Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.     

In search of AKT kinase inhibitors as anticancer agents: structure-based design,docking, and molecular dynamics studies of 2,4,6-trisubstituted pyridines

The AKT isoforms are a group of key kinases that play a critical role in tumorigenesis. These enzymes are overexpressed in different types of cancers, such as breast, colon, prostate, ovarian, and lung. Because of its relevance the AKT isoforms are attractive targets for the design of anticancer molecules. However, it has been found that AKT1 and AKT3 isoforms have a main role in tumor progression and metastasis; thus, the identification of AKT isoforms specific inhibitors seems to be a challenge. Previously, we identified an ATP binding pocket pan-AKT inhibitor, this compound is a 2,4,6-trisubstituted pyridine (compound 11), which represents a new interesting scaffold for the developing of AKT inhibitors. Starting from the 2,4,6-trisubstituted pyridine scaffold, and guided by structure-based design technique, 42 new inhibitors were designed and further evaluated in the three AKT isoforms by multiple docking approach and molecular dynamics. Results showed that seven compounds presented binding selectivity for AKT1 and AKT3, better than for AKT2. The binding affinities of these seven compounds on AKT1 and AKT3 isoforms were mainly determined by hydrophobic contributions between the aromatic portion at position 4 of the pyridine ring with residues Phe236/234, Phe237/235, Phe438/435, and Phe442/439 in the ATP binding pocket. Results presented in this work provide an addition knowledge leading to promising selective AKT inhibitors.     

Novel benzofuran-based sulphonamides as selective carbonic anhydrases IX and XII inhibitors: synthesis and in vitro biological evaluation

Abstract

Pursuing on our efforts toward searching for efficient hCA IX and hCA XII inhibitors, herein we report the design and synthesis of new sets of benzofuran-based sulphonamides (4a,b, 5a,b, 9a–c, and 10a–d), featuring the zinc anchoring benzenesulfonamide moiety linked to a benzofuran tail via a hydrazine or hydrazide linker. All the target benzofurans were examined for their inhibitory activities toward isoforms hCA I, II, IX, and XII. The target tumour-associated hCA IX and XII isoforms were efficiently inhibited with K _Is spanning in ranges 10.0–97.5 and 10.1–71.8?nM, respectively. Interestingly, arylsulfonehydrazones 9 displayed the best selectivity toward hCA IX and XII over hCA I (SIs: 39.4–250.3 and 26.0–149.9, respectively), and over hCA II (SIs: 19.6–57.1 and 13.0–34.2, respectively). Furthermore, the target benzofurans were assessed for their anti-proliferative activity, according to US-NCI protocol, toward a panel of sixty cancer cell lines. Only benzofurans 5b and 10b possessed selective and moderate growth inhibitory activity toward certain cancer cell lines.     

Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed as ROCK inhibitors

In this study, we synthesized a new congener series of N-sulphonylhydrazones designed as candidate ROCK inhibitors using the molecular hybridization of the clinically approved drug fasudil (1) and the IKK-β inhibitor LASSBio-1524 (2). Among the synthesized compounds, the N-methylated derivative 11 (LASSBio-2065) showed the best inhibitory profile for both ROCK isoforms, with IC₅₀ values of 3.1 and 3.8?µM for ROCK1 and ROCK2, respectively. Moreover, these compounds were also active in the scratch assay performed in human breast cancer MDA-MB 231 cells and did not display toxicity in MTT and LDH assays. Molecular modelling studies provided insights into the possible binding modes of these N-sulphonylhydrazones, which present a new molecular architecture capable of being optimized and developed as therapeutically useful ROCK inhibitors.     

Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy

Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors.     

