Dual FGF-2 and Intergrin α5β1 Signaling Mediate GRAF-Induced RhoA Inactivation in a Model of Breast Cancer Dormancy
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Interactions with the bone marrow stroma regulate dormancy and survival of breast cancer micrometastases. In an in vitro model of dormancy in the bone marrow, we previously demonstrated that estrogen-dependent breast cancer cells are partially re-differentiated by FGF-2, re-express integrin α5β1 lost with malignant transformation and acquire an activated PI3K/Akt pathway. Ligation of integrin α5β1 by fibronectin and activation of the PI3K pathway both contribute to survival of these dormant cells. Here, we investigated mechanisms responsible for the dormant phenotype. Experiments demonstrate that integrin α5β1 controls de novo cytoskeletal rearrangements, cell spreading, focal adhesion kinase rearrangement to the cell perimeter and recruitment of a RhoA GAP known as GRAF. This results in the inactivation of RhoA, an effect which is necessary for the stabilization of cortical actin. Experiments also demonstrate that activation of the PI3K pathway by FGF-2 is independent of integrin α5β1 and is also required for cortical actin reorganization, GRAF membrane relocalization and RhoA inactivation. These data suggest that GRAF-mediated RhoA inactivation and consequent phenotypic changes of dormancy depend on dual signaling by FGF-2-initiated PI3K activation and through ligation of integrin α5β1 by fibronectin.
KeywordsBreast cancer Dormancy Fibronectin GRAF Integrin α5β1 Micrometastases PI3 kinase Rho GTPases
Breast cancer cells form micrometastases to the bone marrow in about a third of patients with localized disease . These cells become dormant in the bone marrow microenvironment and survive chemotherapy administered with the specific intent of eliminating them . Very little is known about mechanisms that keep these cells in a dormant state. Direct data from individual breast cancer cells, outnumbered more than 108-fold by hematopoietic progenitors, is not available. One of the limited options in obtaining molecular data defining the behavior of these cells is by development of models, initially with a limited number of key components that define the in vivo system. Such models can be expanded subsequently to include additional key components in order to determine their effects on the model and validate the data obtained.
Towards understanding basic elements of dormancy, we developed an in vitro model incorporating three key elements affecting breast cancer cell dormancy in the bone marrow microenvironment . The components of our system consist of estrogen-dependent human breast cancer cell lines MCF-7 and T-47D, fibronectin and basic fibroblast growth factor (FGF-2) 10 ng/ml. Estrogen-dependent breast cancer cell lines model estrogen-dependent human tumors, which are likely to remain dormant for extended periods and are least likely to have distant metastatic recurrences [4, 5, 6]. In the clinical setting, recurrent estrogen receptor positive cells continue to be estrogen sensitive and susceptible to hormonal blockade [7, 8].
The second element of our system is fibronectin, a structural protein of the bone marrow microenvironment in physical contact with the dormant cells. Fibronectin is found throughout the bone marrow and particularly in the endosteum where homing hematopoietic stem cells have a high affinity . Fibronectin is produced in high amounts with a characteristic cellular matrix formation in an extensive network  by two types of bone marrow stromal cells, the subendosteal reticulocytes and osteoblasts . Both have functional roles in hematopoiesis, with the latter inducing low proliferation and high maintenance of early haemopoietic progenitors, while reticulocytes promote proliferation and differentiation in an in vitro co-culture model . Evidence suggests that metastatic breast cancer cells usurp the hematopoietic niche and respond to signals from the stromal elements . Fibronectin is upregulated in this pre-metastatic niche primed to receive metastatic cancer cells . In an in vitro co-culture system, tumor cells binding to bone marrow stromal cells exclusively depended on the fibronectin receptor integrin α5β1 .
The third element of our model is basic fibroblast growth factor (FGF-2). FGF-2 is a morphogenic differentiation factor in mammary epithelial cells . It inhibits the proliferation of estrogen-dependent breast cancer cells  and promotes their partial re-differentiation . This includes a diminished malignant potential in vitro, including decreased motility and invasion [15, 16] and anchorage independent growth  and decreased tumor growth in murine xenografts . Breast cancer cells transfected with FGF-2 also form branching duct-like stuctures in Matrigel . FGF-2 is synthesized and exported by the stromal cells [18, 19] and deposited in high concentrations in the bone marrow microenvironment [18, 20, 21, 22].
