Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC 2012 Mar 1.
Published in final edited form as:
PMCID: PMC3076137

Steroid Receptor Coactivator-1 Upregulates Integrin α5 Expression to Promote Breast Cancer Cell Adhesion and Migration


Metastatic breast cancer (BC) remains a lethal disease with poorly understood molecular mechanisms. Steroid receptor coactivator 1 (SRC-1 or NCOA1) is overexpressed in a subset of BCs with poor prognosis. SRC-1 potentiates gene expression by serving as a coactivator for nuclear receptors and other transcription factors. We previously reported that SRC-1 promotes BC metastasis without affecting primary mammary tumor formation. Herein, we found that SRC-1 deficiency in mouse and human BC cells substantially reduced cell adhesion and migration capabilities on fibronectin and significantly extended the time of focal adhesion disassembly and re-assembly. In agreement with this phenotype, SRC-1 expression positively correlated with integrin α5 (ITGA5) expression in estrogen receptor-negative breast tumors while SRC-1 deficiency decreased ITGA5 expression. Furthermore, ITGA5 reduction in SRC-1 deficient/insufficient BC cells or knockdown of ITGA5 in SRC-1-expressing BC cells was associated with a disturbed integrin-mediated signaling. Critical downstream changes included reduced phosphorylation and/or dampened activation of focal adhesion kinase, paxillin, Rac1 and Erk1/2 during cell adhesion. Finally, we found that SRC-1 enhanced ITGA5 promoter activity through an AP-1-binding site proximal to the transcriptional initiation site; both SRC-1 and c-Jun were recruited to this promoter region in BC cells. These results demonstrate that SRC-1 can promote BC metastasis by directly enhancing ITGA5 expression and thus promoting ITGA5-mediated cell adhesion and migration. Therefore, targeting ITGA5 in SRC-1-positive BCs may result in inhibition of SRC-1-promoted BC metastasis.

Keywords: nuclear receptor, SRC-1, coactivator, ITGA5, breast cancer


Steroid receptor coactivator 1 (SRC-1) boosts gene expression by serving as a transcriptional coactivator for nuclear hormone receptors and other transcription factors (TFs) such as estrogen receptor α (ERα), progesterone receptor (PR), PEA3, AP-1, HIF-1 and Ets-2 (16). SRC-1 expression in human breast cancer (BC) positively correlates with Her2 expression, endocrine therapy resistance, and poor prognosis (5, 7, 8). Knockout of SRC-1 in the MMTV-polyoma middle T antigen (PyMT) mammary tumor-prone mice dramatically suppresses lung metastasis without affecting primary tumor formation (9). These studies indicate that SRC-1 strongly promotes BC metastasis.

SRC-1 upregulates the expression of several key regulators for BC progression. In particular, SRC-1 deficiency in mouse mammary tumors reverses HER2 overexpression and reduces Akt activity (9). Knockout of SRC-1 in these tumors suppresses the expression of colony stimulating factor (CSF-1) (9), a chemoattractant that recruits macrophages to the tumor site. In turn, the macrophages secret EGF to stimulate tumor cell motility. SRC-1 also serves as a coactivator of PEA3 to enhance Twist1 expression in BC cells. Elevated Twist1 promotes breast tumor cell epithelial mesenchymal transition (EMT), invasion and metastasis by recruiting the NuRD protein complex to repress E-cadherin expression (2, 10). Furthermore, SRC-1 works with Ets-2 to induce c-Myc expression and with HOXC11 to induce calcium-binding protein S100beta expression, both of which are positively associated with acquired resistance to endocrine therapy (5, 7).

Recently, we discovered that the number of mammary tumor cells in the blood of SRC-1 wild type (WT);PyMT mice is significantly higher than that in the blood of SRC-1 knockout (KO);PyMT mice, suggesting a contribution of SRC-1 to BC cell migration and invasion from the primary tumor to the blood vessels (9). Local migration and invasion of tumor cells are early events partially induced by the tumor microenvironment in metastasis. Resident fibroblasts not only secret TGFβ to induce tumor cell EMT, but also produce abundant collagen and fibronectin (FN) extracellular matrix (ECM) proteins to provide anchorages for tumor cell adhesion and migration (1113). Integrins consist of 18 α and 8 β glycoprotein subunits, which form 24 distinct heterodimeric transmembrane receptors. These receptors bind to ECM proteins such as FN to transport signals bidirectionally across the cell membrane, allowing cells to respond to environmental changes (14). Multiple integrins, including αvβ3, αvβ5, α5β1, α6β4, α4β1 and αvβ6, are detected in cancer cells and their expression levels are associated with tumorigenesis and cancer progression (15). In BC, integrin β4 amplifies HER2 signaling to potentiate mammary tumorigenesis (16). Activation of integrin αvβ3 supports BC cell adhesion to the vascular wall and promotes metastasis (17), while knockout of integrin β1 inhibits mammary tumorigenesis in mice (18). In addition, integrins also regulate tumor cell survival, growth and metastasis in an anchorage-independent manner (15).

