Isoliquiritigenin modulates miR-374a/PTEN/Akt axis to suppress breast cancer tumorigenesis and metastasis

Breast cancer is one of the most frightful causes of death among females worldwide. Accumulating evidence attached the importance of microRNAs negative regulation to tumorigenesis in breast cancer, suggesting novel cancer therapies targeting microRNAs modulation. Recent studies demonstrated that isoliquiritigenin could inhibit breast cancer cells proliferation and migration, but the underlying mechanism is still limited. In this study, the anti-cancer effects as well as the detailed mechanisms of isoliquiritigenin were explored. The results proved that isoliquiritigenin could negatively regulate breast cancer growth through the induction of apoptosis. We also verified the anti-cancer effect of isoliquiritigenin on migration and invasion, and identified highly expressed miR-374a as one of the main microRNAs down-regulated by isoliquiritigenin treatment in breast cancer. Further study displayed that isoliquiritigenin increased PTEN expression through the decrease of miR-374a expression to inhibit the aberrant Akt signaling. Our findings suggest isoliquiritigenin as a novel anti-cancer candidate significantly regulating miR-374a/PTEN/Akt axis in microRNA-based breast cancer therapies.

Since miRNAs, with capacity of single miRNA interacting with multiple mRNAs, are the most substantial regulatory genes on the gene expression network, reports in increasing number initially concentrate on developing miRNA-based strategies for cancer therapies [15][16][17] . Well-known tumor suppressor miRNAs have been gradually assessed in clinical trials is of benefit to cancer patients. For an instance, miR MRX34 used in a phase-I clinical trial (NCT01829971) for liver cancer therapy is underway 18 . Recently, attention had shifted towards traditional Chinese medicine (TCM) for the treatment of multiple malignancy-related processes because of the low toxicity and high tolerability, and single compounds from TCM are gaining acceptance as potently potential anticancer agents targeting oncogenic miRNAs (e.g., berberine, curcumin, and resveratrol) [19][20][21] . Isoliquiritigenin (ISL), a natural flavonoid majorly derived from the root of licorice, possesses anti-cancer activities at multistage carcinogenesis processes, including proliferation suppression, cell cycle arrest, angiogenesis inhibition, metastasis obstruction and apoptosis induction in various types of cancer [22][23][24] . In our pervious work, ISL showed an essential inhibitory effect on breast cancer through the inhibition of angiogenesis, the activation of autophagy, and the repression of breast cancer stem cells with minimal effects on the proliferation of the MCF-10A normal human mammary epithelial cell line and normal tissues [25][26][27][28] . Additionally, its inhibitory effect on breast cancer growth and migration has been reported currently. ISL induced growth suppression and apoptosis in vitro and in vivo via repressing Arachidonic acid metabolic network and inactivating Akt pathway 29 . Its anti-migratory effect was confirmed in MDA-MB-231 cells through the reduction of VEGF secretion and the inhibition of PI3K/Akt expression 30 . Although ISL limited breast cancer proliferative and migratory ability, its suppressive effect on breast cancer metastasis and underlying mechanisms on PTEN/Akt signaling deserve further investigation.
In the present study, we addressed the role of ISL on mitochondria-based apoptosis induction and metastasis suppression of breast cancer in vitro and in vivo. Microarray analysis further revealed that miR-374a was one of the primary targets of ISL and negatively correlated to the pro-apoptotic effect and anti-metastatic effect of ISL. Notably, ISL-induced apoptosis and metastatic suppression was partly dependent on the increase of PTEN, which was the direct target of miR-374a ensured by luciferase assay. Taken together, our results unearthed a novel function of ISL as a natural miR-374a inhibitor to suppress breast cancer tumorigenesis and metastasis by regulating the aberrant Akt signaling.

