Molecular Carcinogenesis

Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China Key Laboratory for Carcinogenesis and Invasion, Chinese Ministry of Education, Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Cancer Research Institute and School of Basic Medical Science, Central South University, Changsha, China Department of Oncology, Second Affiliated Hospital of Soochow University, Suzhou, China Department of Pathology, Guangzhou Women and Children’s Medical Center, Guangzhou, China Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai, China Shanghai Center for Bioinformation Technology, Shanghai Academy of Science and Technology, Shanghai, China Department of Pathology, Second Xiangya Hospital of Central South University, Changsha, China

relapse and early metastasis and a poor prognosis. Moreover, there are few treatment options for TNBC patients resistant to standard chemotherapy. 3,4 Therefore, there is an urgent need to identify additional drug targets for TNBC patients with chemoresistance and poor prognosis.
Decitabine (DCA), a demethylating agent approved for the treatment of hematological malignancies by many international regulatory agencies, including the US Food and Drug Administration and the European Commission, has become part of the standard of care for patients with myelodysplastic syndrome. 5,6 Recent studies have shown that DCA-based chemotherapy is the most commonly used treatment for BC. 7 However, DCA resistance is an important limitation of its therapeutic efficacy. 8 Recent studies have shown that the presence of cancer stem cells (CSCs) is associated with chemical resistance. 9 CSCs are a group of tumor cells with the potential for self-renewal and multilineage differentiation. 10 Since CSCs can differentiate, producing a heterogeneous population of cells that compose tumors, they are thought to be the seed cells of tumors. 11 CSCs are also a key factor in the resistance of tumors to chemotherapeutic drugs. 12 The discovery of BC stem cells (BCSCs) provided new ideas for the study of mechanisms of increased metastasis and chemoresistance in TNBC. Since CD24 low CD44 high tumor cells exhibit stem cell-like properties, including self-renewal and a high differentiation potential, CD24 − CD44 + lineage cells are thought to be the malignant counterparts of BC cells. 13 Increasing evidence in recent years indicates that CD24 − CD44 + CSC populations in BC are important factors leading to increased tumor metastasis and chemoresistance. 14 MicroRNAs (miRNAs) are a class of short, noncoding RNAs that regulate their target genes. 15 Numerous studies have shown that miRNAs play an important role in the occurrence and metastasis of many cancers. As miRNA regulation can alter the expression of resistance-related genes, abnormal expression of miRNAs can also induce drug resistance in cancer. 16 Furthermore, miRNAs play a critical role in the differentiation and self-renewal of CSCs. 17 For instance, miR-5100 promotes the self-renewal and cisplatin resistance of lung CSCs by targeting Ras-related protein 6. 18 Recent studies have reported that miR-155 promotes the growth and invasion of a variety of tumors, including lung cancer, BC, and colorectal cancer, suggesting its association with tumorigenesis, development, and drug resistance. [19][20][21] For instance, exosome-derived miR-155 mediates BC chemoresistance via the epithelial-mesenchymal transition pathway. 22 In our previous study, we found and confirmed miR-155 overexpression in BC by miRNA expression profiling microarray. 23 However, the role and molecular mechanism of miR-155 in BCSCs have not been confirmed.
In the present study, we first found that miR-155 was upregulated in BCSCs. We demonstrated that miR-155 increased the percentage of stem cells, DCA resistance and colony-forming ability in BC cells. In addition, molecular mechanistic studies showed that miR-155 directly regulated the target gene tetraspanin-5 (TSPAN5) by binding the 3′-untranslated region (UTR) of its messenger RNA (mRNA). Moreover, TSPAN5 could abolish the miR-155-mediated increase in stemness and DCA resistance in BCSCs. Taken together, our findings suggest that miR-155 is a novel CSC marker and therapeutic target that plays a key role in BCSC self-renewal and DCA resistance.

| Cell isolation and culture
MDA-231 and BT-549 cells (human BC cell lines) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All cell lines were maintained in a humidified incubator containing 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM; 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin).
DCA-resistant MDA-231 cells (MDA-231/DCA) and DCA-resistant BT-549 cells (BT-549/DCA) were prepared as previously described. 24 MDA-231 cells and BT-549 cells were exposed to gradually increasing concentrations of DCA (from 2 to 256 μmol/L). Finally, the cells acquired resistance to DCA through a series of selection steps.
The screened MDA-231/DCA and BT-549/DCA cells were cultured in the same manner as MDA-231 and BT-549 cells, respectively.
BCSCs were enriched by surface marker sorting as previously described. 25 MDA-231 and BT-549 cells were incubated with anti-CD44-PE antibody and anti-CD24-FITC antibody (BD Biosciences) for 30 minutes at 4°C (while protected from light), and CD24 − CD44 + cells were then enriched by flow cytometry. The enriched BCSCs were cultured in a humidified incubator containing 5% CO 2 at 37°C in BCSC culture medium (DMEM/F-12, 20 ng/mL bFGF, 10 ng/mL EGF, 4 μg/mL insulin, B-27 supplement) containing 0.4% bovine serum albumin prepared as previously described. 26 2.2 | RNA isolation and quantitative real-time PCR Cells were lysed with a TRIzol kit (Qiagen), and RNA was isolated according to the manufacturer's instructions. 27   First, MDA-231 or BT-549 cells were seeded at a suitable density and treated with DCA. CCK-8 solution (10 µL) was then added to the cells, which were incubated for 3 hours. Finally, the absorbance (450 nm) was read with a microplate reader. 28 2.5 | Colony-formation assay MDA-231 or BT-549 cells were seeded in a six-well plate, cultured for 2 weeks, fixed and then stained with a 0.1% crystal violet solution. Photographs were taken under a microscope, and colonies consisting of more than 50 cells were counted. Colony-forming ability was indicated by the number of cellular clones. 27

