Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

E‐cadherin determines Caveolin‐1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin‐1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E‐cadherin in CAV1‐dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E‐cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co‐expression of E‐cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav‐1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E‐cadherin expression in B16F10 (E‐cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co‐expression of CAV1 and E‐cadherin in B16F10 (cav‐1/E‐cad) cells abolishes tumor formation, lung metastasis, increased Rac‐1 activity, and cell migration observed with B16F10 (cav‐1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac‐1 activation in these cells.     

Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin alpha(5) in promoting melanoma metastasis. The alpha(5) expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin alpha(4), alpha(v), and beta(1) subunits. Neutralization of alpha(5) with anti-alpha(5) antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between alpha(5) and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin alpha(5) is positively correlated with melanoma metastasis and might be an anti-melanoma target.     

IGF-I enhances α5β1 integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. Insulin-like growth factor-I (IGF)-I plays an important role in regulating cell growth, proliferation, survival, and metabolism. However, the effects of IGF-I in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IGF-I increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Pretreatment of cells with IGF-I receptor antibody reduced IGF-I-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor-κB (NF-κB) pathways after IGF-I treatment were demonstrated, and IGF-I-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and NF-κB cascades. Taken together, our results indicated that IGF-I enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the IGF-I receptor/PI3K/Akt/NF-κB signal transduction pathway.     

Elevation of intracellular cyclic AMP inhibits NF-κB-mediated thymosin β4 expression in melanoma cells

Thymosin β4 (Tβ4) is a major actin-sequestering protein that has been implicated in the growth, survival, motility, and metastasis of certain tumors and is considered an indicator for malignant progression. Therefore, identifying compounds that can downregulate Tβ4 expression is very important for the development of anti-cancer chemotherapies. In this study, we investigated the effects of elevated cAMP on Tβ4 expression and the metastatic potential of murine B16 melanoma cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tβ4 suppression. We found that treatment with the cAMP-inducing compounds α-MSH (α-melanocyte stimulating hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tβ4 expression and regulated EMT-associated genes through the suppression of NF-κB activation in B16F10 cells. Along with decreased Tβ4 expression, the in vitro invasiveness and anchorage-independent growth in a semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of the α-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tβ4 expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover, suppression of NF-κB activation by α-MSH or IBMX is critical for inhibiting Tβ4 expression.     

Loss of the α2β1 integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro

Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.     

CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). αvβ3 or αvβ5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF-κB inhibitors also suppressed the cell migration and MMP-13 expression enhanced by CCN3. Moreover, CCN3 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the αvβ3/αvβ5 integrin receptor, FAK, PI3K, Akt, p65, and NF-κB signal transduction pathway.     

Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells

The human homologue of NG2, the human melanoma proteoglycan (HMP), is expressed on most human melanomas. To investigate the role of this proteoglycan in melanoma progression, we have attempted to identify functionally important molecular ligands for NG2. Immunohistochemical analysis of cell lines that endogenously express NG2/HMP suggests that NG2/HMP associates with CD44 and α₄β₁ integrin, two molecules previously implicated in melanoma progression. Transfection of rat NG2 into the NG2-negative B16 mouse melanoma cell line also resulted in a highly colocalized pattern of expression between the transfected rat NG2 and the endogenously expressed mouse CD44 and α₄β₁ integrin molecules. In functional assays, expression of NG2 decreased the adhesion of B16 melanoma cells to CD44 monoclonal antibodies, hyaluronic acid, the C-terminal 40-kDa fibronectin fragment, and the CS1 fibronectin peptide, suggesting that NG2 may negatively modulate CD44- and α₄β₁-mediated binding events. Expression of NG2 increased the proliferation of melanoma cells in culture and increased tumorigenicity in vivo. Moreover, NG2 expression led to increased lung metastasis of B16F1 and B16F10 melanoma cells in experimental metastasis studies. Together, these studies demonstrate that NG2 is capable of modulating the adhesion, proliferation, and metastatic potential of melanoma cells. J. Cell. Physiol. 177:299–312, 1998. © 1998 Wiley-Liss, Inc.     

Macrophage migration inhibitory factor increases cell motility and up-regulates αvβ3 integrin in human chondrosarcoma cells

The macrophage migration-inhibitory factor (MIF) is a pro-inflammatory cytokine first known for its effect on macrophage migration and activation. Recent studies have shown that MIP plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of MIF on human chondrosarcoma cells are largely unknown. In the present study, MIF was found to increase the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by MIF treatment, and the MIF-induced expression of integrin and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. In addition, migration-prone sublines demonstrated that increased cell migration ability was correlated with increased expression of MIF and αvβ3 integrin. Taken together, our results indicate that MIF enhanced the migration of the chondrosarcoma cells by increasing αvβ3 integrin expression through the PI3K/Akt/NF-κB signal transduction pathway.     