ISL inhibits cell proliferation and induces apoptosis of breast cancer cells.
To determine whether ISL suppressed breast cancer cells, we tested the effects of ISL on the growth of human breast carcinoma cells using MTT assay. After ISL 24 h, 48 h, 72 h treatment, the cell viability was assessed. ISL treatment resulted in a dose-dependent inhibition of cell viability, with IC 50 of 32.66 μM and 22.36 μM for 24 h treatment on MCF-7 and MDA-MB-231, respectively. At the dose of 6.25 μM, ISL did not affect the viability of MCF-7 and MDA-MB-231 cells. ISL at 25 μM dramatically inhibited the breast cancer cells growth and induced a time-dependent decrease of cell numbers (P = 0.002, and P < 0.0001, respectively) (Fig. 1A). Further study demonstrated that ISL also displayed an inhibitory effect on primary culture of breast cancer proliferation (see Supplementary Fig. S1). The results from flow cytometry (i.e, Annexin V-FITC and propidium iodide (PI) staining) determined that ISL at low concentration did not manifest significant effect on apoptosis in breast cancer cells, while the effect of ISL at 25 μM and 50 μM was, at least mostly, through inducing apoptosis (For early apoptosis, P = 0.0038, and P = 0.0031, respectively, and for late apoptosis, P = 0.127, and P < 0.0001, respectively with ANOVA test) (Fig. 1B,C). 24 h ISL treatment significantly decreased Bcl-2 expression and increased Bax expression in a dose-dependent manner of in a range from 6.25 to 50 μM (For Bcl-2, P = 0.0302 and P = 0.0032, respectively, and for Bax, P = 0.003 and P = 0.0471, respectively with ANOVA test). We also measured cleavage of caspase-9 and the release of cytochrome c (Cyt C) from the mitochondria. The content of cytoplasmic Cyt C and cleaved caspase-9 were dose-dependently increased in MCF-7 and MDA-MB-231 cells by ISL treatment for 24 h (Fig. 1D,E). Thus, we chose ISL for further mechanism exploration of cell motility study at 6.25 μM and cell apoptosis study at 25 μM.

ISL suppresses breast cancer growth and lung metastasis in vivo.
We used MMTV-PyMT mice model to further elucidate the in vivo anti-cancer effect of ISL on breast cancer growth and metastasis. Since it has been acknowledged that MMTV-PyMT female mice could develop palpable luminal-type mammary tumors metastasizing to the lung in or after the 10 th week 31, 32 , ISL (50 mg/kg/d) were administered to the mice at 4 th week from birth through oral intake, and at the 11 th week of ISL treatment, mice had been sacrificed and tumors had been dissected from mice. Mammary tumors in vehicle group demonstrated a more hemorrhagic appearance than ISL treatment group, while the average size of ISL-treated tumors was dramatically smaller than that of vehicle group ( Fig. 2A). Further histopathologic analysis indicated that both groups displayed typical features of malignancy, although less metastatic nodules were visible in ISL-treated group (Fig. 2B). The mean of tumor weight and tumor burden were considerably reduced after ISL administration for 11 weeks (Fig. 2C,D), and the survival span and ratio of MMTV-PyMT mice were prolonged with ISL treatment (Fig. 2E). No significant change of morphology was found out on normal tissues treated with ISL (see Supplementary Fig. S2). MMP7 is a matrix metallopeptidase involved in invasion and metastasis in multiple cancers including breast cancer [33][34][35] . Further IHC staining using anti-Bax and anti-MMP-7 revealed that the expression of Bax was substantially increased with ISL treatment, and MMP-7 expression was remarkably decreased, respectively (Fig. 2F). Collectively, these data manifest that ISL has a remarkable inhibitory effect on breast cancer tumorigenesis and pulmonary metastasis.
ISL reduces overexpressed miR-374a in breast cancer cells. Microarray was performed to analyze the miRNA expression at least 1.5 fold change regulated by ISL. 24 miRNAs expression were markedly altered in both MCF-7 and MDA-MB-231 cells cultured with ISL (25 μM) for 24 h (Fig. 3A). Out of the miRNAs screened, 1 miRNA was consistently up-regulated and 20 miRNAs were steadily down-regulated after ISL interference (Fig. 3B). Data downloaded from array express (https://www.ebi.ac.uk/arrayexpress/) and developed in Università di Ferrara demonstrated that miR-374a was reliably remarkably up-regulated in breast cancer patient tissues compared with the marched normal tissues among 20 miRNA differences analyzing with Morpheus (https:// software.broadinstitute.org/morpheus/) (see Supplementary Fig. S3). In situ hybridization analysis further confirmed the up-regulation of miR-374a in breast cancer patients (Fig. 3C). The expression of miR-374a expression was examined in 39 breast cancer tissue samples, and high level of miR-374a was associated with the clinical   stage (P = 0.037) ( Table 1). Findings from qRT-PCR analysis displayed that miR-374a was highly expressed in breast cancer cell lines and breast, especially for highly metastatic MDA-MB-231 ( Fig. 3D, P = 0.0323, P = 0296, and P = 0.001, respectively). Using qRT-PCR, we validated that ISL decreased miR-374a expression in a dose-dependent manner (Fig. 3E), indicating miR-374a was a highly breast cancer-associated oncogenic miRNA modulated by ISL intervention.