Dynamics and Binding Modes of Free cdk2 and its Two Complexes with Inhibitors Studied by Computer Simulations

Abstract

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

Discovery of novel pyruvate dehydrogenase kinases inhibitors by screening of an in-house small molecule library for anti-lung cancer therapeutics

Pyruvate dehydrogenase kinases (PDKs) are widely over-expressed in various human solid cancers, making them attractive therapeutic targets for cancer treatment. Herein, we report the identification of structurally novel PDKs inhibitors by screening of an in-house small molecule library. Biochemical assay indicated that the identified compounds 1–4 inhibited PDK1 activity with EC₅₀ values of 0.50, 1.99, 4.64, and 0.42?µM, respectively. The ITC analysis suggested that the identified compounds 1–4 were pan-isoform PDK inhibitors, which bound to and inhibited the four PDK isoforms. Moreover, 1–4 dose-dependently reduced pyruvate dehydrogenase complex phosphorylation in NCI-H1975 cell. Molecular docking suggested that the most potent compound 4 docked well in the ATP binding pocket of the four PDK isoforms, forming direct hydrogen bond interactions with the conserved amino acids Thr and Asp in ATP binding pocket of PDKs. The cell viability assay demonstrated that 4 potently blocked NCI-H1975 cell proliferation (IC₅₀?=?3.32?µM), but had little effect on human normal lung cell MRC-5 even with the tested concentration up to 40?µM. All the data demonstrated that 4 was a promising lead for the development of structurally novel PDKs inhibitor for the cancer treatment.     

Aquaporin-9 protein is the primary route of hepatocyte glycerol uptake for glycerol gluconeogenesis in mice

It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9(-/-) knock-out mice remained normoglycemic. The reason for this improvement in AQP9(-/-) mice was not established before. Here, we show increased glucose output (by 123% ± 36% S.E.) in primary hepatocyte culture when 0.5 mM extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9(-/-) cells. Likewise, the increase was abolished by 25 μM HTS13286 (IC(50) ~ 2 μM), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate- and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9(-/-) mice.     

Halting ionic shuttle to disrupt the synthetic machinery—Structural and molecular insights into the inhibitory roles of Bedaquiline towards Mycobacterium tuberculosis ATP synthase in the treatment of tuberculosis

Therapeutic targeting of the adenosine triphosphate (ATP) machinery of Mycobacterium tuberculosis (Mtb) has recently presented a potent and alternative measure to halt the pathogenesis of tuberculosis. This has been potentiated by the development of bedaquiline (BDQ), a novel small molecule inhibitor that selectively inhibits mycobacterial F₁F_o-ATP synthase by targeting its rotor c-ring, resulting in the disruption of ATP synthesis and consequential cell death. Although the structural resolution of the mycobacterial C₉ ring in co`mplex with BDQ provided the first-hand detail of BDQ interaction at the c-ring region of the ATP synthase, there still remains a need to obtain essential and dynamic insights into the mechanistic activity of this drug molecule towards crucial survival machinery of Mtb. As such, for the first time, we report an atomistic model to describe the structural dynamics that explicate the experimentally reported antagonistic features of BDQ in halting ion shuttling by the mycobacterial c-ring, using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Surface Area methods. Results showed that BDQ exhibited a considerably high ΔG while it specifically maintained high-affinity interactions with Glu65_B and Asp32_B, blocking their crucial roles in proton binding and shuttling, which is required for ATP synthesis. Moreover, the bulky nature of BDQ induced a rigid and compact conformation of the rotor c-ring, which impedes the essential rotatory motion that drives ion exchange and shuttling. In addition, the binding affinity of a BDQ molecule was considerably increased by the complementary binding of another BDQ molecule, which indicates that an increase in BDQ molecule enhances inhibitory potency against Mtb ATP synthase. Taken together, findings provide atomistic perspectives into the inhibitory mechanisms of BDQ coupled with insights that could enhance the structure-based design of novel ATP synthase inhibitors towards the treatment of tuberculosis.     

Design,synthesis and molecular modelling studies of some pyrazole derivatives as carbonic anhydrase inhibitors

Abstract

In this study, newly synthesised compounds 6, 8, 10 and other compounds (1–5, 7 and 9) and their inhibitory properties against the human isoforms hCA I and hCA II were reported for the first time. Compounds 1–10 showed effective inhibition profiles with K _I values in the range of 5.13–16.9?nM for hCA I and of 11.77–67.39?nM against hCA II, respectively. Molecular docking studies were also performed with Glide XP to get insight into the inhibitory activity and to evaluate the binding modes of the synthesised compounds to hCA I and II. More rigorous binding energy calculations using MM-GBSA protocol which agreed well with observed activities were then performed to improve the docking scores. Results of in silico calculations showed that all compounds obey drug likeness properties. The new compounds reported here might be promising lead compounds for the development of new potent inhibitors as alternatives to classical hCA inhibitors.     

