MALAT-1/p53/miR-155/miR-146a ceRNA circuit tuned by methoxylated quercitin glycoside alters immunogenic and oncogenic profiles of breast cancer

Triple-Negative Breast Cancer (TNBC) is one of the most aggressive and hot BC subtypes. Our research group has recently shed the light on the utility of natural compounds as effective immunotherapeutic agents. The aim of this study is to investigate the role of a methoxylated quercetin glycoside (MQG) isolated from Cleome droserifolia in harnessing TNBC progression and tuning the tumor microenvironment and natural killer cells cytotoxicity. Results showed that MQG showed the highest potency (IC50 = 12 µM) in repressing cellular proliferation, colony-forming ability, migration, and invasion capacities. Mechanistically, MQG was found to modulate a circuit of competing endogenous RNAs where it was found to reduce the oncogenic MALAT-1 lncRNA and induce TP53 and its downstream miRNAs; miR-155 and miR-146a. Accordingly, this leads to alteration in several downstream signaling pathways such as nitric oxide synthesizing machinery, natural killer cells' cytotoxicity through inducing the expression of its activating ligands such as MICA/B, ULBP2, CD155, and ICAM-1 and trimming of the immune-suppressive cytokines such as TNF-α and IL-10. In conclusion, this study shows that MQG act as a compelling anti-cancer agent repressing TNBC hallmarks, activating immune cell recognition, and alleviating the immune-suppressive tumor microenvironment experienced by TNBC patients.


Introduction
By the end of 2020, the number of women diagnosed with breast cancer (BC) reached 7.8 millions worldwide, ranking it as the most prevalent malignancy among females [1]. One of the most influential factors in terms of BC survival rates 1 3 is the molecular subtyping of tumors. Hormone receptorpositive (HR + ) BC correlates to the most forgiving prognoses followed by the human epidermal growth factor receptor-2 positive (HER2 + ) BC subtype [2,3]. Bleaker still is the onset of triple-negative breast cancer (TNBC) which is characterized by the lack of hormonal receptors as well as HER2, which are powerful therapeutic targets within the context of the former two BC subtypes [4][5][6]. It is this challenge that has prompted immunotherapy as the method of choice when dealing with TNBC, whereby a variety of immune checkpoint blockers are in the late stages of clinical trials [7]. These agents do, however, harbor with them several serious and potentially fatal side effects [8][9][10]. In light of this, our research group has previously demonstrated that natural compounds could be utilized in an immunotherapeutic capacity, possibly providing a means to avoid the side effects of synthetic chemotherapeutic and immunotherapeutic agents [11][12][13][14][15][16]. Quercetin-3`-methoxy-3-O-(4``-acetylrhamnoside)-7-O-α-rhamnoside (a methoxylated quercetin glycoside (MQG) has been previously isolated by our group from the Egyptian medicinal shrub Cleome droserifolia [17,18]. MQG showed highly inducing effects on the tumor suppressor triad TP53, miR-15a, and miR-16 expression levels with potent antitumor activity in liver cancer cell lines [18]. However, its impact on TNBC progression has never been investigated.
One of the most well-defined aspects of cancer is the myriad of epigenetic events associated with it [19]. A recent layer of complexity has been added to the epigenetic circuit tuning BC progression which is the association of long noncoding RNAs (lncRNAs) in the regulation of BC hallmarks and downstream targets [20][21][22][23]. MALAT-1 is an oncogenic lncRNA that has been validated to modulate BC progression [24]. However, its role in tuning the immunological profile has rarely been investigated. The dysfunction of the TP53 gene, in particular, brings about the dysregulation of a series of modulatory microRNAs (miRNAs) which in turn shepherd the mutant cell towards an immortal malignant phenotype [5,22,[25][26][27][28]. Two of the most particularly influential players in this scenario are miR-155-5p and miR-146a-5p. MiR-155-5p and miR-146a-5p are well-known for their roles as an immunostimulant, or pro-inflammatory miRNAs [29][30][31] and hence are relevant in eliciting a proper immune surveillance response against the malignant transformation process. Within the tumor immune microenvironment (TIME), miR-155-5p and miR-146a-5p were found to alter cytotoxic T cells activity. However, their role in altering the recognition of BC cells towards the innate arm of the immune system and its impact on the cytokine storm at the TIME is yet to be investigated. Therefore, this study aims to unravel the impact of MQG on the oncological or the immunological profiles of BC cells and to further investigate the underlying molecular mechanism underneath such alterations.

