The RhoA pathway mediates MMP‐2 and MMP‐9‐independent invasive behavior in a triple‐negative breast cancer cell line

Breast cancer is a heterogeneous disease that varies in its biology and response to therapy. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk among patients diagnosed with triple‐negative and/or basal‐like breast cancers. A greater understanding of the molecular mechanisms underlying cancer cell spreading is needed as 90% of cancer‐associated deaths result from metastasis. We previously demonstrated that the Tamoxifen‐selected, MCF‐7 derivative, TMX2‐28, lacks expression of estrogen receptor α (ERα) and is highly invasive, yet maintains an epithelial morphology. The present study was designed to further characterize TMX2‐28 cells and elucidate their invasion mechanism. We found that TMX2‐28 cells do not express human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR), in addition to lacking ERα, making the cells triple‐negative. We then determined that TMX2‐28 cells lack expression of active matrix metalloproteinases (MMPs)‐1, MMP‐2, MMP‐9, and other genes involved in epithelial–mesenchymal transition (EMT) suggesting that TMX2‐28 may not utilize mesenchymal invasion. In contrast, TMX2‐28 cells have high expression of Ras Homolog Gene Family Member, A (RhoA), a protein known to play a critical role in amoeboid invasion. Blocking RhoA activity with the RhoA pathway specific inhibitor H‐1152, or a RhoA specific siRNA, resulted in inhibition of invasive behavior. Collectively, these results suggest that TMX2‐28 breast cancer cells exploit a RhoA‐dependent, proteolytic‐independent invasion mechanism. Targeting the RhoA pathway in triple‐negative, basal‐like breast cancers that have a proteolytic‐independent invasion mechanism may provide therapeutic strategies for the treatment of patients with increased risk of metastasis. J. Cell. Biochem. 114: 1385–1394, 2013. © 2013 Wiley Periodicals, Inc.     

The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process.     

Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling

Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β₃- and β₆-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.     

Rho小G蛋白相关激酶的结构与功能

Rho小G蛋白家族是Ras超家族成员之一,人类Rho小G蛋白包括20个成员,研究最清楚的有RhoA、Rac1和Cdc42。Rho小G蛋白参与了诸如细胞骨架调节、细胞移动、细胞增殖、细胞周期调控等重要的生物学过程。在这些生物学过程的调节中,Rho小G蛋白的下游效应蛋白质如蛋白激酶（p21-activated kinase,PAK）、ROCK（Rho-kinase）、PKN（protein kinase novel）和MRCK（myotonin-related Cdc42-binding kinase）发挥了不可或缺的作用。迄今研究发现,PAK可调节细胞骨架动力学和细胞运动,另外,PAK通过MAPK（mitogen-activated protein kinases）参与转录、细胞凋亡和幸存通路及细胞周期进程;ROCK与肌动蛋白应力纤维介导黏附复合物的形成及与细胞周期进程的调节有关;哺乳动物的PKN与RhoA/B/C相互作用介导细胞骨架调节;MRCK与细胞骨架重排、细胞核转动、微管组织中心再定位、细胞移动和癌细胞侵袭等有关。该文简要介绍Rho小G蛋白下游激酶PAK、ROCK、PKN和MRCK的结构及其在细胞骨架调节中的功能,重点总结它们在真核细胞周期调控中的作用,尤其是在癌细胞周期进程中所发挥的作用,为寻找癌症治疗的新靶点提供理论依据。     

Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin‐mediated signalling pathway