ISL triggers apoptosis through the down-regulation of miR-374a in breast cancer cells. Using
Real-time PCR, we confirmed the transfection of miR-374a into breast cancer cells and the down-regulation by ISL interference (Fig. 4A). Then, apoptotic cells percentages were analyzed through TUNEL staining. The results showed that the suppression of miR-374a by ISL treatment resulted in a significant increase of the number of TUNEL positive cells (P = 0.0303, and P = 0.0156, respectively), while the overexpression of miR-374a decreased the percentage of TUNEL positive cells (Fig. 4B,C). In addition, PCR results further revealed that miR-374a mimic decreased BAX expression, and increased BCL-2 expression on the mRNA level, while ISL reversed the anti-apoptotic effect of miR-374a in both MCF-7 and MDA-MB-231 cells (Fig. 4D), suggesting that miR-374a plays an essential role in the responses to ISL exposure in breast cancer cells.

ISL inhibits miR-374a expression and suppresses migration and invasion of MDA-MB-231.
Although ISL displayed a significant anti-migration effect on MDA-MB-231 in the pervious reports 25,30 , the detailed mechanisms of its effect on anti-migration and anti-invasion are still unclear. The effect of ISL on migration of MDA-MB-231, with or without the miR-374a interference, was determined by wound healing assay, and confirmed by chamber migration assay. After 24 h exposure, ISL-treated cells, whether transfected with miR-374a mimic or miRNA mimic negative control (miR-NC), demonstrated a noticeable delay of responses to moving into wound area, compared with control group (Fig. 5A,C). The results from chamber migration assay showed the inhibitory effect of ISL were at least partly blocked by miR-374a transfection (Fig. 5B,D). Cells invasiveness assessed by transwell coated with Matrigel at 24 h showed similar inhibition by ISL and similarly revealed the interference of miR-374a in the inhibitory effect of ISL on cell invasive ability (Fig. 5C,E). As for MCF-7, ISL did not display the inhibitory effect on migration and invasion at the dose of 6.25 μM (see Supplementary Fig. S4).
As for primary culture of breast cancer invasion, ISL showed an obvious inhibitory effect (see Supplementary  Fig. S5). Taken together, ISL suppresses the migratory and invasive capacities of MDA-MB-231 through the dramatic suppression of miR-374a.
ISL regulates miR-374a/PTEN/Akt/β-catenin axis. TargetScan and Microrna.org software predicted one of the main targets of miR-374a was PTEN, the potent inhibitor of Akt pathway. Dual luciferase reporter assay confirmed the direct interaction of miR-374a and its predicted binding sites of 3′UTR region of PTEN (Fig. 6A). To evaluate the effect of miR-374a down-regulated by ISL on PTEN expression, we tested PTEN mRNA expression after ISL intervention. As shown in Fig. 6B, the increase of PTEN mRNA level by ISL was remarkable at 24 h point, and the up-regulated effect was at least partly alleviated by miR-374a mimic pretreatment (Fig. 6C). Western blot analyses confirmed the amplified of PTEN expression by ISL and displayed that ISL inhibited Akt and GSK3β phosphorylation, resulting in a decrease of β-catenin expression. Additionally, ISL-induced Akt/ GSK3β/β-catenin inhibition was partly reversed with miR-374a mimic transfection (Fig. 6D). The in vivo data were in consistent with in vitro results. ISL considerably increased PTEN expression and decreased β-catenin expression determined by IHC staining (Fig. 6E). Altogether, miR-374a is the functionally relevant effector of PTEN/Akt/β-catenin modulation by ISL.