Cell culture and treatment
Different cancer cell lines such as TNBC cell lines, MDA-MB-231, HR + BC cell lines, MCF-7, and HCC cell lines Hep-G2 and Huh7 cells were obtained from ATCC and Vacsera, Egypt. Adherent cells were cultured in DMEM (Lonza, Switzerland) media as previously described [18,32,33]. The respective compounds (1-3) were isolated from C.droserifolia as previously described in our group [18] and as shown in Fig. 1. Stock solutions were prepared as 250 µM stock solutions in 0.1% DMSO in culture media. Effective concentrations ranging from 1 µM to 200 µM, depending on the experimental setup, were prepared, and used to treat different cancer cells seeded in 96-well or 24-well plates. Cells were treated for 24-96 h in normal growth conditions (37 °C in a 5% CO 2 atmosphere). 5-Fluorouracil was used as a positive control in this study. In all experiments, cells used as a control were labeled as vehicle control and were exposed to 0.1% DMSO in culture media as previously described [11,14,34,35].

Cellular viability and proliferation experiments
3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was used in the cellular viability experiments. BC cells (10,000) were seeded in 200 µL full media in a 96-well plate. Forty-eight h post-transfection, the media was replaced by a 20 µL working solution. After 6 h, the absorbance of the formed purple formazan crystals, solubilized in 200 µL lysis buffer, was measured [18,32,33,36]. For the cellular proliferation experiments, a bromodeoxyuridine (BrdU) incorporation assay was used. BC cells were seeded into black 96-well plates at a cell density of 5 × 10 4 cells/well. According to the Cell Proliferation ELISA kit protocol (Roche Applied Science, Penzberg, Germany), BC cells were incubated with BrdU for 4 h, then fixed for 30 min using Fix-Denate and finally incubated with Anti-BrdU POD for 90 min [18,32,33,36]. All experiments were performed in triplicates and repeated three times or more.

Cellular migration and invasion
BC migration capacity was assessed using the wound-healing/scratch assay. Treated cells were left to grow to a confluency of 90-95%. Post-treatment, 3 scratches were performed 1 3 in each well using a 10-µL pipette tip. BC cells were washed using PBS and replenished with new low-serum media (1% FBS). After 24 h, the surface areas of the scratches were measured and wound closure was quantified with Zen2012 software [18,32,33,36]. While for the invasion experiments, the modified Boyden chamber assay (BD Bioscience, Bedford, USA) was performed. In 24-well plates, BC cells were treated with MQG, and then 6 × 10 4 cells were re-suspended in 200 μL low-serum media (1% FBS) and were seeded in the upper well. Yet, the lower well contained high-serum media (20% FBS). Cells were washed from the upper surface using a cotton swab 8 h after seeding. Then the invaded cells were fixed and stained using 1% crystal violet (Sigma Chemical Co., California, USA) and counted under an inverted light microscope. All experiments were performed in triplicate and repeated three times or more [18,32].

Colony-forming assay
For the colony-forming experiment, treated cells were harvested and seeded post-treatment in a 6-well plate at 800 cells/well. BC cells were incubated in full DMEM under normal conditions (37 °C and 5% CO 2 ) for 15 days. Colonies were fixed using 6% glutaraldehyde, stained by 0.05% crystal violet, and then counted [18,32].

Cell cycle analysis
Expression vectors containing response elements for vital cell cycle proteins such as TP53 (pp53-TA-Luc), c-Myc (pMyc-TA-Luc), RB (pRB-TA-Luc), E2F (pE2F-TA-Luc; Clontech, France) were used. Similarly, BC cells were treated with a plus vector containing an unspecific binding site (Clontech, France). BC cells were seeded and transfected with the respective vectors using Superfect Transfection Reagent (Qiagen, Germany) according to the manufacturer's protocol. After 24 h post-transfection of the plasmid DNA, cells were treated with MQG. After 72 h, BC cells were then lysed, and luciferase expression/luminescence measurement was quantified using Steady-GLO Luciferase Kit (Promega, Germany) according to the manufacturer's instructions. Luminescence was plotted as % luciferase activity relative to cells transfected with the vector alone. Unspecific luminescence detected by the reagents and the empty plug vector (baseline luminescence) was subtracted from all values before plotting as previously illustrated [5,32].