The mesenchymal integrins α5 (ITGA5) and β1 form heterodimers to mediate cell adhesion to FN (15). Knockout of ITGA5 in mice results in embryonic lethality (19). In human hepatocarcinoma cells, ITGA5 promotes cell adhesion and migration on FN through activating focal adhesion kinase (FAK) (20). In transformed mammary epithelial cells, ITGA5 expression is increased along with the EMT process (21). These findings indicate that ITGA5 expression correlates with cancer progression and plays an important role to enhance cancer cell adhesion to and migration along FN.

In this study, we found that SRC-1 works with AP-1 to potentiate ITGA5 expression. The increased ITGA5, in turn, significantly accelerates BC cell adhesion and migration on FN. The identification of ITGA5 as a target gene of SRC-1 and AP-1 in BC cells uncovered a new molecular pathway: SRC-1 regulates ITGA5 expression to promote BC metastasis.

Materials and Methods

Cell adhesion and migration assays

The primary and stable SRC-1 WT;PyMT (WT) and KO;PyMT (KO) mouse mammary tumor cell lines were generated as described previously (22). Adhesion assay was performed on FN or laminin (LN)-coated plates as described previously (23). Individual cell migration was tracked for 18 hours in 96-well plate pre-coated with fluorescent beads and track areas were analyzed using NIH image software as described previously (2, 22).

Western blot analysis of human breast tumors

A total of 24 human BC specimens were collected from surgically removed tumor tissues at Luzhou Medical College Affiliated Hospital in 2009. All patients were Asian women and aged 33–65 years old. No patient survival data were available at this stage. A portion of the specimen was used for clinical diagnosis of tumor pathology and immunohistochemistry for ERα, PR and HER2. The remaining tumor tissues were immediately frozen in liquid nitrogen and stored at −80° C. Tumor tissue lysates were prepared after homogenizing the tissues in a lysis buffer containing the protease inhibitor cocktail. The tissue lysates with 50 µg protein were analyzed by Western blot using antibodies against SRC-1, ITGA5 and β-actin. Band intensities were determined by densitometry and normalized to the β-actin band intensity.

Other Methods

Immunostaining, knockdown and expression of SRC-1, focal adhesion disassembly assay, Western blotting, quantitative RT-PCR (qPCR), cell transfection, luciferase assay and chromatin immunoprecipitation (ChIP) assay were performed as descried in the Supplementary Methods.


SRC-1 deficiency reduces mammary tumor cell adhesion and migration on FN-coated plate

To define the role of SRC-1 in BC cell adhesion to ECM, we compared the adhesion capabilities of two SRC-1 KO (KO1 and KO2) to two WT (WT1 and WT2) mouse mammary tumor cell lines. As expected, SRC-1 protein was detected in both WT cell lines but was absent in both KO cell lines (Fig. 1A). After seeded in a FN-coated plate, about 90% of WT1 and 75% of WT2 cells adhered and spread within 1 hour and these percentages increased to nearly 100% by 2.5 hours. On the contrary, only about 20% of KO1 and 36% of KO2 cells adhered and spread on FN-coated plate by 1 hour, increasing to only 60% and 70%, respectively, by 2.5 hours (Fig. 1A and data not shown). After culturing overnight, both SRC-1 WT and KO cell lines appeared to adhere and spread well on the FN-coated plate (Fig. 1A). Next, we repeated these assays using 5 WT and 5 SRC-1 KO primary cell preparations isolated from individual mammary tumors in WT;PyMT and KO;PyMT mice. About 60–80% of WT primary cells adhered to FN-coated plate in one hour, while only 40–50% of KO primary cells adhered under the same conditions (Fig. 1B). These results demonstrate that SRC-1 is required for effective adhesion of mammary tumor cells to FN.

Fig. 1
SRC-1 deficiency reduces BC cell adhesion and migration on FN-coated plates

Next, the migration of individual SRC-1 WT and KO cells was tracked on FN-coated plates for 18 hours. The average track areas swept by the two WT cell lines were 60,000 and 90,000 pixels, respectively, while those of the SRC-1 KO cell lines were only 25,000 and 20,000 pixels (Fig. 1C). Knockdown of SRC-1 using siRNAs in both mouse WT cell lines effectively reduced their migration areas by 50%. (Fig. 1C). Similarly, knockdown of SRC-1 mRNA in MDA-MB-231 human BC cells using three different shRNAs also significantly reduced cell migration on FN-coated plate by 50% (Fig. 1D). These results indicate that SRC-1 expression facilitates the migration of mammary tumor cells along FN and SRC-1 deficiency attenuates the tumor cell migration.