Modulation of PTEN expression affects ISL-induced apoptosis and invasion inhibition.
We used qRT-PCR to confirm the down-regulation of PTEN by siRNA PTEN and up-regulation of PTEN by ISL, and siRNA negative control (siRNA-NC) was used as the siRNA transfection control. (Fig. 7A). The results of qRT-PCR analysis also demonstrated that the decrease of PTEN would considerably block the BAX mRNA increase by ISL treatment (Fig. 7B). The pro-apoptotic effect and anti-invasive effect of ISL were also affected after the decrease of PTEN expression (Fig. 7C,D,E,F). Additionally, after transfected with siRNA PTEN, the effect of ISL on biomarkers in apoptosis on protein levels were also partly reversed (Fig. 7G). Collectively, PTEN, the suppressor of Akt pathway, is the essential responder to ISL induced apoptosis and invasion inhibition on breast cancer cells.

Discussion
MiR-374a was first characterized as an oncogene in primary small cell lung cancer (NSCLC), and miR-374a overexpression could promote cell migration and invasion, positively correlating to the poor disease-free survival in NSCLC 10,36 . The oncogenic effect of miR-374a on tumor invasion and metastasis contributed to its identification as cancerogenesis promoter in breast cancer progression by activating WNT/β-catenin signaling 12 . Recently, miR-374a has emerged as one of the major proliferative factors in carcinogenetic signaling. High expression of miR-374a promoted cell proliferation in vitro and tumor growth in vivo in in gastric cancer by targeting SRCIN1 37 .
Overexpressed miR-374a in osteosarcoma remarkably accelerated cell proliferation by directly targeting AXIN2 and FOXO1, and silencing miR-374a could induce G 0 /G 1 and G 1 /S arrest in cell cycle 38,39 , suggesting its role as a potential therapeutic target. Based on our study, we also found high expression of miR-374a in breast cancer cells and tumors from breast cancer patients. However, the effect of miR-374a on breast cancer tumorigenesis was still unclear. Thus, we conducted a legitimate study to explore the functional ability of miR-374a in breast cancer growth. Using microarray screening, we demonstrated that miR-374a was one of the major miRNAs down-regulated by ISL treatment. The results were confirmed by RT-qPCR in a dose-dependent manner in both breast cancer cell lines and primary culture of breast cancer (see Supplementary Fig. S6). The decrease of miR-374a by ISL interference was in accordance with the inhibition of breast cancer growth and invasion. Also, our data supported that overexpressing miR-374a could at least partly reverse the inhibitory effect of ISL on cell mobile ability and the pro-apoptotic effect on cell dearth. Hence, our study clarified for the first time that natural compound ISL could induce apoptosis and inhibit metastasis by down-regulating miR-374a. Akt pathway promotes cell survival as one of the major anti-apoptotic factors through the block of extracellular signal induced apoptosis 40 . Likewise, Akt signaling serves as a key activator of cell migration and invasion by phosphorylating a range of intracellular proteins 41 . Also, overexpression of pAkt was negatively associated with overall survival and disease-free survival of breast cancer patients 42 . Recently, ISL was reported as a promising inhibitor on Akt signaling in breast cancer 29,30 . Therefore, we designed rationale experiments to investigate the underlying mechanisms involved Akt signaling pathway regulated by ISL. We collected ISL-treated MCF-7 and MDA-MB-231 cells and examined the phosphorylation of Akt through western blot analysis. Based on the results, Akt inhibition by ISL was in consistent with miR-374a down-regulation by ISL, indicating the role of miR-374a as one of the up-streams regulating Akt pathway. Our data manifested that ISL could promote PTEN expression in vitro and in vivo. Further study displayed that miR-374a could negatively regulate PTEN, the tumor suppressor of Akt signaling, through the direct binding with 3′ UTR of PTEN mRNA. Whether miR-374a can post-transcriptionally modulate PTEN needs to be further studied. The PTEN down-regulation by miR-374a and up-regulation by ISL were also confirmed through RT-qPCR and western blotting. Our findings indicated that ISL could down-regulate miR-374a, the negative regulator of PTEN, to suppress the significant oncogenic Akt signaling pathway.
