The tumor microenvironment has been implicated in tumor progression and therapeutic resistance (Kharaishvili
The transmembrane molecule, cluster of differentiation 44 (CD44), senses tumor microenvironmental changes by binding to ECM components, such as HA, and transduces extracellular signals to regulate tumor progression. For example, HA-activated CD44 signaling was observed to provoke tumor progression by inducing oncogenic events, including RhoGTPase and matrix metalloproteinase (MMP) signaling (Yu and Stamenkovic, 1999; Bourguignon, 2008; Orgaz
Nuclear factor erythroid 2-like 2 (NFE2L2; NRF2) is a key transcription factor that protects cells against oxidative stress by enhancing cytoprotective genes harboring antioxidant response elements (AREs) on their promoters. Under oxidative/electrophilic stress, NRF2 is sequestered from the Kelch-like ECH-associated protein (KEAP1), and transported to the nucleus, where it regulates the expression of phase 2 detoxifying enzymes (e.g., aldo-keto reductase 1C1 [AKR1C1], NAD(P)H quinone oxidoreductase-1 [NQO1]), antioxidant proteins (e.g., glutamate-cysteine ligase modulatory subunit [GCLM]), and drug efflux transporters (e.g., MDR1, breast cancer resistance protein [BCRP]) (Cho and Kleeberger, 2020; Otsuki and Yamamoto, 2020). During the last decade, accumulating evidence has indicated that aberrant activation of NRF2 facilitates tumor growth and survival by inducing cytoprotective genes in cancer cells (Shibata
Recently, our findings revealed an association between CD44/NRF2 and cancer stem cell (CSC)-like properties of breast cancer cells (Ryoo
Doxorubicin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), geneticin, hyaluronic acid sodium salt with extra-low molecular weight (8,000-15,000 Da), 4-MU, Mission Lentiviral Packaging mix, and hexadimethrine bromide were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Fluorescein isothiocyanate (FITC)-conjugated CD44 antibody was purchased from BioLegend (San Diego, CA, USA). Additionally, 6-Carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) was purchased from Life Technologies (Carlsbad, CA, USA). Antibodies recognizing NRF2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MDR1 and CD44 were from Cell Signaling Technology (Danvers, MA, USA). Anti-AKR1C1 was purchased from Abnova (Walnut, CA, USA), and anti-HAS-2 antibody was purchased from Abcam (Cambridge, MA, USA). The SYBR premix ExTaq system was obtained from Takara Bio Inc. (Otsu, Japan). The fluorescent dye Hoechst 33342 was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
The human breast cancer cell line MCF7 was obtained from the American Type Culture Collection (Rockville, MD, USA). Doxorubicin-resistant MCF7-DR cell lines were gifted by Dr. Keon Wook Kang (Seoul National University, Seoul, Korea). SNU620 and SNU620-DR cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). MCF7/MCF7-DR cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), and SNU620/SNU620-DR cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium. Both media were supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and penicillin/streptomycin (WelGene Inc., Daegu, Korea). The cells were maintained at 37°C in an atmosphere of 5% carbon dioxide (CO2).
The HAS2 cDNA open reading frame (ORF) clone (cat. RG224227), and empty control pCMV6-AC-GFP vector (catalog number: PS100010) were purchased from OriGene (Rockville, MD, USA). Lentiviral particles were produced in HEK293T cells following transfection with HAS2-subcloned pCMV6-AC-GFP (HAS2-ov) or the empty vector, and a lentiviral packaging kit (Sigma-Aldrich), as described by the manufacturer. MCF7-DR cells were infected with lentiviral particles in the presence of 8 µg/mL hexadimethrine bromide. Transduction was continued for 48 h, followed by 24 h recovery in complete medium. For the selection of stable transgene-expressing cells, geneticin (G418, 500 μg/mL) was incubated for up to 1 month.