| Mammosphere-formation assay
A mammosphere-formation assay was performed as we previously described. 18 Briefly, cells were plated in a single-cell suspensions in ultra-low attachment plates (24-well plates) at a density of 100 cells/ well. Cells were grown in serum-free cell culture medium for 10 days.
Images of the mammospheres were taken using a microscope, and the number of mammospheres ≥ 60 μm in size in each well was counted.

| Immunophenotyping analysis
Immunophenotyping analysis was performed as previously described. 18 MDA-231 or BT-549 cells treated with or without miR-155 (miR-155 mimics and miR-155 inhibitor) were resuspended in buffer and incubated with antibodies (FITC-CD24, PE-CD44, and corresponding isotype controls) for 30 minutes at 4°C (while protected from light).
The stained cells were analyzed by flow cytometry (FACSA, BD).

| Western blotting
Western blotting was performed as we previously described. 29 Proteins collected from MDA-231 or BT-549 cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Subsequently, blots were incubated with anti-TSPAN5 primary antibody and secondary antibody (1:300; Abcam). The blots were analyzed using a gel imaging system (Bio-Rad). The grayscale values of the bands were quantified with ImageJ2× software. 29

| Luciferase reporter assay
The TSPAN5 3′-UTR was cloned into the psiCHECK vector. The miR-155 "seed"-targeted sequence in the TSPAN5 3′-UTR was mutated from AAUUACGA to AAUACUGA using a Site-Directed Gene Mutagenesis Kit (Beyotime, China). MDA-231 or BT-549 cells were seeded and cotransfected with miR-155 mimics and TSPAN5 vector. Dual-luciferase activities were detected with the Dual-Luciferase Reporter Assay System (Promega) 2 days after transfection. The relative luciferase activity was calculated according to the operating instructions of the system. 27

| Statistical analysis
Data are shown as the mean ± standard deviation. GraphPad Prism 6 (GraphPad, Inc) and SPSS 19.0 software (IBM, Inc) were used for statistical analysis of all data. The Student t test was used for comparisons between two groups. Statistical data were considered significant when the P < .05.

| miR-155 is upregulated in TNBC tissues and TNBC cell lines
Our previous microarray data showed that miR-155 is upregulated in BC. 23 Through further analysis of the data, we found the elevated expression of miR-155 in TNBC tissues ( Figure 1A). To further test    while the knockdown of miR-155 increased TSPAN5 protein levels ( Figure 5D,E). Overall, these findings showed that TSPAN5 is a direct target of miR-155 in MDA-231 and BT-549 cells.

| TSPAN5 is downregulated in TNBC tissues and BCSCs
Based on these results, we evaluated TSPAN5 protein expression in TNBC tissues and BCSCs. TSPAN5 was highly expressed in non-TNBC tissues but not in TNBC tissues ( Figure 6A,B). The results of Western blot analysis showed that TSPAN5 protein levels were significantly lower in TNBC tissues than in non-TNBC tissues ( Figure 6C). Finally, we examined TSPAN5 protein levels in BCSCs and non-BCSCs. We observed a similar significant decrease in TSPAN5 protein levels in BCSCs compared to non-BCSCs ( Figure 6D).

