAGGAAA-3′ on an agarose gel as described previously [25, 29, 38, 60].
RNase protection assay analysis of mature miRNAs
Expression of miRNAs was qualitatively analyzed by RNase protection assay. For RNase protection assay, enriched small RNA isolated from MDA-MB-468 cells [transfected with scrambled sequence siRNA with (or without) anti-CD44 antibody or JNK Inhibitor I, 420116 (20 μM)/vehicle control or transfected with c-Jun siRNA or siRNA or anti-miR-21 inhibitor or miRNA-negative control in the presence or absence of HA for various time intervals (e.g., 0, 5 min, 10 min, 15 min, 30 min or 2 h) at 37°C] was enriched and purified using the mirVana miRNA Isolation kit (Ambion). RNA concentrations were verified by measuring absorbance (A260) on the NanoDrop Spectrophotometer ND-1000 (NanoDrop). The mirVana miRNA probe construction kit (Ambion) was used to synthesize the 32P-labeled miR-21 antisense probe and miR-191 probe loading control as described previously [25, 29, 38].
MDA-MB-468 cells (untreated or pretreated with anti-CD44 antibody) were incubated with HA (50 μg/ml) at 37°C for 30 minutes or with no HA. These cells were then fixed with 2% paraformaldehyde. Subsequently, these cells were rendered permeable by ethanol treatment followed by incubating with Texas Red-conjugated anti-phospho-c-Jun[pS63] antibody or fluorescein (FITC)-conjugated anti-c-Jun antibody followed by DAPI staining (a marker for nucleus). These fluorescence-labeled samples were then examined with a confocal laser scanning microscope.
Tumor cell growth and apoptosis assays
MDA-MB-468 cells were either untreated or pretreated with anti-CD44 antibody or treated with JNK inhibitor, 420116 (20 μM) or transfected with c-Jun siRNA or siRNA with scrambled sequences or anti-miR-21 or miRNA-negative control] in the presence or absence of 50 μg/ml HA, as above. These cells were then plated in 96-well culture plates in 0.2 ml of Dulbecco’s modified Eagle’s medium/F12 medium supplement (GIBCO, Grand Island, NY) containing no serum for 24 h at 37°C in 5% CO2/95% air. These cells (5 × 103 cells/well) were then incubated with various concentrations of Doxorubicin (4 × 10−9 M-1.75 × 10−5 M) with no HA or with HA (50 μg/ml). After 24h incubation at 37°C, MTT-based growth assays were analyzed as described previously [25, 30]. The percentage of absorbance relative to untreated controls (i.e., cells treated with neither HA nor chemotherapeutic drugs) was plotted as a linear function of drug concentration. The 50% inhibitory concentration (IC50) was identified as a concentration of drug required to achieve a 50% growth inhibition relative to untreated controls. For apoptosis assay, FITC-conjugated Annexin V (for measuring apoptotic cells) using Apoptosis Detection Kit (Calbiochem, San Diego, CA) was used according to the manufacturer’s protocol.
We gratefully acknowledge the assistance of Dr. Gerard J. Bourguignon in the preparation and review of this manuscript. This work was supported by Veterans Affairs (VA) Merit Review Awards (RR & D-1I01 RX000601 and BLR & D-5I01 BX000628), United States Public Health grants (R01 CA66163; P01 CA0052925) and DOD grant. L.Y.W.B is a VA Senior Research Career Scientist.