SRC-1 deficiency attenuates disassembly and reassembly of focal adhesion complexes (FACs)

Focal adhesions connect the cell cytoskeleton and the ECM through integrins. In motile cells, the rate of focal adhesion assembly at the leading edge and disassembly at the trailing edge determines the speed of cell movement (24). Immunostaining of vinculin, an adaptor protein located in FACs, showed that focal adhesions distributed in a polarized pattern in most WT cells, suggesting an active migration status of these cells. However, the FAC distribution in most (~70%) of SRC-1 KO cells was non-polarized and, instead, distributed evenly over the cell membrane. This suggested the majority of SRC-1 KO cells remained in an inactive migration status (Fig. 2A). Accordingly, F-actin filaments in SRC-1 KO cells were much more abundant than that in WT cells (Fig. 2A).

Fig. 2
The disassembly and reassembly of FACs and ITGA5 expression in SRC-1 KO and WT mammary tumor cells

To define the role of SRC-1 in focal adhesion turnover, we examined FAC disassembly and reassembly in WT and SRC-1 KO cells by immunostaining vinculin. Cells were treated with nocodazole to stimulate FAC formation (Fig. 2B). After removing nocodazole, most WT cells showed FAC disassembly in 1 hour and newly formed FACs in 2 hours. However, most SRC-1 KO cells took as long as 2 hours for FAC disassembly and 4 hours for FAC reassembly (Fig. 2B). Furthermore, FACs in WT cells transfected with non-targeting siRNAs disassembled in 1 hour and reassembled in 2 hours, while FACs in the same cells transfected with SRC-1 siRNAs disassembled in about 2 hours and reassembled in about 4 hours (Fig. 2C). These results suggest that SRC-1 is required for faster FAC turnover in the mammary tumor cells.

Integrins are essential components of FACs. They connect ECM with their extracellular domains to cytoskeleton through interaction of their intracellular domains with multiple proteins such as talin, paxillin and FAK (14). Integrins also transfer signals across the cell membrane from both sides (14). Since SRC-1 deficiency reduced cell adhesion to FN, we measured the expression levels of integrins β1, β3, αv and α5, which form αvβ3 and α5β1 heterodimers necessary for binding FN. We found no consensus changes in mRNA levels of integrins β1, αv and β3 in WT and SRC-1 KO cells (Fig. 2D). However, we found that both mRNA and protein levels of integrin α5 in the two SRC-1 KO cell lines were significantly lower than that in the two WT cell lines (Fig. 2D). These results suggest that the decreased adhesion capability of SRC-1 KO cells may be related to the lower ITGA5 in these cells.

SRC-1 expression positively correlates with ITGA5 expression

To explore the possibility that SRC-1 may regulate ITGA5 expression, we ectopically expressed SRC-1 in SRC-1 KO cell lines. We found that SRC-1 restoration in these cells promoted ITGA5 expression (Fig. 3A). Conversely, knockdown of SRC-1 in WT cell lines dramatically reduced ITGA5 mRNA and protein levels (Fig. 3A and data not shown). Knockdown of SRC-1 in MDA-MB-231 human BC cells also reduced ITGA5 expression (Fig. 3B). These results indicate that SRC-1 expression levels positively correlate with ITGA5 expression levels in mammary tumor cell lines.

Fig. 3
SRC-1 expression positively correlates with ITGA5 expression

Next, we examined ITGA5 expression levels in SRC-1 WT;PyMT and KO;PyMT mouse mammary tumors. In all four samples of each tumor type, ITGA5 immunoreactivity was detected on the membrane and in the cytoplasm of tumor cells. However, both the immunostaining signals of ITGA5 and the number of ITGA5-positive tumor cells in WT;PyMT tumors were much higher than those in KO;PyMT tumors isolated from 8- and 14-week-old mice (Fig. 3C and data not shown). The average ITGA5 immunoreactivity detected in WT;PyMT tumors (n = 12) was about two folds higher than that detected in KO;PyMT tumors (n = 13). About 40–60% of WT;PyMT tumor cells were ITGA5 positive, while only 20–30% of KO;PyMT tumor cells were ITGA5 positive. These results suggest that ITGA5 expression is also positively associated with SRC-1 expression in mouse mammary tumors.

Finally, we examined the association between SRC-1 and ITGA5 expression levels in human breast tumors by Western blot analysis. The levels of both SRC-1 and ITGA5 proteins were low in ERα- and PR-positive tumors, as well as in ERα-positive and PR-negative tumors. However, the average levels of both SRC-1 and ITGA5 proteins were significantly elevated in ERα- and PR-negative tumors (Fig. 3, D1 and Supplementary Fig. S1). In agreement with the protein levels, the expression levels of SRC-1 and ITGA5 mRNAs were also positively correlated in 264 human ductal breast carcinomas according to the data provided to Oncomine Database by M. Bittner (Fig. 3, D2 and Supplementary Fig. S2).