Total RNA was extracted from the seeded cells using TRIzol reagent (Thermo Fisher Scientific Inc.). For cDNA synthesis, RT reactions were performed by incubating 200 ng of the total RNA with a reaction mixture, which contained 0.5 µg/µL oligo dT12-18 and 200 U/μL Moloney murine leukemia virus RT (Life Technologies). Real-time PCR was performed using a Roche Light Cycler (Mannheim, Germany) with the Takara SYBR Premix ExTaq System (Takara Bio Inc.) (Ryu
Cells (5×105) were incubated in media without serum for 48 h. Then, the supernatant was collected for enzyme-linked immunosorbent assay (ELISA) analysis and particulates were removed by centrifugation. Plasma HA levels were determined by ELISA using a Quantikine Hyaluronan Immunoassay Kit (cat. DHYAL0; R&D Systems, Abingdon, UK), according to the manufacturer’s protocol. Optical density values were determined using SPECTRO StarNano (BMG LABTECH GmbH, Allmendgruen, Ortenberg, Germany) to read the absorbance at 450 and 570 nm.
Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris [pH 7.4], 150 mM sodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% nonidet P-40 [NP-40]) containing a protease inhibitor cocktail (Sigma-Aldrich). The protein concentration was determined using a bicinchoninic acid assay (BCA) kit (Thermo Scientific, Middletown, VA, USA). The protein samples were separated by electrophoresis on 6-12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany) using a Trans-Blot Semi-Dry Cell (Bio-Rad). The membrane was blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with the antibodies. Following the addition of the enhanced chemiluminescence reagent (Thermo Scientific), images were acquired using a GE Healthcare LAS-4000 mini imager (GE Healthcare Sciences, Piscataway, NJ, USA).
Cells were seeded at a density of 5×103 cells/well in 96-well plates. After 24 h of incubation, the cells were treated with doxorubicin, with or without 4-MU. Then, 2 mg/mL of MTT solution (for MCF7/MCF7-DR cells) or 20 µL of MTS reagent (for SNU620/SNU620-DR cells) was added to each well, and cells were incubated for an additional 3 h. MTT reagent was removed after incubation and 100 μL of dimethyl sulfoxide was added to each well (Lee
Predesigned siRNAs for
Cells were plated at a density of 5×103 cells/well on cover glass slides. The cells were washed with cold phosphate-buffered saline (PBS) three times and fixed in cold methanol for 10 min. Then, anti-HAS2 antibody (1:200) was incubated in fixed cells at 4°C overnight. The next day, cells were washed with PBS and incubated with Alexa Fluor 488 conjugate-DAM IgG anti-mouse antibody (1:500) at room temperature for 1 h. For nuclear staining, incubation with Hoechst 33342 was performed for 10 min. Fluorescence images were obtained using LSM 710 confocal microscope (Carl Zeiss, Jena, Germany) (Jung
Cells were detached using 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and resuspended in cold PBS containing 2% FBS. Cells were stained with FITC-conjugated CD44 antibody for 30 min. After washing with PBS, cells were analyzed using a Becton-Dickinson FACSCanto (Becton-Dickinson, Milan, Italy) or CytoFLEX (Beckman-Coulter, CA, USA) as described previously (Ryoo
Cellular ROS levels were determined using fluorescent carboxy-H2DCFDA, as described previously (Ryoo
Cells were seeded at a density of 2×104 cells/mL in 96-well ultralow attachment plates (Corning Costar Corp., Cambridge, MA, USA) and grown in a serum-free DMEM and Nutrient Mixture F-12 medium supplemented with B27 (1:50, Life Technologies), 20 ng/mL epithelial growth factor (EGF), 20 ng/mL basic fibroblast growth factor (R&D System, Minneapolis, MN, USA), 5 µg/mL bovine insulin (Cell Application Inc., San Diego, CA, USA), 0.5 µg/mL hydrocortisone (Sigma-Aldrich), and penicillin/streptomycin (HyClone) as described previously (Ryoo
To evaluate the
Statistical significance was analyzed using Student’s t-test or one-way analysis of variance followed by the Student Newman-Keuls test for multiple comparisons using Prism software (GraphPad Prism, La Jolla, CA, USA).