Although our pervious study demonstrated that ISL and its derivatives inhibited breast cancer proliferation significantly 43 , studies examining the anti-cancer effect on mitochondrial-based apoptosis involving the release of Cyt C are very limited. Our data showed that ISL could dose-dependently increase the ratio of apoptotic cells after 24 h treatment. Interestingly, early apoptosis accounted for the majority of MCF-7 apoptotic cells, while ISL could induce a dramatic late apoptosis in MDA-MB-231, implying that MDA-MB-231 was more sensitive to response to ISL administration. Whether ISL could serve as a novel anti-cancer drug candidate with a more effective inhibitory effect on TNBCs needs to be further investigated. The western blot analysis exhibited that ISL could enlarge the percentage of released Cyt C in the cytoplasm with an increase of cleaved caspase-9 expression. The activated Akt could directly suppress proteolytic activity of caspase-9 via phosphorylating the protein 44 . These findings indicated that ISL could inactivate Akt pathway to enhance the activity of caspase-9, the positive regulator of Cyt C release in intrinsic apoptosis pathway.
In summary, our study exerted that ISL, a natural flavonoid, is a potent inhibitor of miR-374a in breast cancer. ISL inhibited cell proliferation and migration by down-regulating miR-374a expression, which negatively regulated   Supplementary  Fig. 10. Data represent the mean ± s.d. *P < 0.05, **P < 0.01. PTEN, resulting in the induction of apoptosis and invasive inhibition through the inactivation of Akt pathway. These data provide novel insights into the function of miR-374a regulating breast cancer tumorigenesis, and further suggest a potential application of ISL as a miR-374a naturally inhibition candidate in breast cancer therapy.

Materials and Methods
Chemicals and Reagents. ISL was purchased from Alpha Aesar (MA, USA). Bovine serum albumin (BSA) Eosin Y, and Hematoxylin were purchased from Sigma (St. Louis, MO). All other reagents were obtained from standard commercial sources.
Cell culture. MDA-MB-231, MCF-7, BT474, 4T1, MCF-10A and 293 T were obtained from the American Type Culture Collection (ATCC, USA) and maintained in a humidified incubator with 5% CO 2 at 37 °C. MDA-MB-231, BT474 and 293 T were cultured in high glucose DMEM media, while MCF-7 and 4T1 were cultured in RPMI 1640 media, supplemented with 10% FBS and 1% penicillin and streptomycin (Gibco, Life Technologies, Lofer, AU). Spontaneously immortalized MCF10-A was cultured in keratinocyte serum-free medium (Gibco, Life Technologies, USA). Primary breast cancer was isolated from the distant metastatic position of a Luminal A breast cancer patient (Ethic approval obtained from Ethics Committee of Sun Yat-sen University Cancer Center, YB2016-002-03). The written informed consent was obtained. The corresponding experimental protocols were performed in accordance with guidelines of the Sun Yat-sen University Cancer Center (Guangzhou, China).
Cell viability analysis. 5  in 70 μl cell suspensions. After cell attachment, Culture-Insert was gently removed and cells were treated with different reagents for another 24 h. Images were taken before the treatment and after the treatment. The migratory inhibitory effect of ISL was determined by measuring the closure of the wound between cells. Triplicate experiments were performed independently. The images were detected and analyzed by EVOS XL Core Imaging System (Invitrogen, Life Technologies, USA).