SRC-1 potentiates ITGA5-mediated signaling and tumor cell migration

Integrins bind to ECM and integrin clustering activates downstream signaling cascades involving phosphorylation or activation of FAK, Src, Paxillin, Rac1 and Erk1/2 (14). To examine the role of SRC-1 in the integrin-signaling pathway, we incubated SRC-1 WT and KO cells on FN-coated plates for 0.5 or 1 hour and assessed adhesion-induced FAK activation by measuring pY397-FAK, an autophosphorylation site for Src association (14). Western blot analysis revealed that total FAK levels were similar in SRC-1 WT and KO cell lines, while the pY397-FAK levels in the two SRC-1 KO cell lines were significantly lower than that in the two WT cell lines at both time points examined. Knockdown of SRC-1 or ITGA5 in WT cells also reduced the pY397-FAK levels at both or one of the time points examined (Fig. 4A). Similarly, knockdown of SRC-1 in MDA-MB-231 human BC cells consistently decreased the pY397-FAK levels (Fig. 4B).

Fig. 4
Effects of ITGA5 reduction in SRC-1 deficient cells on integrin-signaling pathways and cell migration

It is known that a decreased pY397-FAK level should be associated with a reduced assembly of FAK and Src complex, and the formation of this tyrosine kinase complex is responsible for phosphorylating Y118-paxillin and several other phosphorylation sites of FAK including pY925 for binding GRB2 and activating Erk1/2 (14). In agreement with a lower assembly/activity of the FAK/Src complex, we found that pY118-Paxillin, active Rac1, pY925-FAK and p-Erk1/2 levels in SRC-1 KO cells were significantly reduced when compared with WT cells at both 30- and 60-minute adhesion time points (Fig. 4, A and C). Accordingly, knockdown of SRC-1 or ITGA5 in SRC-1 WT tumor cells or knockdown of SRC-1 in MDA-MB-231 human BC cells also reduced the levels of p-Paxillin (Fig. 4, A and B). Furthermore, p-Erk1/2 levels in SRC-1 KO cell lines were much lower than that in WT cells, which was consistent with the reduced levels of pY925-FAK for GRB2 recruitment (Fig. 4C). Taken together, these results suggest that SRC-1 deficiency and ITGA5 insufficiency disturbed integrin-mediated signaling, leading to reduced activation of FAK, Src, Rac1 and Erk1/2, the important players of cell adhesion and migration.

To assess the requirement of ITGA5 in cell migration, ITGA5 mRNA was knocked down in the two SRC-1 WT tumor cell lines and these cells were subjected to migration assays on a fluorescent bead-coated plate. The WT cells transfected with ITGA5 siRNAs reduced 60–70% of cell motility on the FN-coated plate when compared with WT cells transfected with non-targeting siRNA (Fig. 4D). These results suggest that the elevated ITGA5 resulted from SRC-1 overexpression in the mouse and human mammary tumors plays an important role in promoting BC cell migration.

SRC-1 promotes AP-1-mediated ITGA5 expression

To examine whether SRC-1 can enhance transcriptional activity of the ITGA5 gene, we constructed three luciferase reporter constructs containing different fragments (F1, F2 and F3) of the ITGA5 promoter (Fig. 5A). Expression of SRC-1 in HeLa cells significantly activated all three reporters in a SRC-1 dose-dependent manner, suggesting that SRC-1 may coactivate certain TFs associated with all three fragments of the ITGA5 promoter (Fig. 5B). Sequence analysis identified potential binding sites for several TFs known to use SRC-1 as a coactivator, including PEA3, NF-κB, C/EBPα/β?and AP-1 (2, 3, 2527) (Fig. 5A). Expression of PEA3 or NF-κB, either alone or combined with SRC-1, had no significant effects on the activities of all three promoter-reporters. Expression of C/EBPα alone also showed no obvious activation of these promoter-reporters, while coexpression of SRC-1 slightly increased the activities of F1-Luc and F3-Luc promoter-reporters. Expression of C/EBPβ alone activated F2-Luc and F3-Luc promoter-reporters, but coexpression of SRC-1 only slightly increased the activities of these promoter-reporters in cells transfected with high concentrations of SRC-1 plasmids (Fig. 5C). These results suggest that PEA3, NF-κB and C/EBPα/β are not the major TFs that work with SRC-1 to enhance ITGA5 promoter activity.

Fig. 5
SRC-1 regulates ITGA5 promoter activity

Interestingly, although expression of c-Jun alone did not increase the activity of the three ITGA5 promoter-reporters, coexpression of c-Jun and SRC-1 robustly activated these constructs in a SRC-1 dose-dependent manner, suggesting that the transcriptional activation function of c-Jun and SRC-1 is associated with AP-1 binding site(s) located in the F3 common region of the ITGA5 promoter (Fig. 5, A and D). Furthermore, deletion of the single AP-1 binding site at bp -9 position significantly reduced the basal activity of all three ITGA5 promoter-reporters in cells transfected with or without c-Jun plasmids and completely abolished SRC-1-enhanced activities of these ITGA5 promoter regions (Fig. 5D).