In our model, breast cancer cells are incubated on fibronectin-coated tissue culture plates in the presence of FGF-2 10 ng/ml at clonogenic density, where their primary interaction is with the substratum and not with each other . In the model, cells form dormant clones of 2–12 cells over a 6-day period in contrast to cells incubated without FGF-2, which form proliferating clones of greater than 30 cells. The dormant cells become growth arrested, re-express integrins α2, α5, β1 β3 and β4, lost with transformation [23, 24, 25], and adopt a characteristic morphogenic trait of large size and a highly spread out conformation with large cytoplasm to nucleus ratios . They undergo sustained activation of the phosphoinositol 3-kinase (PI3K) pathway  and extracellular receptor kinase (ERK) pathway , which, along with ligation of integrin α5β1, contribute to their survival .
We wanted to determine the steady-state molecular events that sustained dormancy in these cells. Specifically, we wanted to discern whether the signaling mediating these effects was initiated by FGF-2 directly or through integrin α5β1, which is induced by FGF-2 incubation as the cells reach a dormant steady-state. The phenotypic appearance of the estrogen-dependent cells in the dormancy model was reminiscent of that of FGF-2-transfected MDA-MB-231 cells. MDA-MB-231 cancer cells enforced to express FGF-2 acquired a spread appearance, cortical redistribution of fibrillar actin (F-actin), omnidirectional focal complex activation , and decreased motility, invasiveness and in vivo tumorigenicity . We investigated the characteristics of the dormant cells in the context of our prior observations to determine if the partial re-differentiation of the dormant cells was due to potential inhibitory effects on the activation state of small GTPases, specifically RhoA, implicated in actin polymerization and cancer progression . Our observations suggest that inhibition of RhoA, which trends to higher expression with tumor grade and nodal metastasis in breast cancer , may play a functional role in the partial re-differentiation of breast cancer dormancy in the bone marrow microenvironment.
Materials and Methods
Cells and Cell Culture
MCF-7 cell were obtained from ATCC (American Type Culture Collection) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum (DMEM/10% FCS) (standard medium) as before . MCF-10A cells (ATCC) were cultured in MCF-10 medium in standard tissue culture plates, as before . Cells were incubated at clonogenic densities of 50,000–75,000 cells per 10 cm fibronectin pre-coated plates purchased from BIOCOAT, BD Biosciences, or 15,000 cells per well in 6 well plates (day -1) and supplemented with 10 ng/ml basic FGF (FGF-2) (Invitrogen) the following day (day 0). DMEM/10% FCS with FGF-2 10 ng/ml was replenished on day 3, along with addition of inhibitors and antibodies in specific experiments (LY 294002 25 μM (Calbiochem), integrin α5β1 or α2β1 blocking antibodies 2 μg/ml (Chemicon), integrin blocking peptides to fibronectin (GRGDSP (P1)), to collagen (CQDSETRTFY (P3), and a non-binding control (GRGESP (P2)) (American Peptide Co., Sunnyvale, CA) 100 nM in 10% DMEM), as previously described . For all experiments except transfections, cells were harvested and analyzed on day 6.
Indirect Immunofluorescence and Phallacidin Staining
Cells were cultured on FN coated 22 mm slides (BD Biosciences) which were situated in 6 well tissue culture plates, and inhibitors and blocking agents were added on day 3, as above. Slides were removed on day 6 and cells were fixed in 3.7% formaldehyde in PBS for 10 min. For phalloidin staining, slides were blocked in 1% bovine serum albumin (BSA) for 30 min and incubated in BODIPY-Phallacidin (green) or rhodamine phalloidin (red) (Molecular Probes) at room temperature for 20 min. For indirect immunofluorecence staining, cells were blocked in 5% BSA for one h, incubated with primary antibodies diluted in PBS 0.1% TRITON X-100, with overnight incubation at 4°C (Santa Cruz Biotechnology). Coverslips were washed and incubated with ALEXA FLOUR conjugated antibodies (Molecular Probes) and mounted on glass slides using Prolong Gold with Dapi (Molecular Probes). Cells were viewed and photographed using a ZEISS Axiovert 200 M microscope fitted with an ApoTome Imaging system. Phalloidin staining was quantitated on 400 x magnification. RhoA, GRAF and p190 Rho GAP indirect immunofluorescence slides were photographed at 630 x. Phospho-FAK Y397 quantitation was performed on an Olympus BX20 fluorescence microscope and an Olympus MagnaFire digital photographic system at 1,000x magnification.