Induced fit docking,free energy calculation and molecular dynamics studies on Mycobacterium tuberculosis alanine racemase inhibitor

Alar, a Pyridoxal 5′-phosphate (PLP)-dependent bacterial enzyme is responsible for the racemisation of L-alanine into D-alanine which is essential for the peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. In the present study, we performed induced fit docking, binding free energy calculation and molecular dynamics simulation to elucidate the Alar inhibition potential of 1,2,4-thiadiazolidine-3,5-dione-based inhibitor 1. The inhibitor binds to the hydrophobic groove of Alar and the binding was found to be stable throughout 20-ns MD simulation. Induced fit docking result showed that Lys42, Tyr46, Tyr175 and Tyr364 residues are primarily responsible for the stabilisation of inhibitor–protein complex. Further, high negative van der Waals binding free energy value of –38.88 kcal/mol, indicated it as the main driving force for the inhibitor binding. Based on the information obtained from this study, we designed few molecules as potent Alar inhibitor. In order to gain structural insight and to validate the stability of complex, we performed 20-ns MD simulation of the designed molecule D1. Results obtained from this study can be used for the design of M. tuberculosis Alar potent inhibitors lacking affinity for the co-factor PLP.     

Nitrobenzoxadiazole-based GSTP1-1 inhibitors containing the full peptidyl moiety of (pseudo)glutathione

Context: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice.

Objective: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers.

Materials and methods: Synthesis of derivatives 1 and 5 and in vitro evaluation on human GSTP1-1 and M2-2 are reported.

Results: Conjugates 1 and 5 were found to be low micromolar inhibitors of both isoforms. Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1 isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol (NBDHEX).

Discussion and conclusions: Spectroscopic data are congruent with the formation of a stable sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5 is suitable for in vivo modulation of GST activity in cancer treatment.     

Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure–activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in K_i value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.     

Combined pharmacophore modeling, 3D-QSAR and docking studies to identify novel HDAC inhibitors using drug repurposing

Abstract

Histone deacetylases (HDACs), a critical family of epigenetic enzymes, has emerged as a promising target for antitumor drugs. Here, we describe our protocol of virtual screening in identification of novel potential HDAC inhibitors through pharmacophore modeling, 3D-QSAR, molecular docking and molecular dynamics (MD) simulation. Considering the limitation of current virtual screening works, drug repurposing strategy was applied to discover druggable HDAC inhibitor. The ligand-based pharmacophore and 3D-QSAR models were established, and their reliability was validated by different methods. Then, the DrugBank database was screened, followed by molecular docking. MD simulation (100?ns) was performed to further study the stability of ligand binding modes. Finally, results indicated the hit DB03889 with high in silico inhibitory potency was suitable for further experimental analysis.

Communicated by Ramaswamy H. Sarma     

Structural improvement of LidNA: delta-type LidNA is a potent miRNA inhibitor constructed with unmodified DNA

ABSTRACT

Many miRNA inhibitors have been developed, including chemically modified oligonucleotides, such as 2′-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA has not yet been reported as a miRNA inhibitor due to relatively low DNA/miRNA binding affinity. We designed a structured DNA, LidNA, which was constructed with unmodified DNA, consisting of a complementary sequence to the target miRNA flanked by two structured DNA regions, such as double-stranded DNA. LidNA inhibited miRNA activity more potently than 2′-O-methylated RNA or LNA. To optimize LidNA, two double-stranded regions were joined, causing the molecule to assume a delta-like shape, which we termed delta-type LidNA. Delta-type LidNAs were developed to target endogenous and exogenous miRNAs, and exhibited potent miRNA inhibitory effects with a duration of at least 10 days. Delta-type LidNA-21, which targeted miR-21, inhibited the growth of cancer cell lines. This newly developed LidNA could contribute to miRNA studies across multiple fields.