Since SRC-1 interacted with c-Jun (3) to activate the ITGA5 promoter (Fig. 5D), we performed ChIP assays to examine whether SRC-1 and c-Jun are recruited to the ITGA5 promoter. ChIP assays demonstrated that the endogenous SRC-1 protein is associated with ITGA5 promoter regions d (bp −420 to −220) and e (bp −115 to +73) in both MDA-MB-231 and MDA-MB-435 cancer cells (Fig. 6, A and B). Region e contains the functional AP-1 site (Fig. 5A). In contrast, SRC-1 was not associated with ITGA5 promoter regions a, b and c (Fig. 6, A and B). In agreement with the presence of the functional AP-1 binding site in region e of the ITGA5 promoter, c-Jun was found strongly associated with region e in both MDA-MB-231 and MDA-MB-435 cells, but only weakly associated with region d in MDA-MB-435 cells (Fig. 6, A and C). These results suggest that both c-Jun and SRC-1 are recruited to the e region of the ITGA5 promoter. The association of SRC-1 with region d might be due to either SRC-1 interaction with another TF or a cross PCR reaction from an extended template of region e, which is associated with both SRC-1 and c-Jun.

Fig. 6
SRC-1 and AP-1 are associated with a proximal region of the ITGA5 promoter


The three transcriptional coactivators in the SRC family are gene expression amplifiers regulated by multiple signaling pathways (28). These coactivators are expressed in normal cells at limiting concentrations, so that their changes in concentration and/or activity can effectively modulate gene expression (28). These coactivators are commonly overexpressed in cancers, acting to promote carcinogenesis and/or metastasis. Specifically, SRC-3 (AIB1) is amplified and overexpressed in a subset of BC and its overexpression is associated with HER2 expression and resistance to endocrine therapy (29, 30). In mouse BC models, knockout of SRC-3 suppresses oncogene and carcinogen-induced carcinogenesis and metastasis, while SRC-3 overexpression is sufficient to induce high frequency of mammary tumors (22, 3133). Recent studies also showed that SRC-3 is required for EGFR and HER2 phosphorylation and activation in BC cells (33). SRC-3Delta4, a splicing isoform of SRC-3, also can serve as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylation of FAK and c-Src to enhance cell migration (34). SRC-2 (NCOA2) is identified as an overexpressed oncogene in prostate cancer (35). SRC-1 is overproduced in a subset of BC and its expression is positively associated with HER2 expression, tamoxifen resistance and poor prognosis (6, 8, 28). In mouse models, knockout of SRC-1 does not affect primary mammary tumor formation but effectively suppresses metastasis (9). These findings highlight the crucial roles of the SRC family coactivators in cancer initiation, progression and metastasis.

Because SRCs work as transcriptional co-regulators for many nuclear receptors and other TFs, it has been difficult to identify key SRC-associated TFs and their target genes responsible for promoting tumorigenesis and metastasis. Although recent studies have made progress in understanding the mechanisms responsible for SRC-1 to promote BC metastasis (2, 5, 9), it is just the beginning to identify key genes and gene networks regulated by SRC-1.

In this study, we have found that SRC-1 deficiency slows BC cell adhesion and migration on FN, which correlates well with the attenuated disassembly and reassembly of FACs in SRC-1 KO and knockdown cells. Furthermore, we identified ITGA5 as a new SRC-1 target gene based on multiple lines of evidence. First, SRC-1 expression levels are associated with ITGA5 expression levels in both mouse and human BC cell lines and primary tumors. Knockout or knockdown of SRC-1 reduces ITGA5 expression, while SRC-1 expression enhances ITGA5 expression. Second, SRC-1 is a known coactivator of AP-1 (3), and we found that both AP-1 and SRC-1 associate with the ITGA5 promoter to enhance ITGA5 promoter activity. Although experiment to knock down c-Jun was not performed to further validate the functional contribution of c-Jun to ITGA5 expression in this study, previous studies have shown that AP-1 strongly enhances ITGA5 expression (36, 37). Finally, the reduced ITGA5 expression caused by SRC-1 deficiency or insufficiency in BC cells partially impairs the function of the FN-integrin-FAK-cell migration pathway. In this pathway, the heterodimers of α5β1 integrins bind FN to induce phosphorylation of pY397-FAk and formation of the active FAK/Src complex, followed by further phosphorylation of FAK and phosphorylation/activation of downstream signaling components including Paxillin, RAC1, and Erk1/2 (14, 3840). In agreement with an important role of SRC-1-mediated ITGA5 expression in the FN and integrin interaction-initiated signaling and cell migration, the FAK phosphorylation at Y397, paxillin phosphorylation and activation of Rac-1 and Erk1/2 during cell adhesion process are significantly reduced in SRC-1 knockout and SRC-1 or ITGA5 knockdown BC cells. Taken together, these findings demonstrate that SRC-1-mediated ITGA5 expression is partially responsible for SRC-1-promoted adhesion, migration and metastasis of BC cells.

