What are the effective inhibitors for treating cancer and fibrosis?---Part 1

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Small Molecule Inhibitors Of Epithelial‐mesenchymal Transition For The Treatment Of Cancer And Fibrosis

Ya‐Long Feng¹ | Dan‐Qian Chen¹ | Nosratola D. Vaziri² | Yan Guo^1,3 |Ying‐Yong Zhao¹

Abstract

Tissue fibrosis and cancer both lead to high morbidity and mortality worldwide; thus, effective therapeutic strategies are urgently needed. Because drug resistance has been widely reported in fibrotic tissue and cancer, developing a strategy to discover novel targets for a targeted drug intervention is necessary for the effective treatment of fibrosis and cancer. Although many factors lead to fibrosis and cancer, pathophysiological analysis has demonstrated that tissue fibrosis and cancer share a common process of epithelial‐mesenchymal transition (EMT). EMT is associated with many mediators, including transcription factors (Snail, zinc‐finger E‐box‐binding protein and signal transducer and activator of transcription 3), signaling pathways (transform- ing growth factor‐β1, RAC‐α serine/threonine‐protein kinase, Wnt, nuclear factor‐kappa B, peroxisome proliferator-activated receptor, Notch, and RAS), RNA‐binding proteins (ESRP1 and ESRP2) and microRNAs. Therefore, drugs targeting EMT may be a promising therapy against both fibrosis and tumors. A large number of compounds that are synthesized or derived from natural products and their derivatives suppress the EMT by targeting these mediators in fibrosis and cancer. By targeting EMT, these compounds exhibited anticancer effects in multiple cancer types, and some of them also showed antifibrotic effects. Therefore drugs targeting EMT not only have both antifibrotic and anticancer effects but also exert effective therapeutic effects on multiorgan fibrosis and cancer, which provides effective therapy against fibrosis and cancer. Taken together, the results highlighted in this review provide new concepts for discovering new antifibrotic and antitumor drugs.

KEYWORDS: cancer, epithelial‐mesenchymal transition, fibrosis, natural product, small molecule, transcription factor, tumor

cistanche can treat fibrosis and cancer

1 | INTRODUCTION

Tissue fibrosis and cancer are two major causes of high human morbidity and mortality worldwide. Although there are multiple therapies for cancer, including chemotherapy, oncologic surgery, and radiation therapy, an effective therapeutic strategy is needed.1 Among these therapeutic strategies, chemotherapy is the main tool for curing various cancers. The therapeutic resistance of anticancer drugs, such as 5‐fluorouracil, gemcitabine, gefitinib, and trastuzumab, has been widely observed in the clinic.2 However, due to the lack of effective therapeutic drugs, tissue fibrosis still threatens human health. Although some drugs exhibit antifibrotic effects, including angiotensin‐converting enzyme inhibitors, aldosterone inhibitors, statins, angiotensin II type 1 receptor blockers, endothelin receptors, β‐blockers, acetylsalicylic acid, and matrix metalloproteinase (MMP) inhibitors, none of them are specifically designed to target fibrosis, and the related side effects limit their clinical use for treating fibrosis.3-6Thus, antifibrotic and anticancer treatments are extremely urgent, and new therapeutic drugs should be designed based on specific targets that contribute to the progression of fibrosis and tumors.

Epithelial‐mesenchymal transition (EMT) is a reversible terminal differentiation process in which epithelial cells shed their properties and acquire a more mesenchymal phenotype.7 EMT is a fundamental process widely involved in the development and the progression of various diseases, and there are mainly three types of EMT8(Figure 1). Type I EMT is involved in embryonic development and organ formation. Type II EMT is critical for wound healing and fibrosis. Type III EMT contributes to the progression and metastasis of tumors.9 Extensivestudies revealed that EMT profoundly contributed to the production of myofibroblasts, which are the major cells producing massive amounts of collagen that leads to the deposition of collagen in the development of fibrosis.10In addition, EMT confers increased motility and invasiveness in epithelial‐derived tumor cells and promotes tumor metastasis. Therefore, fibrosis and tumors share the common process of EMT, and drugs that specifically target EMT may exhibit both antifibrosis and antitumor effects, which will provide an effective strategy against fibrosis and tumors.