Establishment of infection by facultative intracellular pathogen Mycobacterium tuberculosis (Mtb) requires adherence to and internalisation by macrophages. However, the effector molecules exploited by Mtb for entry into macrophages remain to be fully understood. The mammalian cell entry (Mce) proteins play an essential role in facilitating the internalisation of mycobacteria into mammalian cells. Here, we characterized Mtb Mce3C as a new mycobacterial surface protein that could promote mycobacterial adhesion to and invasion of macrophages in an RGD motif‐dependent manner. We then further demonstrated that β2 integrin was required for Mce3C‐mediated cell entry. In addition, we found that binding of Mce3C recruited β2 integrin‐dependent signalling adaptors and induced local actin rearrangement at the site of mycobacterial invasion. By using specific antibodies and pharmacological inhibitors, we further demonstrated the involvement of Src‐family tyrosine kinases, spleen tyrosine kinase, Vav, Rho, and Rho‐associated kinase in Mce3C‐mediated mycobacterial invasion. Our results reveal a novel mechanism by which Mtb Mce3C exploits integrin‐mediated signalling cascade for Mce, providing potential targets for the development of therapies against Mtb infection.     

Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin , and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly.     

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Despite current advances in cancer research, metastasis remains the leading factor in cancer-related deaths. Here we identify sorting nexin 9 (SNX9) as a new regulator of breast cancer metastasis. We detect an increase in SNX9 expression in human breast cancer metastases compared with primary tumors and demonstrate that SNX9 expression in MDA-MB-231 breast cancer cells is necessary to maintain their ability to metastasize in a chick embryo model. Conversely, SNX9 knockdown impairs this process. In vitro studies using several cancer cell lines derived from a variety of human tumors reveal a role for SNX9 in cell invasion and identify mechanisms responsible for this novel function. We show that SNX9 controls the activation of RhoA and Cdc42 GTPases and also regulates cell motility via the modulation of well-known molecules involved in metastasis, namely RhoA-ROCK and N-WASP. In addition, we find that SNX9 is required for RhoGTPase-dependent, clathrin-independent endocytosis, and in this capacity can functionally substitute to the bona fide Rho GAP, GTPase regulator associated with focal adhesion kinase (GRAF1). Taken together, our data establish novel roles for SNX9 as a multifunctional protein scaffold that regulates, and potentially coordinates, several cellular processes that together can enhance cancer cell metastasis.     

LIPH promotes metastasis by enriching stem‐like cells in triple‐negative breast cancer

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24? stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44⁺/CD24^? stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.     

A novel strategy for specifically down-regulating individual Rho GTPase activity in tumor cells

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.     

α‐parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling

During blood vessel development, vascular smooth muscle cells (vSMCs) and pericytes (PCs) are recruited to nascent vessels to stabilize them and to guide further vessel remodelling. Here, we show that loss of the focal adhesion (FA) protein α‐parvin (α‐pv) in mice leads to embryonic lethality due to severe cardiovascular defects. The vascular abnormalities are characterized by poor vessel remodelling, impaired coverage of endothelial tubes with vSMC/PCs and defective association of the recruited vSMC/PCs with endothelial cells (ECs). α‐pv‐deficient vSMCs are round and hypercontractile leading either to their accumulation in the tissue or to local vessel constrictions. Because of the high contractility, α‐pv‐deficient vSMCs fail to polarize their cytoskeleton resulting in loss of persistent and directed migration. Mechanistically, the absence of α‐pv leads to increased RhoA and Rho‐kinase (ROCK)‐mediated signalling, activation of myosin II and actomyosin hypercontraction in vSMCs. Our findings show that α‐pv represents an essential adhesion checkpoint that controls RhoA/ROCK‐mediated contractility in vSMCs.     

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.     