- Toole BP, Hascall VC: Hyaluronan and tumor growth. Am J Pathol. 2002, 161: 745-747. 10.1016/S0002-9440(10)64232-0PubMed CentralView ArticlePubMedGoogle Scholar
- Bourguignon LY: CD44-mediated oncogenic signaling and cytoskeleton activation during mammary tumor progression. J Mammary Gland Biol Neoplasia. 2001, 6: 287-297. 10.1023/A:1011371523994View ArticlePubMedGoogle Scholar
- Bourguignon LY: Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression. Sem. Cancer Biol. 2008, 18: 251-259. 10.1016/j.semcancer.2008.03.007.View ArticleGoogle Scholar
- Toole BP, Wight TN, Tammi MI: Hyaluronan-cell interactions in cancer and vascular disease. J Biol Chem. 2002, 277: 4593-4596. 10.1074/jbc.R100039200View ArticlePubMedGoogle Scholar
- Auvinen PK, Parkkinen JJ, Johansson RT, Agren UM, Tammi RH, Eskelinen MJ, Kosma VM: Expression of hyaluronan in begnign and malignant breast lesion. Int J Cancer. 1997, 74: 477-481. 10.1002/(SICI)1097-0215(19971021)74:5<477::AID-IJC1>3.0.CO;2-0View ArticlePubMedGoogle Scholar
- Auvinen P, Tammi R, Parkkinen J, Tammi M, Agren U, Johansson R, Hirvikoski P, Johansson R, Hirvikoski P, Eskelinen M, Kosma VM: Hyaluronan in peritumoral stroma and maligmnant cells associated with breast cancer spreading and predicts survival. Am J Pathol. 2000, 156: 529-536. 10.1016/S0002-9440(10)64757-8PubMed CentralView ArticlePubMedGoogle Scholar
- Karihtala P, Soini Y, Auvinen P, Tammi R, Tammi M, Kosma VM: Hyaluronan in breast cancer: correlations with nitric oxide synthases and tyrosine nitrosylation. J Histochem Cytochem. 2007, 55: 1191-1198. 10.1369/jhc.7A7270.2007View ArticlePubMedGoogle Scholar
- Assmann V, Marshall JF, Fieber C, Hofmann M, Hart IR: The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells. J Cell Sci. 1998, 111: 1685-1694.PubMedGoogle Scholar
- Wang C, Thor AD, Moore DH, Zhao Y, Kerschmann R, Stern R, Watson PH, Turley EA: The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression. Clin Cancer Res. 1998, 4: 567-576.PubMedGoogle Scholar
- Turley EA, Noble PW, Bourguignon LY: Signaling properties of hyaluronan receptors. J Biol Chem. 2002, 277: 4589-4592. 10.1074/jbc.R100038200View ArticlePubMedGoogle Scholar
- Bourguignon LY, Gunja-Smith Z, Iida N, Zhu HB, Young LJ, Muller WJ, Cardiff RD: CD44v(3, 8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. J Cell Physiol. 1998, 176: 206-215. 10.1002/(SICI)1097-4652(199807)176:1<206::AID-JCP22>3.0.CO;2-3View ArticlePubMedGoogle Scholar
- Bourguignon LY, Zhu H, Shao L, Zhu D, Chen YW: Rho-kinase (ROK) promotes CD44v(3, 8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells. Cell Motil Cytoskeleton. 1999, 43: 269-287. 10.1002/(SICI)1097-0169(1999)43:4<269::AID-CM1>3.0.CO;2-5View ArticlePubMedGoogle Scholar
- Bourguignon LY, Singleton PA, Zhu H, Diedrich F: Hyaluronan-mediated CD44 interaction with RhoGEF and Rho kinase promotes Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression. J Biol Chem. 2003, 278: 29420-29434. 10.1074/jbc.M301885200View ArticlePubMedGoogle Scholar
- Iida N, Bourguignon LY: Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis. J Cell Physiol. 1997, 171: 152-160. 10.1002/(SICI)1097-4652(199705)171:2<152::AID-JCP5>3.0.CO;2-NView ArticlePubMedGoogle Scholar
- Ip YT, Davis RJ: Signal transduction by the c-JUN N-terminal kinase (JNK)-from inflammation to development. Curr Opin Cell Biol. 1998, 2: 205-219.View ArticleGoogle Scholar