Small molecules have a long history of acting as drugs for the treatment of hypertension, infections, heart failure, diabetes, asthma, rhinitis, and tumors and comprise approximately 75% of the anticancer drugs approved by the FDA from 1981 to 2014.11 Recently, many small molecules target EMT and exhibit good therapeutic effects on retarding progressive tissue fibrosis and cancer in experimental conditions.12-14 In addition, it is worth noting that many small molecules, such as tivantinib, trametinib, sunitinib, nintedanib, and binimetinib, are in ongoing clinical trials for the treatment of tumors.2 These cases show promising prospects for treating fibrosis and cancer, which encourages researchers to find effective small molecule drugs for treating fibrosis and cancer.

In this review, we describe some important transcription factors, signaling pathways, RNA‐binding proteins and microRNAs (miRNAs), and several novel regulators that contribute to EMT and further present some small molecules that exhibit therapeutic effects against EMT. Targeting these mediators with these compounds may be a promising therapeutic strategy to treat fibrosis and cancer.

2 | SMALL MOLECULES AGAINST EMT

Several excellent reviews have discussed the mechanisms of EMT well.15 Briefly, histological and pathological analyses show that epithelial cells are present in single-cell layers or multilayers and show apical‐basal polarity. Epithelial cells not only interact with the basement membrane with integrins but also communicate with each other via specialized intercellular junctions, including desmosomes, subapical tight junctions, adherens junctions, and scattered gap junctions. These interactions help maintain epithelium integrity and function. However, some stimuli drive EMT in pathological conditions. During the process of EMT, epithelial cell‐cell junctions are dissolved, and the epithelial cells lose their apical‐basal polarity and acquire front‐rear polarity. In addition, the cytoskeletal architecture is reorganized, and E‐cadherin expression is replaced by N‐cadherin expression, which enhances cell motility and invasiveness. In fibrosis, mesenchymal‐like cells transform into myofibroblasts, which can be activated to produce excessive collagen, and in tumors, these mesenchymal‐like cells migrate along with the circulatory system to a secondary location where they form a secondary tumor via mesenchymal‐epithelial transition (MET).16It is worth noting that tissue fibrosis and tumors share the common process of EMT, which suggests that targeting EMT may be an effective strategy to treat both fibrosis and tumors (Figure 1). EMT is regulated by various mediators such as transcription factors, signaling pathways, RNA‐binding proteins, and miRNAs. In addition, there are many small molecules that exhibit effective therapeutic effects on tissue fibrosis and tumors by suppressing EMT via targeting these mediators.

2.1 | Inhibition of transcription factors by small molecules

Transcription factors such as Snail, Twist, zinc‐finger E‐box‐binding homeobox (ZEB), and signal transducer and activator of transcription 3 (STAT3) are activated early and play central roles in the EMT. They not only repress the epithelial phenotype but also activate the mesenchymal phenotype individually or by cooperating with other transcription factors. Thus, inhibiting the activation of transcription factors may be an effective way to block EMTto treat fibrosis and tumors. Indeed, many compounds significantly intervene in fibrosis and tumors by suppressing the activation of transcription factors.

2.1.1 | Snail transcription factors

Snail is a zinc‐finger transcriptional repressor that consists of three isoforms, Snail1, Snail2, and Snail3. Among them, Snail1 and Snail2 are activated in the EMT during development, fibrosis, and cancer. The upregulation ofSnail1 is found in many different types of cancer such as gastric, colorectal, and prostate cancer.17-19 In addition, the overexpression of Snail1 enhanced the motility and invasion of prostate cancer cells, and the silencing of Snailremarkedly suppressed the adhesion, migration, and invasion of Hep‐2 cells.19 Further study revealed that the knockdown of Snail blocked the EMT, which was accompanied by the downregulation of the expression of MMP‐2, MMP‐9, vimentin, N‐cadherin, and fibronectin.19 Moreover, Snail expression was demonstrated at different stages of kidney fibrosis, and the reactivation of Snail‐induced renal fibrosis20 (Figure 2). Furthermore, hypoxia‐inducible factor 1α (HIF‐1α) mediated EMT by regulating Snail and the β‐catenin signaling pathway in early pulmonary fibrosis induced by paraquat.21 Based on these findings, it was suggested that Snail played a key role in the EMT during fibrosis and cancer, and the targeted inhibition of Snail expression might be an effective therapy to treat fibrosis and tumors.

