Effective Suppression of Hypoxia Induced Angiogenesis by Synergism of Simvastatin and Metformin via Inhibiting ET-1-ETBR-Hif1α Signaling Axis

Background: Hypoxic stress accelerates metabolic reprogramming to promote malignant progression, which contains huge and interesting drug targets for cancer therapy. According to recently reports and our previously researches, simvastatin (SVA) or metformin (MET), two metabolic relevant drugs, play an important role in inhibiting tumor growth, angiogenesis, improvement hypoxic microenvironment. However, the signal networks of hypoxia induced metabolic reprogramming is enormous and intricacy, blocking a signal target can’t play an effective role in antitumor, whether combination of SVA and MET could exert a more effective antitumor effect is not clear. Methods: The antitumor effect of synergism of SVA and MET were detected in mouse models, breast cancer patient-derived organoid (PDO) and multiple tumor cell lines compared with untreated, SVA or MET alone. Transcriptome sequencing were performed for exploring the possible mechanisms. Inhibition of endothelin 1 (ET-1) induced Hif1α by synergism of SVA and MET were contrasted with bosentan, an ETBR antagonist. Results: Our results show that synergism of SVA and MET can inhibit tumor cell proliferation and promote apoptosis, alleviate hypoxia, decrease angiogenesis and increase vessel normalization within tumor than use of SVA or MET alone. RNA-sequencing result implies that just synergism of SVA and MET can inhibit EDN1 expression, which encodes endothelin 1, an important regulator of angiogenesis and hypoxia-related pathway, but not in use of SVA or MET alone. More specically, synergism of SVA and MET suppresses the ET-1 induced Hif1α expression by inhibiting ET-1-ETBR signaling pathway and increasing PHD2 expression. In addition, the antitumor effect of synergism of SVA and MET is more ecient than bosentan in tumor cells and breast cancer PDO by inhibiting ET-1-ETBR-Hif1α signaling axis. Conclusions: Thus, our data show that synergism of SVA and MET can be a viable therapy for antitumor.

After treatment with SVA and/or MET, cells were collected and followed the Annexin V-PE apoptosis detection kit (BD Biosciences, USA) to analysis the percentage of apoptosis cells.
RNA sequencing assay HepG2 cells were untreated or treated with 2.5μM SVA, 5mM MET and combination of 2.5μM SVA and 5mM MET for 24h, quickly extracted and cryopreserved with liquid nitrogen. Then RNA sequencing were performed by Guangzhou RiboBio (China, Guangzhou). Every group were contain 3 independent samples for gene expression analysis.

Mouse models
All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at Xi 'an Jiaotong University. The 5×10 5 4T1 cells were injected into the fat pad of the 4 th left mammary of four-week-old female BALB/c mice, and 1×10 6 B16 cells were subcutaneously injected into four-week-old female C57BL/6J mice. And the mice were treated with 15mg/kg/day simvastatin and/or 100 mg/kg/day metformin by intragastric administration.

Immunoblotting
After treatment with SVA and/or MET, cells were harvested by RIPA buffer, and the lysates were underwent SDS-PAGE and transferred onto PVDF membrane. Then the membrane were incubated with primary antibody and HRP-conjugated sencondary antibody, and the chemiluminescent signals were detected by ChemiDoc TM XRS+ (Bio-rad, USA). The antibodies anti-p-RB, anti-RB, anti-cleaved caspase3, anti-caspase3, anti-Hif1α were obtained from Cell Signaling Technology (USA), anti-MCM7, anti-ET-1, anti-PHD2 were purchased from Santa Cruz (USA), anti-ETAR and anti-ETBR were purchased from Abcam (UK), and surviving were purchased from Proteintech (China).
The perfusion status of vessels were labeled by Rhodamine-labeled lectin (Vector Labs, USA) for 15 min. 20 mg/kg Lectin were intravenous injected into mice, then intracardiac perfusion of 40 ml 4% PFA with a ux of 10 ml/min. Tumors were extracted and xed with 4% PFA, embedded in OCT (Sakura Finetek, USA), and produced frozen sections. Finally, co-staining with CD31 and imaging with confocal laser scanning microscopy.
Tissues were xed in 10% neutralized formaldehyde and embedded in para n, and then stained with PCNA (Santa Cruz, USA), cleaved caspase3 and Hif1α. Images were taken by Leica SCN400 slide scanner (Leica, Germany). The detailed procedures were similarly performed as described previously [19,20].