- Vlahopoulos S, Zoumpourlis VC: JNK: a key modulator of intracellular signaling. Biochemistry (Mosc). 2004, 69: 844-854.View ArticleGoogle Scholar
- Angel P, Imagawa M, Chiu RJ, Stein B, Inmbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M: Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987, 49: 729-739. 10.1016/0092-8674(87)90611-8View ArticlePubMedGoogle Scholar
- Ritke MK, Bergoltz VV, Allan WP, Yalowich JC: Increased c-JUN/AP-1 levels in etoposide-resistant human leukemia K562 cells. Biochem Pharmacol. 1994, 48: 525-533.View ArticlePubMedGoogle Scholar
- Smith LM, Wise SC, Hendricks DT, Sabichi AL, Bos T, Reddy P, Brown PH, Birrer MJ: cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype. Oncogene. 1999, 18: 6063-6070. 10.1038/sj.onc.1202989View ArticlePubMedGoogle Scholar
- Abdul A, Zada P, Singh SM, Reddy VA, Elsässer A, Meisel A, Haferlach T, Tenen DG, Hiddemann W, Behre G: Downregulation of c-JUN expression and cell cycle regulatory molecules in acute myeloid leukemia cells upon CD44 ligation. Oncogene. 2003, 22: 2296-2308. 10.1038/sj.onc.1206393View ArticleGoogle Scholar
- Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004, 116: 281-297. 10.1016/S0092-8674(04)00045-5View ArticlePubMedGoogle Scholar
- Lewis BP, Burge CB, Bartel DP: Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005, 120: 15-20. 10.1016/j.cell.2004.12.035View ArticlePubMedGoogle Scholar
- Ma F, Liu X, Li D, Wang P, Li N, Lu L, Cao X: MicroRNA-466l upregulates IL-10 expression in TLR-triggered macrophages by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation. J Immunol. 2010, 184: 6053-6059. 10.4049/jimmunol.0902308View ArticlePubMedGoogle Scholar
- Dong J, Zhao YP, Zhou L, Zhang TP, Chen G: Bcl-2 upregulation induced by miR-21 via a direct interaction is associated with apoptosis and chemoresistance in MIA PaCa-2 pancreatic cancer cells. Arch Med Res. 2011, 42: 8-14. 10.1016/j.arcmed.2011.01.006View ArticlePubMedGoogle Scholar
- Bourguignon LY, Spevak CC, Wong G, Xia W, Gilad E: 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. J Biol Chem. 2009, 284: 26533-26546. 10.1074/jbc.M109.027466PubMed CentralView ArticlePubMedGoogle Scholar
- Garzon R, Pichiorri F, Palumbo T, Iuliano R, Cimmino A, Aqeilan R, Volinia S, Bhatt D, Alder H, Marcucci G, Calin GA, Liu CG, Bloomfield CD, Andreeff M, Croce CM: MicroRNA fingerprints during human megakaryocytopoiesis. Proc Natl Acad Sci U S A. 2006, 103: 5078-5083. 10.1073/pnas.0600587103PubMed CentralView ArticlePubMedGoogle Scholar
- Song B, Wang C, Liu J, Wang X, Lv L, Wei L, Xie L, Zheng Y, Song X: MicroRNA-21 regulates breast cancer invasion partly by targeting tissue inhibitor of metalloproteinase 3 expression. J Exp Clin Cancer Res. 2010, 29: 29- 10.1186/1756-9966-29-29PubMed CentralView ArticlePubMedGoogle Scholar
- Lu Z, Liu M, Stribinskis V, Klinge CM, Ramos KS, Colburn NH, Li Y: MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene. Oncogene. 2008, 27: 4373-4379. 10.1038/onc.2008.72View ArticlePubMedGoogle Scholar
- Bourguignon LY, Earle C, Wong G, Spevak CC, Krueger K: 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. Oncogene. 2012, 31: 149-160. 10.1038/onc.2011.222PubMed CentralView ArticlePubMedGoogle Scholar
- Bourguignon LY, Peyrollier K, Xia W, Gilad E: Hyaluronan-CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells. J Biol Chem. 2008, 283: 17635-17651. 10.1074/jbc.M800109200PubMed CentralView ArticlePubMedGoogle Scholar