Metformin is a first‐line drug for treating type II diabetes mellitus that has shown a therapeutic effect on many cancers. Further study revealed that metformin increased the expression of E‐cadherin, miR‐200a, miR‐200c, andmiR‐429 and decreased the expression of miR‐34a, vimentin, Snail1, and ZEB1 in transforming growth factor‐β(TGF‐β)‐induced EMT in the colorectal cancer cell lines SW480 and HCT‐116 (Table 1).57 In addition, some compounds derived from natural products have also shown significant intervention in fibrosis and tumors by targeting Snails (Table 1). Toosendanin, a triterpenoid isolated from Melia toosendan Sieb. et Zucc (Meliaceae), is used to prevent and control agricultural pests.97 Recently, it was reported that toosendanin significantly inhibited TNF‐β1‐induced EMT in lung cancer cells via the extracellular signal‐regulated kinase (ERK)/Snail pathway and suppressed EMT and tumor growth in pancreatic cancer by deactivating the RAC‐α serine/threonine‐protein kinase(Akt)/mechanistic target of rapamycin (mTOR) pathway (Figure 2), which suggests that toosendanin is a promising pharmacological agent for the treatment of cancer.22,23 A recent study revealed that reference enhanced oxaliplatin sensitivity by inhibiting EMT via the Snail pathway.19 In addition, penicillin is a major diterpenoid compound extracted from Rabdosia rubescens (Hemsl.) Hara exhibited a therapeutic effect on TNF‐α‐induced EMT and the metastasis of colorectal cancer by targeting the Akt/glycogen synthase kinase‐3 β (GSK3β)/Snail pathway31 (Figure2). Moreover, ferulic acid is a bioactive component derived from Ligusticum chuanxiong Hort that suppressed EMTinduced by TGF‐β1 via inhibiting the Smad/integrin‐linked kinase/Snail signaling pathway in a renal proximal tubular epithelial cell line (NRK‐52E), suggesting that ferulic acid might be a potent fibrosis antagonist.

2.1.2 | ZEB transcription factors

The two ZEB family members, ZEB1 and ZEB2, are transcriptional inhibitors that are involved in cell proliferation, migration, invasion, and apoptosis. Extensive studies have confirmed that ZEB1 is upregulated in the EMT and plays a key role in the development of tumors and fibrosis.99,100 It was reported that the Np63‐miR‐205 axis increased epithelial marker gene expression and decreased mesenchymal marker gene expression in oral squamous cell carcinoma by downregulating ZEB1 and ZEB2.101 Moreover, the heterogeneous expression of ZEB1 induced by EMT played an important role in metastasis through the regulation of miR‐200c.102 In addition, miR‐302a‐3pexerted has a protective role via inhibiting ZEB1 and EMT in diabetic kidney disease.103 These results suggest that various mediators contribute to EMT via the ZEB signaling pathway, suggesting that ZEB is a promising target for the treatment of fibrosis and tumors.

Decitabine is a drug used to treat myelodysplastic syndromes that inhibited EMT by regulating the miR‐200/ZEB signaling pathway in non–small‐cell lung cancer PC9 cells.58 In addition, a phase Ib/II clinical trial revealed that low‐dose decitabine enhanced the efficacy of immunotherapy in patients with drug‐resistant relapsed/refractory alimentary tract cancer, making it an attractive therapy for cancers.

Silibinin, a bioactive component from Silybum marianum (L.) Gaertn., exhibited anticancer properties in multiple types of cancer, such as bladder and lung cancer (Table 1). Recent studies revealed that silibinin reversed EMT by inhibiting the expression of ZEB1, vimentin, and MMP‐2 as well as the transactivation of β‐catenin in bladder cancer metastasis.39 Furthermore, the combined treatment of silibinin with trichostatin A(an inhibitor of histone deacetylases) or decitabine suppressed EMT in non–small‐cell lung cancer.105 These studies suggest that silibinin is an important candidate anticancer drug. Honokiol was extracted from the seed cones of Magnolia grandiflora L. and showed a therapeutic effect on various cancers. Honokiol effectively inhibited EMT in breast cancer through the STAT3/ZEB1/E‐cadherin axis (Figure 2) and reversed EMT in renal cell carcinoma via miR‐141/ZEB2 (Table 1).