Patient-derived organoid model
Breast cancer tissues were obtained from First A liated Hospital of Xi 'an Jiaotong University with informed consent and approved by the Ethics Review Committee of the First A liated Hospital of Xi 'an Jiaotong University. The breast cancer tissues were chopped up and digested by Collagenase at 37 ℃ for 1h followed by ltering on a 150 mesh lter and centrifuging 10min at 400g. The precipitate was resuspended in organoid medium, and centrifuged one more time, then resuspended with 40μL BME type 2 (Trevigen, USA). After BME was solidi ed, 400μL organoid medium was added for culture. The detailed procedures and reagents were performed as described previously [21] The viability were assayed as followed: the PDOs were dispensed into 384-well microplates and treated with SVA and/or MET for 0, 24h, 48 or 72h, then analyzed by CellTiter-Glo (Promega, USA) accordance with its' technical manual.

Statistical analysis
The quantitative analysis performed by Prism 7.0 software, and the quantitative data were represented by mean ± SEM. Statistical significance was calculated using two-tailed student ' s t test or one-way ANOVA t-test. Each experiment data was represented a minimum of three biological replicates. *P < 0.05, ** P < 0.01, *** P < 0.001.

Results
Combination treatment of simvastatin and metformin inhibits tumor growth in vivo.
Malignancy could reprogram their metabolism and utilize glucose and lipid for their high energy demand [3]. According to our previous research and recently reports, treated by MET [17][18][19][20] or SVA [12][13][14]16] alone could inhibit growth and induce apoptosis of tumor cells both in vivo and in vitro [22]. These two antitumor drugs are mainly clinically used to reduce the glucose and lipid [16, [23][24][25]. Based on this, we found there was an observably inhibition of tumor growth and prolong the survival duration of mice after the treatment with combination of SVA and MET. Not only compared with untreated group, but also used SVA or MET alone, the combination treatment displayed a signi cant antitumor effect ( Fig. 1A-F). The combination treatment decreased the expression of proliferating cell nuclear antigen (PCNA) and increased cleaved caspase 3 expression of the 4T1 and B16 tumors than other group (Fig. 1G, H, sFig1), it suggested that combination treatment could inhibit the proliferation and induce apoptosis of tumor cells.
Combination treatment of simvastatin and metformin is a synergism for antitumor effect Similar to the in vivo, combination treatment of simvastatin and metformin also inhibited proliferation of poly-type of tumor cells in vitro ( Fig. 2A, B, sFig 2, Table 1), which displayed a moderate to strong synergism [26]. According to our preciously study, SVA can inhibit phosphorylated retinoblastoma (RB) and mini-chromosome maintenance protein 7 (MCM7) expression to suppression of tumor cell growth [13]. The synergism of simvastatin and metformin inhibited the tumor cell proliferation via decreasing the p-RB and MCM7 (Fig. 2C). It could induce 2-3 times apoptosis ratio than other treatment, accompanied by upregulating of cleaved caspase 3 and downregulating survivin (Fig. 2D-F). Thus, we found SVA and MET synergistically suppressed cell proliferation and induced apoptosis.