Total RNA and miRNAs extraction
Total RNA and miRNAs were isolated using a Biozol RNA extraction reagent. Extracted RNA was then quantified spectrophotometrically. RNA integrity was examined by 18 s rRNA bands detection on 1% agarose gel electrophoresis. RNA samples with 260/280 optical density > 2 were excluded [18,32,33,36].

Quantification of NO production
NO production was measured using Griess reagent assay (Promega, USA) according to the manufacturer protocol [5,32]. Briefly, 50 μL of cells' supernatant were mixed with 50 μL sulfanilamide solution and incubated for 10 min. Then, another 50 µL of N-1-naphthyl ethylenediamine dihydrochloride (NED) solution is added and absorbance was measured at 540 nm using Wallac 1420 Victor 2 Multilabel Counter (Perkin Elmer, USA). Experiments were performed in triplicates and repeated 3 times or more [20,32].

Lactate dehydrogenase (LDH) assay
Treated BC cells were seeded in a 96-well plate at a cell density of 15,000 cells/well. After 2 h, primary NK cells were added to the target BC cells at a 5:1 effector to target ratio (E: T) and incubated for 8 h. Later, the lactate dehydrogenase (LDH) activity assay kit (MAK066-1K1-Sigma-Aldrich, St. Louis, MO, USA) was used to measure the in vitro NK cells cytotoxic potential following the manufacturer's instructions. The lysis % was calculated according to the following equation: % cytotoxicity = (target maximum release − experimental release)/(target maximum release) × 100. The experiment was done in triplicate and repeated more than 3 times [16,32,33,36,39]

Statistical analysis
Data are presented as mean ± standard error of the mean (SEM) for 3 different experiments. Non-parametric unpaired student-t-test was executed to compare between every two independent groups. One-way analysis of variance with post hoc analysis was adopted for multiple comparisons. P-value of < 0.05 was considered statistically significant, and the threshold of significance is denoted by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were analyzed using GraphPad Prism 8.2.1 software as previously described [11].

Preferential selectivity of flavonol glycosides towards MDA-MB-231 TNBC cell lines
The cytotoxic profiles of flavonol glycosides isolated from C. droserifolia (Compounds 1-3) were screened against different cancer cell lines. Flavonol glycosides showed a preferential selectivity towards the TNBC cell lines, MDA-MB-231 when compared to HR + BC cells and HCC cell lines as shown in Table 1 and Fig. 2. Therefore, the other functional analysis experiments were performed on MDA-MB-231 TNBC cells. The IC 50 values of the respective compounds were calculated using the corresponding dose-response curves of each compound in each cell line after 3 days of treatment.

Impact of serial dilutions of flavonol glycosides isolated from C. droserifolia on MDA-MB-231 cellular viability and proliferation rates
Serial dilutions of flavonol glycosides (1-10 µM) were prepared and applied to MDA-MB-231 cells for 72 h. Compounds (1-3) showed a concentration-dependent reduction in the cellular viability (Fig. 3a-c) and cellular proliferation rate ( Fig. 3d-f). Compound 2 was the most potent compound (lowest IC 50 as indicated in Table 1) and showed the most

Impact of flavonol glycosides isolated from C. droserifolia on MDA-MB-231 colony-forming ability, migration and invasion capacities
To investigate the long-term effects of serial dilutions of C. droserifolia flavonol glycosides (1-3) on MDA-MB-231 cell lines, an anchorage-independent growth assay was performed. In a similar pattern to cellular viability and proliferation assays, compounds (1-3) showed a concentrationdependent reduction in the clonogenic properties also with compound 2 (MQG) showing the most potent inhibitory impact as shown in (Fig. 4a-c) (Fig. 4d).