2.1.3 | STAT3STAT3

a member of the STAT protein family can be phosphorylated as a transcription factor and plays a key role in various cellular processes, including cell growth, apoptosis, and differentiation. Hypoxia is a key mediator that induces EMT during tumor initiation and metastasis, and STAT3 promotes HIF‐1α expression to induce EMT by binding to the promoter of HIF‐1α. In addition, STAT3 was identified as a positive regulator that aggravated TGF‐β1‐induced EMT.106 In particular, the JAK2/STAT3 signaling pathway had a significant effect on the EMT in multiple cancers.107,108 In addition, STAT3 cooperated with other transcription factors promoting EMT, such as Twist and ZEB1.109 For example, the expression of p‐STAT3 (Tyr‐705) and ZEB1 was positively associated with metastasis, and they cooperatively enhanced EMT in colorectal carcinoma.110 Moreover, STAT3 was upregulated in TGF‐β1‐induced EMT during renal fibrosis, implying that STAT3 is an innovative target for the prevention of fibrosis.111 Furthermore, there are many mediators that induce EMT through the activation ofSTAT3, such as Pin1, HOXB8, miR‐30d, and IL‐6.

Sepantronium bromide is an inhibitor of survivin that exhibits anticancer properties in multiple cancer types. A recent study revealed that sepantronium bromide reduced the invasion of glioblastoma induced by radiation and reversed EMT by targeting STAT3.

Tanshinone IIA is a major bioactive component from the famous medical herb Salvia miltiorrhiza that was reported to attenuate the proliferation of bladder cancer cells (Table 1). Further study revealed that tanshinoneIIA suppressed EMT in bladder cancer cells via the modulation of the STAT3‐CCL2 signaling pathway.32 In addition, tanshinone IIA blocked EMT by regulating the TGF‐β/Smad pathway in peritoneal fibrosis.116 From these data, it was suggested that compounds that target EMT might have both antifibrotic and anticancer properties. In addition, polyphyllin I, a steroidal saponin derived from Paris polyphylla, has shown anti-inflammatory and anticancer properties. It was suggested that polyphyllin I, which reversed EMT by modulating the IL‐6/STAT3 pathway, served as a novel solution to conquer EGFR‐TKI resistance in non–small‐cell lung cancer 82 (Figure 2). Therefore, combined treatment with polyphyllin I and erlotinib is a promising therapy for lung cancer patients to strengthen drug efficacy and reduce drug resistance.82 The other compound, quercetin, is a flavonoid widely distributed in fruits, vegetables, and beverages that show antioxidative anti-inflammatory, and anticancer properties. Quercetin reversed IL‐6‐induced EMT in pancreatic cancer cells through STAT3.44Collectively, the data indicate that the use of compounds targeting STAT3 might be an effective approach for curing fibrosis and cancer.

2.2 | Targeting signaling pathways against EMT by small molecules

Many signaling pathways, such as the TGF‐β1, nuclear factor‐κB (NF‐κB), Wnt, Akt, peroxisome proliferator‐activated receptor (PPAR), and Notch pathways, and the renin‐angiotensin system (RAS) contribute to the EMT. Targeting these signaling pathways may be effective for the treatment of fibrosis and tumors, and many small molecules that target these signaling pathways reverse EMT in fibrosis and tumors.

2.2.1 | TGF‐β1 signaling pathway

TGF‐β1 plays a crucial role in various cellular functions, including cell growth, proliferation, differentiation, and apoptosis, and is considered a key mediator in EMT during the processes of tumor formation and fibrosis.117 Many components contribute to EMT through the TGF‐β1 signaling pathway, which indicates that the inhibition of the theTGF‐β1 signaling pathway may be effective in the treatment of cancer and fibrosis. Several excellent reviews have discussed the role of the TGF‐β1 signaling pathway in tumors and fibrosis well.117,118 Here, we present several important small molecules that suppress EMT in tumors and fibrosis by targeting the TGF‐β1 signaling pathway(Table 1).