Chamber migration and invasion assays.
Cell migratory ability was assessed by 6-well transwell chambers (Corning Inc., Coring, USA) with 8-μm pore size. Briefly, 3 × 10 5 cells/well were seeded on the upper side with different concentrations of ISL and lower chamber was filled with media (10% FBS). After 24 h, migrated cells were stained by 0.5% crystal violet. Cell invasive ability was assessed by 6-well matrigel-coated transwell chambers (Corning Inc., Coring, USA) with 8-μm pore size as similar previous description. Triplicate experiments were performed independently. The images were detected and analyzed by EVOS XL Core Imaging System (Invitrogen, Life Technologies, USA). Real-time RT-PCR. MiRNA was prepared using mirVana miRNA Isolation Kit (Ambion, Life Technologies, USA) and total RNA was prepared with RNAiso Plus reagent based on the manufacturer's protocol. For miRNA reverse transcription, cDNA was synthesized using miRCURY LNA microRNA cDNA synthesis kit II (Exiqon, Vedbaek, DK). For mRNA reverse transcription, cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Bio Inc., Shiga, JP). Real-time PCR for miRNA and RNA were performed using ExiLENT SYBR Green master mix (Exiqon, Vedbaek, DK) and SsoFast EvaGreen Supermixes (Bio-Rad, Kidlington, UK), respectively. Relative quantification was determined by normalization to GAPDH or U6. The primers are shown in Table S1.
In situ hybridization (ISH) analysis. Expression of miR-374a in formalin-fixed paraffin-embedded archive tissues tissues and normal tissues from 39 breast cancer patients (TMA) (US Biomax, Inc., Rockville, USA) collected in 2004 was determined by ISH with probes for miR-374a. Each tissue spot was accompanied with cases material including sex, age, pathologic type, pathologic grade and clinical stage. The staining intensity was defined as described previously 45 . The study was conducted in accordance with the guidelines of Ethics Committee of Sun Yat-sen University Cancer Center (Guangzhou, China) and Tongxuxian People's Hospital (Henan, China) (Ethic approval, 081116). A written informed consent was obtained from all participants involved in this study. The corresponding experimental protocols were approved by Ethics Committee of Sun Yat-sen University Cancer Center (Guangzhou, China). The images were detected by B203LED microscopy (Optec, Chongqing, CN) and analyzed by Image J (National Institutes of Health, Maryland, USA).  Table S2.
Luciferase reporter assay. The amplified PTEN wide-type/mutant fragments were cloned into the Sac I and HindIII restriction sites of the pMIR-REPORT Luciferase plasmid (Ambion, Life Technologies, USA) using In-Fusion Dry-Down PCR Cloning Kit (TaKaRa Bio Inc., Shiga, JP). The primers are shown in Table S3. 293 T cells were seeded into 96-well plates with transfection mixture. The luciferase activity was recorded using the Dual-Glo Luciferase Assay System (Promega, Madison, WI) by normalization to Renilla luciferase activity according to manufacturer's instructions.

Statistical analysis.
All the data were expressed as means ± standard deviations (SD). Two-tailed student's t test, one way ANOVA, and Chi-square test were used to determine the significant difference of different experiment results by 13.0 SPSS (SPSS Inc., Chicago, USA) software. Survival curves were plotted using the Kaplan-Meier method. Data with P < 0.05 were considered as statistical significance. Supplementary Fig. S3 during the current study are available in the array express repository, (https://www.ebi.ac.uk/arrayexpress/).