AEG‐1 induces gastric cancer metastasis by upregulation of eIF4E expression

Gastric cancer is the third leading cause of cancer‐related deaths worldwide, and patients with lymph node, peritoneal and distant metastasis have a poor prognosis. Overexpression of Astrocyte‐elevated gene‐1 (AEG‐1) has been reported to be correlated with the progression and metastasis of gastric cancer. However, its mechanisms are quite unclear. In this study, we found that elevated expression of AEG‐1 was correlated with metastasis in human gastric cancer tissues. Moreover, gain‐ or loss‐of‐function of AEG‐1, respectively, promoted or suppressed epithelial–mesenchymal transition (EMT), migration and invasion of gastric cancer cells. AEG‐1 positively regulated eIF4E, MMP‐9 and Twist expression. Manipulating eIF4E expression by transfection of overexpression constructs or siRNAs partially eliminated AEG‐1‐regulated EMT, cell migration and invasion. In addition, overexpression or knockdown of eIF4E promoted or suppressed EMT, cell migration and invasion in parallel with upregulation of MMP‐9 and Twist expression, while manipulating eIF4E expression partially abrogated AEG‐1‐induced MMP‐9 and Twist. Finally, silencing of AEG‐1 expression not only inhibited tumour growth in parallel with downregulation of eIF4E, MMP‐9 and Twist expression in a xenograft nude mouse model, but also suppressed lymph node and peritoneal metastasis of gastric cancer in an orthotopic nude mouse model. These findings suggest that AEG‐1 promotes gastric cancer metastasis through upregulation of eIF4E‐mediated MMP‐9 and Twist, which provides new diagnostic markers and therapeutic targets for cancer metastasis.     

Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappa B-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells

Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and play critical roles in tissue repair, tumor invasion, and metastasis. MMPs are regulated by different cytokines, ECM proteins, and other factors. However, the molecular mechanisms by which osteopontin (OPN), an ECM protein, regulates ECM invasion and tumor growth and modulates MMP activation in B16F10 cells are not well defined. We have purified OPN from human milk and shown that OPN induces pro-MMP-2 production and activation in these cells. Moreover, our data revealed that OPN-induced membrane type 1 (MT1) MMP expression correlates with translocation of p65 (nuclear factor-kappaB (NF-kappaB)) into the nucleus. However, when the super-repressor form of IkappaBalpha (inhibitor of NF-kappaB) was transfected into cells followed by treatment with OPN, no induction of MT1-MMP expression was observed, indicating that OPN activates pro-MMP-2 via an NF-kappaB-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with alpha(v)beta(3) integrin, but these effects were reduced drastically when the MMP-2-specific antisense S-oligonucleotide was used to suppress MMP-2 expression. Interestingly, when the OPN-treated cells were injected into nude mice, the mice developed larger tumors, and the MMP-2 levels in the tumors were significantly higher than in controls. The proliferation data indicate that OPN increases the growth rate in these cells. Both tumor size and MMP-2 expression were reduced dramatically when anti-MMP-2 antibody or antisense S-oligonucleotide-transfected cells were injected into the nude mice. To our knowledge, this is the first report that MMP-2 plays a direct role in OPN-induced cell migration, invasion, and tumor growth and that demonstrates that OPN-stimulated MMP-2 activation occurs through NF-kappaB-mediated induction of MT1-MMP.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

TNF‐α increases αvβ3 integrin expression and migration in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumour with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Tumour necrosis factor (TNF)‐α is a key cytokine involved in inflammation, immunity, cellular homeostasis and tumour progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. However, the effects of TNF‐α in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that TNF‐α increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of MAPK kinase (MEK), extracellular signal‐regulating kinase (ERK) and nuclear factor‐κB (NF‐κB) pathways after TNF‐α treatment were demonstrated, and TNF‐α‐induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK and NF‐κB cascades. Taken together, our results indicated that TNF‐α enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the MEK/ERK/NF‐κB signal transduction pathway. J. Cell. Physiol. 226: 792–799, 2011. © 2010 Wiley‐Liss, Inc.     

Serum osteopontin, an enhancer of tumor metastasis to bone, promotes B16 melanoma cell migration

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti-metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, alphavbeta3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild-type mice induced cell migration of B16 melanoma cells, while serum from OPN-deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN-induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN-induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN-induced signal might be effective in suppression of tumor metastasis.     

Laminins 411 and 421 differentially promote tumor cell migration via α6β1 integrin and MCAM (CD146)

α4-laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by blood and lymphatic vessels and some tumor cells. Laminin-411 promotes migration of leukocytes and endothelial cells, but the effect of this laminin and laminin-421 on tumor cells is poorly understood. In the present study, we demonstrate that laminin-411 and, to a greater extent, laminin-421 significantly promote migration of tumor cells originated from melanomas, gliomas and different carcinomas via α6β1 integrin. In solid-phase binding assays, both laminins similarly bound α6β1 integrin but only laminin-421, among several laminin isoforms, readily bound MCAM (CD146), a cell-surface adhesion molecule strongly associated with tumor progression. Accordingly, a function-blocking mAb to MCAM inhibited tumor cell migration on laminin-421 but not on laminins 411 or 521. In tumor tissues, melanoma cells co-expressed MCAM, laminin α4, β1, β2 and γ1 chains, and integrin α6 and β1 chains. The present data highlight the novel role of α4-laminins in tumor cell migration and identify laminin-421 as a primary ligand for MCAM and a putative mediator of tumor invasion and metastasis.     

Extra domain A-containing fibronectin expression in Spin90-deficient fibroblasts mediates cancer-stroma interaction and promotes breast cancer progression

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4β1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4β1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4β1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.