MCF7-DR cells demonstrated increased cell viability following a 72 h incubation with doxorubicin (Supplementary Fig. 1). Similar to our previous report (Ryoo
Activated NRF2 signaling contributes to chemoresistance in many types of cancer cells (Choi and Kwak, 2016). Similar to our previous study (Ryoo
Next, to elucidate the effect of HA on NRF2 signaling, we incubated MCF7-DR cells with HA. Immunoblotting analysis revealed that the protein levels of NRF2, AKR1C1, and MDR1 were further increased by HA in a concentration-dependent manner (Fig. 2E). However, there was no significant change in CD44 protein expression after HA treatment. These results suggest that the HA/CD44 axis is involved in NRF2 activation in MCF4-DR cells.
To investigate the direct role of CD44 on NRF2 signaling, we silenced
Next, to determine whether increased HAS2 expression is involved in NRF2 activation and doxorubicin resistance in MCF7-DR cells, HAS2 expression was silenced in MCF7-DR cells (Fig. 3E). Immunoblotting analysis showed that CD44 expression was not affected by
Next, to confirm the role of HAS2 in NRF2 activation and doxorubicin resistance in MCF7-DR cells, we applied 4-MU, a pharmacological inhibitor of HAS2. When MCF7-DR cells were treated with 4-MU (0.5 mM) for 24 h, HAS2 protein levels were significantly decreased (Fig. 4A). In line with this, the protein levels of NRF2, AKR1C1, and MDR1 were reduced in 4-MU-treated MCF7-DR cells (Fig. 4B). As a result of decreased NRF2 target gene expression, cellular ROS levels were found to be higher in 4-MU-treated MCF7-DR cells than the vehicle-treated MCF7-DR cells (Fig. 4C). As a phenotypic effect, 4-MU treatment suppressed the sphere formation capacity of MCF7-DR cells (Fig. 4D). The average diameters of the vehicle-treated and 4-MU- treated MCF7-DR cells were 67 and 32 μm, respectively. Additionally, when MCF7-DR cells were co-incubated with 4-MU and doxorubicin (2 μM) for 24 h, doxorubicin-induced cytotoxicity was significantly enhanced compared to the doxorubicin-treated group (Fig. 4E). These results show that the pharmacological inhibition of HAS2 could render drug-resistant cancer cells more susceptible to doxorubicin treatment via the inhibition of NRF2 signaling.
Considering the relationship between HAS2 and doxorubicin resistance, we attempted to elucidate the effect of HAS2 overexpression. Forced expression of HAS2 in MCF7-DR cells (HAS2-ov) resulted in increased HAS2 mRNA levels and HA concentration (Fig. 5A, 5B), and led to the increase in NRF2, AKR1C1, and MDR1 levels, without altering CD44 levels (Fig. 5C), thereby confirming the relationship between HAS2 expression and NRF2 activation.
Whether HAS2high cells affect neighboring cancer cells for the enhanced aggressive phenotype is still unknown. As we observed that a significant amount of HA was synthesized by HAS2 in HAS2-overexpressed cells (HAS2-ov), we cultured MCF7-DR cells using HA-enriched medium (conditioned medium from HAS2-ov culture) and the effect on NRF2 and drug sensitivity was monitored. When MCF7-DR cells were incubated with the HA-enriched conditioned medium, the protein levels of NRF2 increased (Fig. 5D), and doxorubicin-induced cytotoxicity was reduced (Fig. 5E). These observations imply the potential interaction of HAS2high cells with neighboring cancer cells for the acquisition of drug resistance through NRF2 activation within the tumor microenvironment.