| DISCUSSION
Accumulating data suggest that TNBC has a poor prognosis due to the presence of cells resistant to DCA. Although a number of studies have been conducted to explore the underlying mechanisms associated with DCA resistance in BC patients, the target and molecular mechanisms of DCA resistance remain unclear. 30 Generally, cancer tissues are maintained by CSCs with the ability to undergo self- prognosis. 32 In recent years, we and other teams have found that miR -155 plays the role of a tumor promoter in many cancers. 23 However, F I G U R E 5 TSPAN5 is the target of miR-155 in BC cells. TSPAN5 was predicted to be a target of miR-155 by the online databases TargetScan and miRDB (A). Luciferase assays revealed that the miR-155 mimics significantly decreased luciferase activity in cells transfected with the wild-type TSPAN5 3′-UTR, whereas mutation of the putative miR-155 target sites in the TSPAN5 3′-UTR abrogated the responsiveness of luciferase to miR-155 (B). Expression of the TSPAN5 protein in MDA-231 (D) and BT-549 cells (E) transfected with miR-NC, miR-155 mimics, or miR-155 inhibitor was detected by Western blotting (representative images are shown, n = 3). The results are presented as the means of three independent experiments ± SDs. Bars, SDs, **P < .01 by unpaired t test. SD, standard deviation; TSPAN5, tetraspanin-5; UTR, untranslated region [Color figure can be viewed at wileyonlinelibrary.com] the underlying mechanism of miR-155 in DCA resistance in BCSCs has not been elucidated.
In this report, we show that miR-155 was upregulated in TNBC tissues, cell lines, and stem cells. In addition, miR-155 induced DCA resistance and stem cell characteristics, as it upregulated SOX2 and NANOG, increased CD24 − CD44 + cell numbers, and enhanced cloneformation ability in TNBC cells. Furthermore, knockdown of miR-155 reduced stem cell characteristics and DCA resistance in TNBC cells.
As a specific DNA methyltransferase (DNMT) inhibitor, DCA can induce the differentiation of tumor cells into normal cells and tumor cell apoptosis by reversing the DNA methylation process. 5 The mechanism involved in DCA resistance in TNBC is complex. Our previous studies showed that miR-155 is upregulated in TNBC, but whether miR-155 is involved in stemness and DCA resistance in BC has not been studied.
Studies have shown that DNMT expression in TNBC is associated with sensitivity to DCA. 33 Studies have also shown that miR-155 is a novel marker of TNBC chemoresistance. 15 Our findings support the notion that miR-155 promotes the stem cell characteristics, the colony-forming ability, and the DCA resistance of TNBC cells.
Although tumor cells exhibit the same level of self-renewal, which is limited by genetic programs, shown by normal stem cells, the continuous expansion of self-renewing cells in cancer leads to tumors and subsequent cancer metastasis and resistance. 34 BC cells with a CD24 − CD44 + phenotype have BCSC characteristics and exhibit a high self-renewal capacity and high level of proliferation. We sorted CD24 − CD44 + BCSCs by which miR-155 expression had been upregulated and found that the CD24 − CD44 + BCSCs were resistant to DCA, which is consistent with the results of other studies.
Moreover, overexpression of miR-155 promoted SOX2 and NANOG levels while increasing the number of CD24 − CD44 + BCSCs. Indeed, SOX2 and NANOG are transcription factors essential for stem cell self-renewal. 35 We and other researchers have found that the transcription factors NANOG and SOX2, which are upregulated in CSCs, can promote cell self-renewal by regulating genes involved in proliferation and differentiation. Diseases associated with SOX2 and NANOG include teratocarcinoma and germ cell and embryonic cancers. 36,37 Here, we show that the miR-155-mediated promotion  38 Previous studies demonstrated that TSPAN5 can regulate the function of ADAM10 on the cell surface and promote ADAM10-mediated degradation of CD44. 39 Studies have also shown that TSPAN5 acts as a proapoptotic factor that inhibits tumor growth in gastric cancer. 40 On the other hand, TSPAN5 is positively regulated by the Notch signaling pathway. 41 In this report, TSPAN5 was found to be poorly expressed in BCSCs, and overexpression of TSPAN5 abrogated miR-155-induced changes in stemness and DCA resistance in TNBC cells. These findings indicate that miR-155 promotes cellular stemness and proliferation by targeting TSPAN5 in TNBC cells and that miR-155 affects DCA resistance through TSPAN5. The way in which TSPAN5 regulates TNBC cell stemness and DCA resistance remains unclear. Studies have shown that TSPAN5 is involved in the degradation of the BCSC surface marker protein CD44. 42 However, elucidation of the specific molecular mechanism requires further study. Finally, we cannot completely rule out the possibility that miR-155 regulates DCA resistance via other potential targets, such as colony-stimulating factor 1 receptor, interleukin 17 receptor B, mitogen-activated protein kinase 10, and tyrosinase-related protein 1, all of which were predicted as targets by online databases.
In summary, the data in this report indicate a strong molecular link between miR-155, DCA resistance, and self-renewal in BCSCs.
The effects of miR-155 upregulation suggest that DCA resistance and stem cell characteristics in BCSCs are regulated via common molecular mechanisms, such as extensive proliferation and self-renewal.
The roles of the miR-155/TSPAN5 axis in BCSCs support the use of miR-155 inhibitors to enhance sensitivity to DCA as a potential strategy to target BCSCs.

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
LY, XW, and YL drafted the manuscript; YC, XW, and XZ participated in clinical data collection; HF and XF carried out immunohistochemical experiments; LY carried out data analysis; and XW and JX supervised the research program and edited the manuscript. All authors read and approved the final manuscript.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are available from the corresponding author upon request.

ETHICS STATEMENT
This study was designed and carried out in accordance with the guidelines of the Experimental Center of GDMU and the laws of China.
F I G U R E 9 Schematic illustration of the possible mechanism by which miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5. TSPAN5, tetraspanin-5 [Color figure can be viewed at wileyonlinelibrary.com]