Galunisertib, also known as LY2109761, is a TGF‐β receptor kinase inhibitor that specifically downregulated the phosphorylation of Smad2 and upregulated E‐cadherin expression in cultured human hepatocellular carcinoma cell lines, implying that it could suppress the EMT.119 Galunisertib is now in ongoing clinical trials in patients with glioblastoma, pancreatic cancer, and hepatocellular carcinoma.62 Nobiletin is a flavonoid compound isolated from Citrus depressa that suppressed EMT by antagonizing the TGF‐β1/Smad signaling pathway in human non–small‐cell lung cancer cells.42 In addition, oridonin is a diterpenoid from Rabdosia rubescens (Hemsl.) Hara inhibited EMT via blocking TGF‐β1/Smad2/3 in osteosarcoma.35 Moreover, GW788388 is a novel inhibitor of the TGF‐β type I receptor that inhibited TGF‐β‐induced EMT and fibrogenesis in db/db mice that showed significant diabetic nephropathy.63 Furthermore, oxymatrine, a bioactive alkaloid from Sophora japonica L., alleviated EMT induced by high glucose by inhibiting the TGF‐β1/Smad signaling pathway in NRK‐52E cells, indicating that oxymatrine might be a therapeutic agent for diabeticnephropathy.64 Isoviolanthin, a flavonoid isolated from Dendrobium officinale Kimura et Migo, suppressed TGF‐β1‐induced EMT through inhibiting the TGF‐β1/Smad and phosphatidylinositol 3‐kinase (PI3K)/Akt/mTOR signaling pathways in hepatocellular carcinoma cells.46 In addition, vitamin D, a well‐known inhibitor of EMT, inhibited EMT by negatively regulating the TGF‐β1 signaling pathway in human bronchial epithelial cells, and MART‐10 (a vitamin D analog) suppressed metastasis via downregulating EMT in pancreatic cancer cells.

2.2.2 | NF‐κB signaling pathway

The NF‐κB signaling pathway is widely involved in multiple cellular activities, such as cell proliferation, apoptosis, invasion, and inflammation. NF‐κB plays a vital role in the inflammation of tissue fibrosis, and inhibiting NF‐κBactivity reduced inflammation and enhances recovery from CCl4‐induced liver fibrosis.121 In addition, NF‐κBcontributed to inflammation, apoptosis, growth, migration, and invasion of cancer cells.122,123 Recently, it was found that the mediator of RNA polymerase II transcription, subunit 28 (MED28) modulated EMT through NF‐κB inhuman breast cancer cells, which suggests that the NF‐κB signaling pathway also plays a key role in the process of EMT.124 Therefore, targeting the NF‐κB signaling pathway may be a good choice to treat fibrosis and tumors.

Osthole, a dominant component in Cnidium monnieri (L.) Cuss. exhibited various biological activities including neuroprotective, osteogenic, cardiovascular protective, immunomodulatory, hepatoprotective, and antimicrobial effects. Osthole was found to block TGF‐β1‐induced EMT, adhesion, migration, and invasion by the inactivation of the NF‐κB/Snail pathways in A549 cells (Table 1).92 In addition, ostiole also attenuated insulin‐like growth factor‐ 1‐induced EMT by the PI3K/Akt pathway and inhibited hepatocyte growth factor‐induced EMT via the c‐Met/Akt/mTOR pathway in human brain cancer cells.125 Pterostilbene is a stilbene containing blueberries that effectively blocked the EMT in breast cancer stem cells by the NF‐κB/miR‐488 circuit.90 Moreover, pterostilbene negatively regulated EMT and inhibited triple‐negative breast cancer metastasis via inducing the expression of miR‐205.

The sclerotia of Polyporus umbellatus (Pers.) Fries are widely used to promote urination and prevent dampness.127Our previous studies systematically demonstrated that ergosta‐4,6,8(14),22‐terrain‐3‐one (ergone) isolated from Polyporus umbellatus (Pers.) Fries showed significant antitumor, diuretic, and renoprotective effects.128-135 Our recent study demonstrated that ergone inhibited NF‐κB signaling and α‐SMA expression in 5/6 nephrectomized and unilateral ureteral obstruction rats (Figure 2 and Table 1).