In the literature, the role of integrins α5β1 in cancer is somewhat controversial. One early study showed that elevated levels of integrin α5β1 suppressed the transformed phenotype of CHO cells (41). Some studies also reported an inhibitory effect of integrins α5β1 on tumorigenesis and metastasis (12, 42). However, multiple lines of evidence suggest that integrin α5β1 expression positively correlates with cancer progression and metastasis. First, integrin α5β1 expression is upregulated in malignant BC cells and its upregulation correlates with poor prognosis (43). Second, SDF1-activated CXCR4 upregulates integrin α5β1 expression and enhances prostate tumor cell adhesion, invasion and metastasis (44). Third, a mutant p53 has been shown to drive cell invasion and metastatic behavior via activating EGFR/integrins α5β1 signaling (45). Fourth, FN and ITGA5 are required for switching cell-cell adhesion to cell-ECM adhesion, and this switch is required for tumor cells to invade into the stromal tissue (46). Fifth, E-cadherin expression in ovarian and breast cancers negatively correlates with ITGA5 expression, suggesting an important role of ITGA5 in EMT (47, 48). Finally, blocking the function of integrins α5β1 by an antibody (Volociximab) or a non-RGD-based peptide inhibitor (ATN-161) significantly inhibits BC growth and metastasis (49, 50). In this study, we have demonstrated that SRC-1 upregulates ITGA5 expression to promote BC cell adhesion and migration on FN, facilitating local invasion of these tumor cells. Based on the important contributions of ITGA5 in cancer cell migration, invasion and metastasis described above, it is reasonable to conclude that ITGA5 functions as one of the key target genes of SRC-1 to mediate SRC-1-promoted BC cell migration and metastasis.

Since SRC-1 serves as a coactivator for multiple TFs such as AP-1, PEA3, Ets-2 and HOXC11 to upregulate the expression of multiple target genes such as ITGA5, Twist1, CSF-1, c-Myc and S100beta in promotion of BC metastasis (2, 5, 7, 9), targeting SRC-1 could be a potential strategy to interfere multiple pathways involved in cancer metastasis.

Supplementary Material


We thank Hongwu Chen for providing SRC-1 expression adenovirus, Brian York, Junjiang Fu and Jun Hong for experimental assistance, and Jean Tien for critical reading of the manuscript. This work is supported by NIH grants (CA112403, CA119689 and DK58242) and an American Cancer Society Scholar Award (RSG-05-082-01-TBE).