- Bourguignon LY, Wong G, Earle C, Krueger K, Spevak CC: Hyaluronan-CD44 interaction promotes c-Src-mediated Twist signaling, microRNA-10b expression and RhoA/RhoC upregulation leading to Rho-Kinase-associated cytoskeleton activation and breast tumor cell invasion. J Biol Chem. 2010, 285: 36721-36735. 10.1074/jbc.M110.162305PubMed CentralView ArticlePubMedGoogle Scholar
- Wang SJ, Wong G, de Heer AM, Xia W, Bourguignon LY: CD44 variant isoforms in head and neck squamous cell carcinoma progression. Laryngoscope. 2009, 119: 1518-1530. 10.1002/lary.20506PubMed CentralView ArticlePubMedGoogle Scholar
- Wang S, Bourguignon LY: Role of hyaluronan-mediated CD44 signaling in head and neck squamous cell carcinoma progression and chemoresistance. Am J Pathol. 2011, 178: 956-963. 10.1016/j.ajpath.2010.11.077PubMed CentralView ArticlePubMedGoogle Scholar
- Jiao X, Katiyar S, Willmarth NE, Liu M, Ma X, Flomenberg N, Lisanti MP, Pestell RG: c-Jun induces mammary epithelial cellular invasion and breast cancer stem cell expansion. J Biol Chem. 2010, 285: 8218-8226. 10.1074/jbc.M110.100792PubMed CentralView ArticlePubMedGoogle Scholar
- Wang J, Kuiatse I, Lee AV, Pan J, Giuliano A, Cui X: Sustained c-Jun-NH2-kinase activity promotes epithelial-mesenchymal transition, invasion, and survival of breast cancer cells by regulating extracellular signal-regulated kinase activation. Mol Cancer Res. 2010, 8: 266-277. 10.1158/1541-7786.MCR-09-0221PubMed CentralView ArticlePubMedGoogle Scholar
- Franklin CC, Sanchez V, Wagner F, Woodgett JR, Kraft AS: Phorbol ester-induced amino terminal phosphorylation of c-Jun but not JunB regulates transcriptional activation. Proc Natl Acad Sci U S A. 1992, 89: 7247-7251. 10.1073/pnas.89.15.7247PubMed CentralView ArticlePubMedGoogle Scholar
- Yu C, Minemoto Y, Zhang J, Liu J, Tang F, Bui TN, Xiang J, Lin A: JNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Mol Cell. 2004, 13: 329-340. 10.1016/S1097-2765(04)00028-0View ArticlePubMedGoogle Scholar
- Bourguignon LY, Wong G, Earle C, Chen L: Hyaluronan-CD44v3 interaction with Oct4-Sox2-Nanog promotes miR-302 expression leading to self-renewal, clonal formation, and cisplatin resistance in cancer stem cells from head and neck squamous cell carcinoma. J Biol Chem. 2012, 287: 32800-32824. 10.1074/jbc.M111.308528PubMed CentralView ArticlePubMedGoogle Scholar
- Fujita S, Ito T, Mizutani T, Minoguchi S, Yamamichi N, Sakurai K, Iba H: miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. J Mol Biol. 2008, 378: 492-504. 10.1016/j.jmb.2008.03.015View ArticlePubMedGoogle Scholar
- Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006, 103: 2257-2261. 10.1073/pnas.0510565103PubMed CentralView ArticlePubMedGoogle Scholar
- Hunter AM: The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis. 2007, 12: 1543-1568. 10.1007/s10495-007-0087-3View ArticlePubMedGoogle Scholar
- Delpech B, Chevallier B, Reinhardt N, Julien JP, Duval C, Maingonnat C, Bastit P, Asselain B: Serum hyaluronan (hyaluronic acid) in breast cancer patients. Int J Cancer. 1990, 46: 388-390. 10.1002/ijc.2910460309View ArticlePubMedGoogle Scholar
- Itano N, Kimata K: Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluronan synthase. J Biol Chem. 1996, 271: 9875-9878. 10.1074/jbc.271.17.9875View ArticlePubMedGoogle Scholar
- Weigel PH, Hascall VC, Tammi M: Hyaluronan synthases. J Biol Chem. 1997, 272: 13997-14000. 10.1074/jbc.272.22.13997View ArticlePubMedGoogle Scholar