Impact of methoxylated quercetin glycoside on cell cycle proteins
After validating the potential selective anticancer activity of MQG in halting the oncogenic profile of human BC cells Data are presented as mean ± SEM of three independent experiments; ***P < 0.001, **P < 0.01, *P < 0.05 compared with control group effectively and being non-toxic to human normal cells (Supplementary Fig. 1), our aim was extrapolated to unravel the mechanism by which MQG could harness BC progression. For that reason, vital cell cycle proteins were screened such as the tumor suppressor TP53, the oncogenic protein cMyc, and the RB/E2F complex proteins. The results showed that MQG led to a significant elevation in TP53 protein levels (P < 0.0001). However, it did not affect c-Myc and RB/E2F complex activity (Fig. 5a-d).

Impact of methoxylated quercetin glycoside on ncRNAs circuit around TP53
Then, it was interesting to further unravel the machinery downstream/upstream of the TP53. TP53 is a well-known upstream regulator for an array of miRNAs previously validated by our group [5]. Nonetheless, it was recently reported that MALAT-1 lncRNA acts as a direct upstream regulator for TP53 [40]. On the other hand, miR-155 and miR-146a are reported to act as downstream miRNAs to TP53 and have a dual role in tuning the oncological and immunological profiles. For that reason, screening of MALAT-1, miR-155, and miR-146a were performed in treated MDA-MB-231 cells by ~ IC 50 value (10 µM) of MQG. The results showed that the oncogenic MALAT-1 lncRNA has been significantly reduced (P < 0.001) while a significant increase of miR-155 (P = 0.0002) and miR-146a (P = 0.0026) levels were observed (Fig. 6); building up a novel axis MALAT-1/TP53/ miR-155/miR-146a drawn downstream MQG in TNBC cells.

Impact of methoxylated quercetin glycoside on NO machinery system
Furthermore, nitric oxide (NO) has been validated as an important cytokine at the TIME and at the same time has an indisputable role in altering the oncological profile of BC cells. Interestingly, the NO synthesizing enzymes (NOS 2 and NOS 3 ) are validated targets for miR-155 and miR-146a. Therefore, to draw the full axis downstream MQG in TNBC cells, the impact of MQG on the NO machinery system was probed. Interestingly, MQG resulted in marked repression of NOS 2 (P = 0.0030), NOS3 (P = 0.0005) mRNA levels, and consequently a marked reduction in the NO produced from MDA-MB-231 (P = 0.0020) (Fig. 7). (threefolds, P < 0.0001), CD155 (twofolds, P = 0.0038), ICAM-1 (threefolds, P = 0.0188) and significant reduction of immune-suppressive cytokines TNF-α (P = 0.0228) and IL-10 (P = 0.0006). Collectively, this resulted in induction in primary NK cells cytotoxicity (P = 0.0001) (Fig. 8a-h).