1. Xu J, Qiu Y, DeMayo FJ, Tsai SY, Tsai MJ, O'Malley BW. Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science. 1998;279:1922–1925. [PubMed]
2. Qin L, Liu Z, Chen H, Xu J. The steroid receptor coactivator-1 regulates twist expression and promotes breast cancer metastasis. Cancer Res. 2009;69:3819–3827. [PMC free article] [PubMed]
3. Lee SK, Kim HJ, Na SY, Kim TS, Choi HS, Im SY, Lee JW. Steroid receptor coactivator-1 coactivates activating protein-1-mediated transactivations through interaction with the c-Jun and c-Fos subunits. J Biol Chem. 1998;273:16651–16654. [PubMed]
4. Carrero P, Okamoto K, Coumailleau P, O'Brien S, Tanaka H, Poellinger L. Redox-regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-1 to hypoxia-inducible factor 1alpha. Mol Cell Biol. 2000;20:402–415. [PMC free article] [PubMed]
5. Al-azawi D, Ilroy MM, Kelly G, Redmond AM, Bane FT, Cocchiglia S, Hill AD, Young LS. Ets-2 and p160 proteins collaborate to regulate c-Myc in endocrine resistant breast cancer. Oncogene. 2008;27:3021–3031. [PubMed]
6. Myers E, Hill AD, Kelly G, McDermott EW, O'Higgins NJ, Buggy Y, Young LS. Associations and interactions between Ets-1 and Ets-2 and coregulatory proteins, SRC-1, AIB1, and NCoR in breast cancer. Clin Cancer Res. 2005;11:2111–2122. [PubMed]
7. McIlroy M, McCartan D, Early SPOG, Pennington S, Hill AD, Young LS. Interaction of developmental transcription factor HOXC11 with steroid receptor coactivator SRC-1 mediates resistance to endocrine therapy in breast cancer [corrected] Cancer Res. 70:1585–1594. [PubMed]
8. Redmond AM, Bane FT, Stafford AT, McIlroy M, Dillon MF, Crotty TB, Hill AD, Young LS. Coassociation of estrogen receptor and p160 proteins predicts resistance to endocrine treatment; SRC-1 is an independent predictor of breast cancer recurrence. Clin Cancer Res. 2009;15:2098–2106. [PubMed]
9. Wang S, Yuan Y, Liao L, Kuang SQ, Tien JC, O'Malley BW, Xu J. Disruption of the SRC-1 gene in mice suppresses breast cancer metastasis without affecting primary tumor formation. Proc Natl Acad Sci U S A. 2009;106:151–156. [PMC free article] [PubMed]
10. Fu J, Qin L, He T, Qin J, Hong J, Wong J, Liao L, Xu J. The TWIST/Mi2/NuRD protein complex and its essential role in cancer metastasis. Cell Res [PMC free article] [PubMed]
11. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–454. [PubMed]
12. Larsen M, Artym VV, Green JA, Yamada KM. The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol. 2006;18:463–471. [PubMed]
13. Bierie B, Moses HL. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer. 2006;6:506–520. [PubMed]
14. Luo BH, Carman CV, Springer TA. Structural basis of integrin regulation and signaling. Annu Rev Immunol. 2007;25:619–647. [PMC free article] [PubMed]
15. Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 10:9–22. [PMC free article] [PubMed]
16. Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, Giancotti FG. Beta 4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell. 2006;126:489–502. [PubMed]
17. Felding-Habermann B, O'Toole TE, Smith JW, Fransvea E, Ruggeri ZM, Ginsberg MH, Hughes PE, Pampori N, Shattil SJ, Saven A, Mueller BM. Integrin activation controls metastasis in human breast cancer. Proc Natl Acad Sci U S A. 2001;98:1853–1858. [PMC free article] [PubMed]
18. White DE, Kurpios NA, Zuo D, Hassell JA, Blaess S, Mueller U, Muller WJ. Targeted disruption of beta1-integrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell. 2004;6:159–170. [PubMed]
19. Watt FM, Hodivala KJ. Cell adhesion. Fibronectin and integrin knockouts come unstuck. Curr Biol. 1994;4:270–272. [PubMed]
20. Wang QY, Zhang Y, Shen ZH, Chen HL. alpha1,3 fucosyltransferase-VII up-regulates the mRNA of alpha5 integrin and its biological function. J Cell Biochem. 2008;104:2078–2090. [PubMed]
21. Maschler S, Wirl G, Spring H, Bredow DV, Sordat I, Beug H, Reichmann E. Tumor cell invasiveness correlates with changes in integrin expression and localization. Oncogene. 2005;24:2032–2041. [PubMed]
22. Qin L, Liao L, Redmond A, Young L, Yuan Y, Chen H, O'Malley BW, Xu J. The AIB1 oncogene promotes breast cancer metastasis by activation of PEA3-mediated matrix metalloproteinase 2 (MMP2) and MMP9 expression. Mol Cell Biol. 2008;28:5937–5950. [PMC free article] [PubMed]
23. Qin L, Zhang M. Maspin regulates endothelial cell adhesion and migration through an integrin signaling pathway. J Biol Chem. 2010;285:32360–32369. [PMC free article] [PubMed]
24. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back. Science. 2003;302:1704–1709. [PubMed]
25. Na SY, Lee SK, Han SJ, Choi HS, Im SY, Lee JW. Steroid receptor coactivator-1 interacts with the p50 subunit and coactivates nuclear factor kappaB-mediated transactivations. J Biol Chem. 1998;273:10831–10834. [PubMed]
26. Yin L, Wang Y, Dridi S, Vinson C, Hillgartner FB. Role of CCAAT/enhancer-binding protein, histone acetylation, and coactivator recruitment in the regulation of malic enzyme transcription by thyroid hormone. Mol Cell Endocrinol. 2005;245:43–52. [PubMed]
27. Dong J, Tsai-Morris CH, Dufau ML. A novel estradiol/estrogen receptor alpha-dependent transcriptional mechanism controls expression of the human prolactin receptor. J Biol Chem. 2006;281:18825–18836. [PubMed]
28. Xu J, Wu RC, O'Malley BW. Normal and cancer-related functions of the p160 steroid receptor co-activator (SRC) family. Nat Rev Cancer. 2009;9:615–630. [PMC free article] [PubMed]
29. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS. AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science. 1997;277:965–968. [PubMed]
30. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SA, Wong J, Allred DC, Clark GM, Schiff R. Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer. J Natl Cancer Inst. 2003;95:353–361. [PubMed]
31. Kuang SQ, Liao L, Zhang H, Lee AV, O'Malley BW, Xu J. AIB1/SRC-3 deficiency affects insulin-like growth factor I signaling pathway and suppresses v-Ha-ras-induced breast cancer initiation and progression in mice. Cancer Res. 2004;64:1875–1885. [PubMed]
32. Kuang SQ, Liao L, Wang S, Medina D, O'Malley BW, Xu J. Mice lacking the amplified in breast cancer 1/steroid receptor coactivator-3 are resistant to chemical carcinogen-induced mammary tumorigenesis. Cancer Res. 2005;65:7993–8002. [PubMed]
33. Fereshteh MP, Tilli MT, Kim SE, Xu J, O'Malley BW, Wellstein A, Furth PA, Riegel AT. The nuclear receptor coactivator amplified in breast cancer-1 is required for Neu (ErbB2/HER2) activation, signaling, and mammary tumorigenesis in mice. Cancer Res. 2008;68:3697–3706. [PMC free article] [PubMed]
34. Long W, Yi P, Amazit L, LaMarca HL, Ashcroft F, Kumar R, Mancini MA, Tsai SY, Tsai MJ, O'Malley BW. SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration. Mol Cell. 37:321–332. [PMC free article] [PubMed]
35. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, Arora VK, Kaushik P, Cerami E, Reva B, Antipin Y, Mitsiades N, Landers T, Dolgalev I, Major JE, Wilson M, Socci ND, Lash AE, Heguy A, Eastham JA, Scher HI, Reuter VE, Scardino PT, Sander C, Sawyers CT, Gerald WL. Integrative genomic profiling of human prostate cancer. Cancer Cell. 18:11–22. [PMC free article] [PubMed]
36. Corbi AL, Jensen UB, Watt FM. The alpha2 and alpha5 integrin genes: identification of transcription factors that regulate promoter activity in epidermal keratinocytes. FEBS Lett. 2000;474:201–207. [PubMed]
37. Han S, Roman J. COX-2 inhibitors suppress integrin alpha5 expression in human lung carcinoma cells through activation of Erk: involvement of Sp1 and AP-1 sites. Int J Cancer. 2005;116:536–546. [PubMed]
38. Cheresh DA, Leng J, Klemke RL. Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells. J Cell Biol. 1999;146:1107–1116. [PMC free article] [PubMed]
39. Cho SY, Klemke RL. Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix. J Cell Biol. 2000;149:223–236. [PMC free article] [PubMed]
40. Mitra SK, Schlaepfer DD. Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr Opin Cell Biol. 2006;18:516–523. [PubMed]
41. Giancotti FG, Ruoslahti E. Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells. Cell. 1990;60:849–859. [PubMed]
42. Fang Z, Yao W, Xiong Y, Zhang J, Liu L, Li J, Zhang C, Wan J. Functional elucidation and methylation-mediated downregulation of ITGA5 gene in breast cancer cell line MDA-MB-468. J Cell Biochem. 110:1130–1141. [PubMed]
43. Nam JM, Onodera Y, Bissell MJ, Park CC. Breast cancer cells in three-dimensional culture display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and fibronectin. Cancer Res. 70:5238–5248. [PMC free article] [PubMed]
44. Engl T, Relja B, Marian D, Blumenberg C, Muller I, Beecken WD, Jones J, Ringel EM, Bereiter-Hahn J, Jonas D, Blaheta RA. CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins. Neoplasia. 2006;8:290–301. [PMC free article] [PubMed]
45. Muller PA, Caswell PT, Doyle B, Iwanicki MP, Tan EH, Karim S, Lukashchuk N, Gillespie DA, Ludwig RL, Gosselin P, Cromer A, Brugge JS, Sansom OJ, Norman JC, Vousden KH. Mutant p53 drives invasion by promoting integrin recycling. Cell. 2009;139:1327–1341. [PubMed]
46. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol. 2004;5:816–826. [PubMed]
47. Sawada K, Mitra AK, Radjabi AR, Bhaskar V, Kistner EO, Tretiakova M, Jagadeeswaran S, Montag A, Becker A, Kenny HA, Peter ME, Ramakrishnan V, Yamada SD, Lengyel E. Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target. Cancer Res. 2008;68:2329–2339. [PMC free article] [PubMed]
48. Wu H, Liang YL, Li Z, Jin J, Zhang W, Duan L, Zha X. Positive expression of E-cadherin suppresses cell adhesion to fibronectin via reduction of alpha5beta1 integrin in human breast carcinoma cells. J Cancer Res Clin Oncol. 2006;132:795–803. [PubMed]
49. Khalili P, Arakelian A, Chen G, Plunkett ML, Beck I, Parry GC, Donate F, Shaw DE, Mazar AP, Rabbani SA. A non-RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo. Mol Cancer Ther. 2006;5:2271–2280. [PubMed]
50. Ricart AD, Tolcher AW, Liu G, Holen K, Schwartz G, Albertini M, Weiss G, Yazji S, Ng C, Wilding G. Volociximab, a chimeric monoclonal antibody that specifically binds alpha5beta1 integrin: a phase I, pharmacokinetic, and biological correlative study. Clin Cancer Res. 2008;14:7924–7929. [PMC free article] [PubMed]
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Gene
    Gene records that cite the current articles. Citations in Gene are added manually by NCBI or imported from outside public resources.
  • GEO Profiles
    GEO Profiles
    Gene Expression Omnibus (GEO) Profiles of molecular abundance data. The current articles are references on the Gene record associated with the GEO profile.
  • HomoloGene
    HomoloGene clusters of homologous genes and sequences that cite the current articles. These are references on the Gene and sequence records in the HomoloGene entry.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...