乳腺癌中Rho A蛋白表达及其与Cyclin D1和P21WAF1/CIP1蛋白表达的相关性

目的探讨Rho A蛋白在人乳腺癌中的表达情况,Rho A蛋白与临床病理因素的关系,及其与细胞周期蛋白Cyclin D1,细胞周期抑制蛋白 P21 WAF1/CIP1表达的相关性.方法应用免疫组化S-P法,检测64例乳腺癌组织及20例正常乳腺组织中Rho A蛋白、Cyclin D1和P21 WAF1/CIP1蛋白的表达情况.结果 (1)Rho A、 Cyclin D1和P21 WAF1/CIP1蛋白在正常乳腺组织中的表达率分别为5.00%、25.00%、15.00%,在乳腺癌组织中的表达率分别为73.44%、59.38%、48.44%,三者在乳腺癌组织中的阳性表达分别与正常乳腺组织相比,均差异有显著性意义(P< 0.01).(2)Rho A蛋白表达与病理组织分级,淋巴结转移相关(P< 0.05),与患者年龄、肿瘤大小及临床分期无关.(3)RhoA蛋白与P21 WAF1/CIP1蛋白表达呈负相关(χ2=4.548,P<0.05),与Cyclin D1蛋白表达无关.结论乳腺癌患者RhoA蛋白过表达与预后不良有关.RhoA蛋白通过下调P21 WAF1/CIP1蛋白参与细胞周期调节,进而与乳腺癌发展及侵袭转移相关.     

Hic‐5 affects proliferation,migration and invasion of B16 murine melanoma cells

Hic‐5 is a shuttling protein between the cell membrane and the nucleus which functions as a focal adhesion adaptor protein and a nuclear receptor coactivator. Although several studies have shown its involvement in other types of cancer, the role of Hic‐5 in melanoma is unknown. Herein, we show for the first time that Hic‐5 is expressed in B16‐F1 murine melanoma cells. To determine its function in melanoma cells, we used shRNA‐mediated RNA interference and established stable clones with down‐regulated Hic‐5 expression. These clones had impaired growth and metastatic potential compared with controls in vivo, which correlated with decreased proliferation, migration and invasion in vitro. Moreover, silencing of Hic‐5 expression in B16‐F1 activated RhoA with an amoeboid phenotypic change, indicating that Hic‐5 is a key regulator of B16‐F1 metastasis in the context of Rho‐dependent motility. These results provide new evidence that Hic‐5 is a possible molecular target for treatment of melanoma.     

KiSS1 suppresses TNFα‐induced breast cancer cell invasion via an inhibition of RhoA‐Mediated NF‐κB activation

Tumor necrosis factor‐alpha (TNFα) induces cancer development and metastasis, which is prominently achieved by nuclear factor‐kappa B (NF‐κB) activation. TNFα‐induced NF‐κB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down‐regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFα‐induced NF‐κB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFα‐induced NF‐κB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin‐10) stimulation inhibited TNFα‐induced NF‐κB activity, suppressed TNFα‐induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFα‐induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFα‐induced NF‐κB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion. J. Cell. Biochem. 107: 1139–1149, 2009. © 2009 Wiley‐Liss, Inc.     

17β-Estradiol enhances breast cancer cell motility and invasion via extra-nuclear activation of actin-binding protein ezrin

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr(567) in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion

Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.     

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.     

A novel synthetic small molecule YH‐306 suppresses colorectal tumour growth and metastasis via FAK pathway

Cell migration and invasion are key processes in the metastasis of cancer, and suppression of these steps is a promising strategy for cancer therapeutics. The aim of this study was to explore small molecules for treating colorectal cancer (CRC) and to investigate their anti‐metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH‐306 significantly inhibited the migration and invasion of CRC cells in a dose‐dependent manner. In addition, YH‐306 inhibited cell adhesion and protrusion formation of HCT116 and HT‐29 CRC cells. Moreover, YH‐306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH‐306 inhibited CRC colonization in vitro and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis in vivo. YH‐306 suppressed the activation of focal adhesion kinase (FAK), c‐Src, paxillin, and phosphatidylinositol 3‐kinases (PI3K), Rac1 and the expression of matrix metalloproteases (MMP) 2 and MMP9. Meanwhile, YH‐306 also inhibited actin‐related protein (Arp2/3) complex‐mediated actin polymerization. Taken together, YH‐306 is a candidate drug in preventing growth and metastasis of CRC by modulating FAK signalling pathway.