Synergism of simvastatin and metformin inhibits angiogenesis and promotes vcessel normalization
To investigate the mechanism of synergism of SVA and MET in antitumor effect, we applied RNA sequencing to computationally investigate transcriptional dynamics after treatment of synergism of SVA and MET, SVA or MET alone and their control in HepG2 cells. Then we predicted the altered biological process of synergism alone, but no distinction in treatment after SVA or MET alone and their control.
Compared with negative control, SVA or MET, synergism of SVA and MET changed lots of angiogenesis related genes (Fig. 3A). Angiogenesis is required for caner progression, thus antiangiogenic therapy a widely used in antitumor therapy [27][28][29]. Platelet endothelial cell adhesion molecule (PECAM-1) (CD31) can marker the endothelial cell of vessel [30]. Synergetic treatment decreased the number and branch points of tumor inside vessel than others by CD31-staining ( Fig. 3B-G). It suggested that synergism of SVA or MET could inhibits tumor angiogenesis.
Inhibition of tumor angiogenesis is the key point of the hypothesis about tumor-starving, vascular endothelial growth factor (VEGF) as a major driver of it, became a notable target for antitumor. However, in preclinical studies anti-VEGF therapy can induce tumor vasculature showing a more "mature" or "normal" phenotype. The vessel normalization display an attenuated hyperpermeability, increased vascular pericyte coverage, and a more normal basement membrane [30]. Here, we found the length of CD31-stained vessels after synergism was longer than other treatments (Fig. 3B, E). NG2 is the marker of pericytes, and vascular endothelial (VE)-cadherin is the strictly endothelial speci c adhesion molecule [30], both of them were increased and colocalization with CD31 after synergetic treatment of SVA and MET ( Fig. 4A-D). Lectin can bind to the surface of endothelial cells accompanied with blood ow. Lectin + vessels also increased after synergism of SVA and MET (Fig. 4E, F). Synergism of SVA and MET inhibited disorganized and nonfunctional vessels and promoted vessel normalization and maturity.
Simvastatin and metformin synergistically promotes vessel normalization via inhibiting ET-1-Hif1α signaling axis KEGG pathway analysis showed that activity of relaxin and Hif1α signaling pathway were changed after synergism of SVA and MET. EDN1 markedly decreased after synergism treatment, which encodes endothelin 1 (ET-1) [31], and play an important role in regulating Hif1α signaling pathway (Fig. 5A). ET-1 and its receptor endothelin receptor B (ETBR) were reduced by synergism treatment, but it had no effect on receptor endothelin receptor A (ETAR) (Fig. 5B-D). Subsequently, we analyzed the hypoxic area by staining of Hif1α in 4T1 tumor and B16 tumor. Synergism treatment decreased more than 50% hypoxic area than untreated group, and 25%-35% than simvastatin or metformin treatment groups in 4t1 tumors; decreased more than 60% hypoxic area than untreated group, and 40%-50% than simvastatin or metformin treatment groups in B16 tumors (Fig. 5E, F). Similar to in vivo, synergism treatment inhibited Hif1α expression during hypoxic condition (1% O 2 ) (Fig. 5G).
Under normoxic conditions, prolyl hydroxylase 2 (PHD2) catalyzes hydroxylation of HIF1α to lead its proteasomal degradation [8]. Endothelin 1 induced Hif1α and inhibited PHD2 expression associated with up-regulated expression of ETBR in HepG2 and MCF7 cells (Fig. 6A). Synergism treatment shown a signi cant inhibition of ET-1 induced ETBR and Hif1α expression, accompanied with promoting PDH2 expression (Fig. 6B). Furthermore, ET-1 induced Hif1α expression shown a nuclear location, which also could be inhibited by synergism treatment (Fig. 6C, sFig 3). Bosentan is an endothelin receptor antagonist, targeted ETAR and ETBR, and have been evaluated in clinical oncology [32]. Synergism treatment could suppress ET-1 induced Hif1α and ETBR expression similar to inhibition of ETBR expression by bosentan or small interfering RNAs (siRNAs) (Fig. 6D, sFig 4). Compared with treatment by bosentan, synergism of SVA and MET displayed a stronger inhibition cell proliferation by decreasing ET-1 induced Hif1α expression and correspondent PHD2 expression (Fig. 6D-E). It suggested that inhibiting ET-1 induced Hif1α expression could become one important pathway to suppress angiogenesis and promoted vessel normalization by synergism of simvastatin and metformin.
Synergism of simvastatin and metformin suppresses growth and ET-1 induced Hif1α expression of breast cancer PDOs PDO models link cancer genetics and patient trial, cover the shortage of cell-line-and xenograft-based drug research, directly and rapidly test drug sensitivity, and promote personalized tumor therapy [21,33]. From 8 tumors, we established breast cancer PDOs that readily expanded. Based on the results of hematoxylin-eosin (H&E) staining of para n section, the histopathological features of breast cancer PDOs shown a strong concordance with of their corresponding parental tumor (Fig. 7A, sTab 1). There were dramatically inhibited the viability and volume of breast cancer PDOs after three days of synergism of SVA and MET treatment than vehicle, simvastatin or metformin alone (Fig. 7B-D). We also found the expression of ET-1 and ETBR were suppressed by synergism treatment (Fig. 7E, F). Similarly to the in vitro, the ET-1 induced Hif1α expression were inhibited by bosentan or synergism (Fig. 7G). Thus, we found that inhibition of ET-1 induced Hif1α expression is a signi cant mechanism of synergism of SVA and MET to anti-tumor effect (Fig. 7H).