Next, to confirm the relationship between HAS2 and NRF2/chemoresistance, we used additional resistant cancer cell lines. SNU620-DR, a gastric cancer cell line harboring doxorubicin resistance, showed increased cell viability after 72 h of incubation with doxorubicin (Fig. 6A). Compared to the parental cell line, basal protein levels of NRF2, AKR1C1, and CD44 were higher in SNU620-DR cells (Fig. 6B), and flow cytometry analysis showed that 85.82% of SNU620 DR cells retained CD44-positive population, while only 26.90 % of SNU620 parental cells were CD44-positive (Fig. 6C). Consistently, HAS2 and HA concentrations were elevated in SNU620-DR cells (Fig. 6D, 6E). Similar to MCF7-DR cells, genetic silencing or pharmacological inhibition of HAS2 decreased NRF2 expression (Fig. 6F, 6G). When SNU620-DR cells were treated with 4-MU for 24 h, these cells showed increased sensitivity to doxorubicin treatment compared to the vehicle-treated group (Fig. 6H). These results confirm that targeting HAS2 might be an effective strategy to overcome the anticancer resistance of NRF2high cancers.
To investigate the clinical importance of HAS2 in tumor patients, we analyzed clinical data using cBioportal platform. TCGA Pan-Cancer Atlas data analysis across 32 types of cancer revealed a higher alteration frequency of
Next, we specifically analyzed breast TCGA data for patients with breast invasive carcinoma. Among 996 breast cancer patients, 22 patients exhibited higher HAS2 mRNA levels (z-score >=2) compared to the unaltered mRNA group. We additionally found that HAS2-high breast cancer patients showed a shorter overall survival rate (median survival months=91.99) than the unaltered HAS2 gene group (median survival months=130.16) (Fig. 7C). Moreover, HAS2 mRNA levels were associated with increased mRNA levels of AKR1C1 and GCLM in these patients. Mean log2 mRNA expressions of AKR1C1 were 6.34, and 7.32 in the unaltered and HAS2-high group, respectively (Fig. 7D). GCLM mRNA levels were also higher in the HAS2-high patients compared to the unaltered group (Fig. 7E). Collectively, these results demonstrate that HAS2 expression is related to activation of NRF2 signaling, and higher HAS2 levels are associated with poor clinical outcomes in breast cancer patients.
Despite tremendous advances in anticancer therapeutic strategies, tumor resistance continues to be a principal limiting factor for successful cancer treatment. Recent studies indicate that the tumor microenvironment can be a promising target for overcoming tumor resistance (Son
CD44 is an important cell surface receptor for HA. Considering that high levels of HA are detected in the tumor stroma, high CD44 expression contributes to HA-mediated cellular signaling in cancer (Bourguignon
Since HAS2 upregulates the expression of mesenchymal markers, such as N-cadherin and vimentin, and the formation of invadopodia, cancer cells expressing a higher level of HAS2 can interact with CD44-positive cancer cells in the tumor microenvironment, which causes these cells to have a more metastatic phenotype (Sheng
Previously, TCGA breast cancer datasets indicated that aberrant amplification of the
It is still not clear how HAS2 is upregulated in drug-resistant cancer cells. Some factors are known to affect HAS2 expression. Estradiol can stimulate HAS2 mRNA expression when combined with insulin-like growth factor-1, insulin, and follicle-stimulating hormone (Chavoshinejad
In summary, our results indicate that increased HAS2 levels lead to doxorubicin resistance in breast and gastric cancer cells via the activation of HA/CD44 and NRF2 signaling. These results provide an in-depth understanding of the interaction between tumor microenvironmental HA and NRF2-driven chemoresistance, and further suggest that HAS2 is a novel target to control the therapeutic resistance of NRF2-high cancers.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2018R1A2A1A05078894, 2017R1A6A3A11030293, and 2021R1C1C1006881).
The authors confirm that there are no conflicts of interest.