Discussion
The current study sheds the light on a novel crosstalk between ncRNAs building up a novel ceRNAs circuit and their respective preys (targets) in TNBC cells. MQG was found to have the lowest IC 50 in repressing the proliferation and viability of MDA-MB-231 TNBC cell lines. This could be directly linked to MQG chemical structure. It was reported that the methoxy and acetyl substitution are responsible for increasing the biological activity of substituted compounds compared to its respective congers [41,42]. TNBC patients are the least fortunate if compared to other BC subtypes. The lack of therapeutic targets renders its patients especially needful of additional treatment options. Hence, TNBC patients comprised the chief scope of our research. Mechanistically, it was important to unravel the mechanism by which MQG acts as a potent selective anticancer agent against MDA-MB-231 cells. TP53 is a vital tumor suppressor protein and the most fundamental orchestrator of apoptosis and cell cycle arrest [43]. TP53 is markedly downregulated in TNBC patients and cell lines [3,44,45]. For that reason, TP53 was our primary target to unravel MQG molecular mechanism in TNCB cell lines. Consistent . Student t test was performed. Data are presented as mean ± SEM of three independent experiments; ***P < 0.001, **P < 0.01, *P < 0.05 compared with control group with our previous study [18], the results showed that TP53 transcript and protein levels are induced by MQG in MDA-MB-231 cells. In the current study, we further assayed the protein levels of the alternative apoptotic genes E2F, RB, and c-MYC, all of which showed no increase upon treatment with MQG. Taken together, these results suggest that the significant cytotoxicity of MQG is partially mediated by p53. Then, it was intriguing to unravel the ncRNAs circuit revolving around the TP53. MALAT-1 is an oncogenic lncRNA that was recently reported to modulate TP53 in mice but it has never been investigated in BC. Nonetheless, miR-155 and miR-146a were recently reported to act  of ncRNAs such as H19 lncRNA, miR-486-5p, miR-548a, miR-20a [14,16,34,35] Further downstream still is the observation of NOS2 and NOS3 downregulation-2 validated target genes of miRNA-155 and miRNA-146a simultaneously [47,48]. Oft dysregulated in cancer, the NO machinery (i.e. NOS 2 /NOS 3 ) is highly implicated in driving tumor migration, angiogenesis, and mutation through radical-based DNA damage [49]. Indeed, high NOS activity is a significant predictor for poor prognosis among TNBC patients [50,51]. Our results about the general mitigation of NO machinery upon MQG treatment thus demonstrate an additional facet of its antitumor activity.
The emergence of immunotherapy during the past decade brought about an entirely new line of thinking in the landscape of cancer therapy prompting a widespread consensus of the immune system as the future of the oncological field. With this in mind, we chose to furthermore investigate the MALAT-1/p53/miR-155/miR-146a axis' significant role in modulating the innate immune recognition. Accordingly, our study showed significant increases in expression of the NK cell ligands MICA/B, ULBP2, CD155, and ICAM-1 as downstream targets for the ceRNA molecules MALAT-1/ miR-155/miR-146a [52][53][54]. These ligands, in a normal context, act as signals for NK cell cytotoxicity during cellular stress. As a result, one mechanism of carcinogenic evolution is the shedding of these ligands, which are found to be especially downregulated in TNBC patients [55]. Their induction in MDA-MB-231 upon treatment with MQG and subsequent increase in NK-mediated cytotoxicity strongly alludes to a counteraction of this immune evasion mechanism. MQG administration also exhibited a significant downregulation of IL-10, an inhibitory cytokine to NK, CD4 + , and dendritic cells among others, and a reportedly negative prognostic factor in BC [56,57]. In doing so, a tentative but promising window is opened towards an entirely new addition to the immunotherapeutic arsenal shifting the focus from the currently dominant adaptive immunity-based agents for which resistance is growing and intense side effects are reported [9,[58][59][60]. MQG's derivation from natural sources moreover represents a toxicologically safe and potentially much cheaper alternative to synthetic immunotherapeutic agents.
Curiously, we observed a concomitant downregulation of TNF-α upon MQG administration, despite reports of a positive correlation between miR-155 and the cytokine [61,62]. High TNF-α production contributes to an inflamed, immunosuppressive TIME and has been reported to mitigate TNBC progression in vitro when knocked out [63]. Although somewhat counterintuitive, the downregulation of TNF-α witnessed in our study hence signifies another favorable modulation to the TIME imposed by MQG. It must be considered that, in inducing p53, the expression of several miRNAs apart from the ones we have tested is overwhelmingly likely to have been affected. The suppression of TNF-α, therefore, could be the result of a differential miRNAs expression such as miR-140-5p/miR-181a-5p [64,65] In conclusion, the current study probes a detailed molecular mechanism detailing the impact of MQG on the oncological and immunological profiles of TNBC cells. Furthermore, this study sheds the light on a novel selective multifaceted anticancer immunotherapeutic nutraceutical that holds great potential for TNBC patients that renders it as a potential candidate for clinical trials. This study also demonstrates a novel ceRNA circuit MALAT-1/miR-155/ miR-146a orchestrated by p53 in TNBC cells and thus possesses potent effectiveness against TNBC progression and aggressiveness and immune-suppressive nature as summarized in Fig. 9.
Author contributions MA-L and RAY contributed to most of the practical work and wrote the manuscript. AR, RS, AE-K, HN contributed to the practical work. MZG, AM co-supervised the work. RAY conceived the original idea, designed the experimental setup, supervised the work. All authors discussed the results and contributed to the final manuscript.
Funding This study was not supported by any funding agency.

Conflict of interest All authors declare no conflict of interest.
Ethical approval This study complies with all Ethical Standards. The current study does not include any human participants or animals so informed consents are not applicable.