Discussion
Our studies demonstrate that simvastatin and metformin shown an effective synergism in anti-tumor, which can inhibit growth of tumor cells, angiogenesis and promote vessel normalization by inhibiting ET-1 expression and ET-1 induced Hif1α expression, than SVA or MET alone. Several researches were focused on the e cacy of simvastatin plus metformin treatment in polycystic ovary syndrome [34].
Others were mainly about anti-tumor effect, such as inhibition of prostate cancer, colorectal cancer, hepatocellular carcinoma and endometrial cancer, etc [35][36][37][38][39][40][41][42]. In our study, we also found that combination of simvastatin and metformin had a wide range of anti-cancer effects in breast cancer, hepatocellular carcinoma, lung cancer and cervical cancer. And the widespread anti-tumor effect of SVA plus MET is not a simple superposition, but a moderate to strong synergism than use of SVA or MET alone.
Study on combination SVA and MET in anti-tumor effect, mainly focused on inducing apoptosis and inhibition proliferation. Similarly to our results, in endometrial cancer [36], combination SVA and MET synergistically induced apoptosis and inhibited cell growth, which could be reversed by silencing Bim expression [36]. In metastatic castration-resistant prostate cancer cells, it inhibits cell viability and metastasis and increases Ripk1-and Ripk3-dependent necrotic cell death after 48-96h treatment by combination of simvastatin and metformin, but not induces apoptosis [35]. The duration of treatment and tissue differential expression may be mainly reason of the different effect of simvastatin and metformin. In hepatoma cells, combination of metformin and simvastatin decreased cell proliferation and arresting cell cycle in G0/G1 by inhibiting translational controlled tumor protein (TCTP) and mammalian target of rapamycin (mTOR) pathway and increasing tumor suppressor phosphatase and tensin homolog (PTEN) and promoting autophagy [42]. Combination of SVA and MET inhibited primary ventral prostate tumor formation, cachexia, bone metastasis, and biochemical failure by ameliorating metabolic aberrations and increasing AMPKα catalytic activity, more effectively than SVA, MET treatment alone or docetaxel (DTX) chemotherapy, the standard-of-care of metastatic castration-resistant prostate cancer (CRPC). They also found SVA and MET combination may have a broader applicability in triplenegative breast cancer [35]. In prostate cancer patients with SVA and MET combination treatment have less aggression and longer free biochemical recurrence survival than treated only with SVA and MET and suggested may regulate multiple signaling pathways, not only modi cation of the AMPK, but also androgen receptor (AR) and cyclin-dependent kinase inhibitors (CKIs) [41]. In our study, we found SVA and MET combination inhibited cell growth by decreasing RB and MCM7 expression, and promoted apoptosis by increasing cleaved-caspase 3 and decreasing survival expression.
Angiogenesis is another hallmark of cancer [28]. According to our previously study, use of SVA or MET alone can inhibit tumor angiogenesis and induce vessel normalization [15,20,43]. In this study, we found synergism of SVA and MET noticeably inhibited angiogenesis of the structure disorder and nonfunctional, and promoted the integrity of vascular structure and functional normalization more than SVA or MET only treatment in tumor of orthotopically transplanted mice. According to our RNA-seq results, ET-1 shown a notable downregulation after treatment of synergism of SVA and MET. ET-1 play an important role in mitosis for blood, endothelial cells, broblasts, vascular smooth muscle cells and pericytes, and its receptor ETBR also regulated proliferation of blood and endothelial. In melanoma cells, ET-1 promotes tubular network formation through ETBR [31,44,45]. Here, we found synergism of SVA and MET could inhibit ET-1 and ETBR expression in vivo and in vitro. Thus, it is an important strategy for anti-tumor by inhibition of ET-1-ETBR axis mediated angiogenesis.
Synergism of SVA and MET inhibited HIF1α expression and decreased hypoxia area. ET-1 can stabilize HIF1α by inhibiting PHD2 to control tumor environment [46,47]. Our results shown an outstanding decline of ET-1 induced ETBR and HIF1α expression, accompanied by an up-regulation of PHD2. However, hypoxia can also upregulate ET-1 expression [48]. Fluvastatin attenuated hypoxia-induced ET-1 transactivation by increasing polyubiquitination of HIF1α and decreasing HIF1α binding to ET-1 promotor [49]. Whether synergism of SVA and MET also could show a more effectively downregulate hypoxiainduced ET-1 expression, consequently inhibit this loop, we will study in our next work.
Bosentan is an antagonist of ETBR, uses in treatment of pulmonary arterial hypertension in clinical, and investigates in Phase II of metastatic melanoma [50, 51]. Synergism of SVA and MET displayed a signi cant in uence of anti-tumor effect by regulation of ET-1 induced HIF1α downregulation and PHD2 upregulation than bosentan both in tumor cells and breast cancer PDO. It is suggested that synergism of SVA and MET might be a more effective therapeutic schedule for inhibition of ET-1-Hif1α axis in antitumor treatment. But we just culture 8 breast cancer PDOs, more tumor cases and more tumor types will be need to collect and culture, and combined genomic sequencing and RNA sequencing to screen the other therapeutic target of synergism of SVA and MET, nally use for patients who suit to treat with synergism of SVA and MET in clinical.

Conclusions
On the hand, our work identi es synergism of SVA and MET as a more effective anti-tumor strategy, compared with ETBR antagonist bosentan. Because of the common problem in development and approval of new drugs, which is a formidably and costly challenge, and often accompanied by a high failure rate. Drug repurposing is an important entry point for drug research and development. Such drugs show a new bene t in medical condition than their original indication [52]. Here, simvastatin and metformin is the rst-line clinical medication for reducing hyperglycemia and hyperlipemia. The safety and tolerability have been tested in humans, it will be a huge advantage for reducing time and cost of research and development.

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Availability of data and materials
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Competing interests
The authors declare that they have no conflict of interest.