2.2.3 | Wnt signaling pathway

The Wnt signaling pathway is an evolutionarily conserved developmental signaling cascade that plays a critical role in regulating organ development and tissue homeostasis. Leucine‐rich repeat‐containing G protein‐coupled receptor 5(LGR5) is a novel functional marker in glioma stem cells that promotes EMT by activating the Wnt/β‐catenin signaling pathway.138 Sry‐like high‐mobility group box 8 regulates cancer stem‐like properties and cisplatin‐induced EMT by the Wnt/β‐catenin signaling pathway in tongue squamous cell carcinoma.139 In addition, bone marrow mesenchymal stromal cells suppressed EMT by inhibiting the Wnt/β‐catenin signaling pathway in silica‐induced pulmonary fibrosis.140 Our previous study showed the activation of the canonical Wnt/β‐catenin signaling pathway accompanied by the upregulation of proinflammatory and pro‐oxidative protein expression in the NF‐κB signaling pathway and downregulation of the anti‐inflammatory Nrf2 signaling pathway in patients with chronic kidney disease compared with the Nrf2 signaling pathway in healthy controls.

Many compounds inhibit the Wnt signaling pathway, and these compounds may suppress EMT in cancer and fibrosis (Table 1).142 FH535, a β‐catenin/Tcf inhibitor, not only increased radio‐sensitivity but also suppressed EMTin the radioresistant KYSE‐150R esophageal cancer cell line, which indicated that inhibitors of the Wnt signaling pathway might be effective anticancer agents with the potential to be anticancer drugs.65 In addition, it was reported that FH535 alleviated multiple types of cancer, including colorectal cancer, gastric cancer, and hepatocellular carcinoma.143-145 Isoquercitrin, a bioactive flavonoid from Bidens bipinnata L., inhibited the theWnt/β‐catenin signaling pathway and hepatocyte growth factor/scatter factor‐induced EMT in NBT‐II cells.146 In addition, salinomycin was previously used as an antibiotic and also showed significant anticancer activity by suppressing EMT.95,147,148 Salinomycin‐inhibited EMT by suppressing the Wnt/β‐catenin signaling pathway in epithelial ovarian cancer cells, indicating that small molecules targeting the Wnt/β‐catenin signaling pathway might have anticancer properties by reversing EMT.95 Poria cocos (Polyporaceae) is a well‐known fungus that exhibits an effective therapeutic effect to improve kidney function in clinic.149,150 Our previous studies have confirmed that extracts of the surface layer of Poria cocos show remarkable antihyperlipidemic, diuretic, and renoprotective effects.151-158 Recently, our group isolated more than 90 triterpenoid compounds from the surface layer of Poria cocos, some of which exhibited significant antifibrotic properties.159-161 New triterpenoids, including poricoic acidZC, periodic acid ZD, and periodic acid ZE, significantly downregulated the expression of Wnt1, active β‐catenin, Snail, Twist, MMP‐7, PAI‐1, and FSP1 in HK‐2 cells induced by TGF‐β1 and angiotensin II and mice with unilateral ureteral occlusion29 (Figure 2 and Table 1). Moreover, new triterpenoids, including poricoic acid ZG and poricoicacid ZH, improved renal fibrosis by targeting the phosphorylation of Smad3 signaling and the Wnt/β‐catenin signaling pathway162 (Figure 2 and Table 1).

Alismatis rhizome (AR), the dried rhizome of Alisma Orientale (Sam.) Julep exhibited diuretic, antihyperlipidemic, and renoprotective effects163 that was also confirmed by our previous studies.164-167 Our recent study demonstrated that triterpenoid was the main component of AR,30 and further study showed that the novel tetracyclic triterpenoid 25‐O‐methylation F inhibited EMT by suppressing the Wnt/β‐catenin signaling pathway as well as the phosphorylation of Smad3 signaling in both NRK‐52E and NRK‐49F cells30 (Figure 2 and Table 1). Additionally, it was also observed that ergone inhibited extracellular matrix accumulation in HK‐2 cells and attenuated podocyte injury by inhibiting the activation of the Wnt/β‐catenin signaling pathway induced by angiotensin II.81 Taken together, these data indicate that tetracyclic triterpenoid and steroid compounds show significant antifibrotic properties in renal fibrosis.