- Spicer AP, Nguyen TK: Mammalian hyaluronan synthases: investigation of functional relationships in vivo. Biochem Soc Trans. 1999, 27: 109-115.View ArticlePubMedGoogle Scholar
- Stern R, Jedrzejas MJ: Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev. 2006, 106: 818-839. 10.1021/cr050247kPubMed CentralView ArticlePubMedGoogle Scholar
- Banerji S, Wright AJ, Noble M, Mahoney DJ, Campbell ID, Day AJ, Jackson DG: Structures of the CD44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction. Nat Struct Mol Biol. 2007, 14: 234-239. 10.1038/nsmb1201View ArticlePubMedGoogle Scholar
- Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI: Genomic structure of DNA coding the lymphocyte homing receptor CD44 reveals 12 alternatively spliced exons. Proc Natl Acad Sci U S A. 1992, 89: 12160-12164. 10.1073/pnas.89.24.12160PubMed CentralView ArticlePubMedGoogle Scholar
- Screaton GR, Bell MV, Bell JI, Jackson DG: The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human and rat. J Biol Chem. 1993, 268: 12235-12238.PubMedGoogle Scholar
- Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003, 100: 3983-3988. 10.1073/pnas.0530291100PubMed CentralView ArticlePubMedGoogle Scholar
- Kollmann K, Heller G, Sexl V: c-JUN prevents methylation of p16INK4a (and Cdk6): the villain turned bodyguard. Oncotarget. 2011, 2: 422-427.PubMed CentralView ArticlePubMedGoogle Scholar
- Schreiber M, Kolbus A, Piu F, Szabowski A, Mohle-Steinlein U, Tian J, Karin M, Angel P, Wagner EF: Control of cell cycle progression by c-JUN is p53 dependent. Genes Dev. 1999, 13: 607-619. 10.1101/gad.13.5.607PubMed CentralView ArticlePubMedGoogle Scholar
- Wisdom R, Johnson RS, Moore C: c-JUN regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 1999, 18: 188-197. 10.1093/emboj/18.1.188PubMed CentralView ArticlePubMedGoogle Scholar
- Chuthapisith S, Eremin JM, El-Sheemy M, Eremin O: Neoadjuvant chemotherapy in women with large and locally advanced breast cancer: chemoresistance and prediction of response to drug therapy. Surgeon. 2006, 4: 211-219. 10.1016/S1479-666X(06)80062-4View ArticlePubMedGoogle Scholar
- Kuo MT: Roles of multidrug resistance genes in breast cancer chemoresistance. Adv Exp Med Biol. 2007, 608: 23-30. 10.1007/978-0-387-74039-3_2View ArticlePubMedGoogle Scholar
- Lønning PE: Study of suboptimum treatment response: lessons from breast cancer. Lancet Oncol. 2003, 4: 177-185. 10.1016/S1470-2045(03)01022-2View ArticlePubMedGoogle Scholar
- Gewirtz DA: A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol. 1999, 57: 727-741. 10.1016/S0006-2952(98)00307-4View ArticlePubMedGoogle Scholar
- Suniti M, Ghatak S, Zoltan-Jones A, Toole BP: Regulation of multidrug resistance in cancer cells by hyaluronan. J Biol Chem. 2003, 278: 25285-25288. 10.1074/jbc.C300173200View ArticleGoogle Scholar
- Suniti M, Ghatak S, Toole BP: Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2. J Biol Chem. 2005, 280: 20310-20315. 10.1074/jbc.M500737200View ArticleGoogle Scholar
- Löffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermüller J, Kretzschmar AK, Burger R, Gramatzki M, Blumert C, Bauer K, Cvijic H, Ullmann AK, Stadler PF, Horn F: Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood. 2007, 110: 1330-1333. 10.1182/blood-2007-03-081133View ArticlePubMedGoogle Scholar
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