Abbreviations: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; 3′-UTR: 3′-untranslated regions; RISC: RNA-induced silencing complex; MBL: Molecular beacon-like LidNA; YMBL: Y-type molecular beacon-like LidNA; TDMD: target-directed microRNA degradation.     

Design, synthesis, and biological activity of novel Magmas inhibitors

Magmas (mitochondria associated, granulocyte-macrophage colony stimulating factor signaling molecule), is a highly conserved and essential gene, expressed in all cell types. We designed and synthesized several small molecule Magmas inhibitors (SMMI) and assayed their effects on proliferation in yeast. We found that the most active compound 9 inhibited growth at the 4 μM scale. This compound was shown by fluorometric titration to bind to Magmas with a K_d = 33 μM. Target specificity of the lead compound was established by demonstrating direct binding of the compound to Magmas and by genetic studies. Molecular modeling suggested that the inhibitor bound at the predicted site in Magmas.     

Virtual screening to identify novel potential inhibitors for Glutamine synthetase of Mycobacterium tuberculosis

Abstract

Glutamine synthetase (GS) of Mycobacterium tuberculosis (Mtb) is an essential enzyme which is involved in nitrogen metabolism and cell wall synthesis. It is involved in the inhibition of phagosome-lysosome fusion by preventing acidification. Targeting GS can be helpful to control the infection of Mtb. In order to identify potential inhibitors, we screened chemical libraries (56,400 compounds of ChEMBL anti-mycobacterial, 1596 FDA approved drugs, 419 Natural product and 916 phytochemical) against this target using the virtual screening approach. Screening by molecular docking identified ten top-ranked compounds as GSMtb inhibitors and they were compared with known inhibitors (as control). Since GS enzyme (GSHs) is also present in human. We have compared the protein sequence of GS from Mtb and human using the P-BLAST in NCBI. We found ～27% identity in between these two sequences, so we also compared the binding affinity of inhibitor between Mtb and human. Finally, we identified top two compounds namely CHEMBL387509, CHEMBL226198 from ChEMBL anti-mycobacterial dataset, and Eriocitrin and Malvidin from phytochemical dataset which showed lees binding affinity towards GSHs whereas Pamidronate, and Phentermine from FDA approved drugs and (-)-Quinic Acid, Hexopyranuronic acid, Quebrachit, and Castanospermine from natural product showed protein-ligand interaction with Mtb protein while no interaction with GSHs. The top two docked complexes were subjected to molecular dynamic simulation to understand the stability of the molecule. Further, we calculated the binding free energy of the docked complex and analyzed hydrogen bond, salt bridge, pie stacking, and hydrophobic interaction in the docking region. These ligands exhibited very good binding affinity GSMtb enzymes. Therefore, these ligands are novel and drug-likeness compounds, and they may be potential inhibitors of M tuberculosis.

Communicated by Ramaswamy H. Sarma     

The Structure of M-Gtfi,An Inhibitor of Glucosyltransferase from S. Mutans,And Its Inhibitory Relationship with Other Sulfate Ester-Containing Inhibitors

Abstract

M-GTFI, an inhibitor of glucosyltransferase from S. mutans was produced by Micromonospora narashinoensis strain No. 731. The isolation procedure for M-GTFI was improved and established for spectro-scopic analyses, and some properties of the inhibitor were investigated. The structure of M-GTFI was shown to be trisodium [2-sulphonato-9-undecenyll-oxacyclotriacont-3-en-2-one, 16, 18-his sulp-honate. The chemical structure of M-GTFI was therefore similar to that of izumenolide which is a β-lactamase inhibitor containing sulfate ester groups in its molecule.

The inhibitory characteristics of M-GTFI were parallel to that of other inhibitory compounds containing sulphate esters but the spectrum of activity was wider.     

Aquaporin3 Is Required for FGF-2-Induced Migration of Human Breast Cancers

Purpose

The aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers.

Methods

Potential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO₄. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression.

Results

FGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO₄, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.

Conclusions

AQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.