Immunoprecipitation, Rho GTP Assays and Western Blots
For Immunoprecipitation, cells were incubated at clonogenic density on 10 cm fibronectin-coated plates, and cultured as above, harvested and lysed on day 6 in 1X modified RIPA buffer (Millipore) (approximately 2.5 million cells or 1 mg total protein). Pre-cleared lysates were subjected to immunoprecipitation using 10ug FAK antibody-conjugated agarose (Millipore) overnight at 4°C. Beads were washed in modified RIPA lysis buffer and collected by centrifugation at 10,000 X g, at 4°C. The beads were boiled in 1X Lamelli buffer (BIORAD) for 5 min and subjected to SDS PAGE for western blot analysis.
For Rho GTP assays, equal numbers of cells were used because the dormant cells exhibited a larger cytoplasm to nucleus ratio and contained approximately 20% more protein than the growing cells (approximately 2.5 million cells, or 1 mg protein). Cells were harvested and lysed using 1X Mg Lysis buffer (Millipore). Rho GTP was immunoprecipitated using rhotekin RBD-agarose slurry (Millipore), which exclusively binds RhoGTP, according to the manufacturer’s instructions. Beads were washed, resuspended in 1X Laemmli buffer (Biorad) and subjected to SDS PAGE and immunoblotting for RhoA (Santa Cruz Biotechnology). For western blots, samples were transferred to PVDF membranes, blocked in 2% BSA 1X TBS-T, followed by addition of primary antibodies (SantaCruz Biotechnology and Millipore) and detected via chemiluminescence (Amersham).
Transfection and RhoA Constructs
RhoA DNA constructs, (kind gifts of Ian Whitehead), were grown as described . Briefly, cDNAs encoding human wild-type RhoA, fused to an NH2-terminal hemagluttinin (HA)-epitope tag were generated and cloned into pAX142. An identical mutant panel was generated for each isoform: RhoA-19N (dominant-inhibitory), RhoAWT (wild type), and RhoA-63L (constitutively active) . DNA was isolated from bacterial cultures using Highspeed Plasmid MAXI Kit, (Qiagen) according to the manufacturer’s instructions. RhoA constructs were transfected using Fugene6 transfection reagent (Roche) according to the manufacturer’s instructions into MCF-7 cells cultured at clonogenic density on FN coated coverslips. Rho constructs were co-transfected with pmaxGFP DNA (AMAXA) at previously optimized concentrations for maximum transfection efficiency at ratios of 10:1 or 3.0 μg RhoA constructs/0.3 μg GFP vector DNA. Medium was replenished at 12 h, and FGF-2 10 ng/ml was added on day 2 after transfection. Cells were stained with rhodamine phalloidin on day 4 following transfection, as described above. Cells were counted as having cortical actin rearrangement when >50% of the cell’s periphery was subtended by cortical actin. GFP positive cells in dormant clones (consisting of < 12 cells) or in growing clones (> 30 cells) were used for quantitation. Triplicate cover slips were independently transfected in two separate experiments. Means and standard deviations for data collected from green fluorescent cells on the three slides were calculated in each experiment and the significance of differences between different vector transfections were determined using Student’s t test.
The Qiagen Qproteome Cell Compartment fractionation kit (Qiagen) was used to isolate plasma membranes and cytoplasmic fractions from cells in dormant or growing clones according to the manufacturer’s protocol. Briefly, equal numbers of cells from dormant (+FGF-2) or growing (-FGF-2) clones cultured on FN-coated plates were subjected to sequential centrifugation during which soluble fractions containing plasma membrane and cytosolic fractions were extracted. Fractions were subjected to SDS PAGE and immunoblotted with anti-GRAF goat polyclonal antibody (Santa Cruz Biotechnology) and anti-BAX antibody as a localization control.
Integrin α5β1 Binding is Necessary for Cortical Actin Redistribution in Dormant Cells
Inactivation of Rhoa is Necessary but not Sufficient for Cortical Actin Redistribution in Dormant Cells
Activation of Focal Adhesion kinase in Dormant Cells is Associated with Membrane Localization of the GTP Activating Protein GRAF
To determine a possible mechanism for the inactivation of RhoA in dormant cells, we analyzed the FAK immunoprecipitates for GTPase Regulator Associated with the Focal Adhesion Kinase pp125(FAK) (GRAF), a protein with demonstrated RhoA GAP activity shown to co-localize with activated FAK in focal complexes. Figure 6a suggests that GRAF becomes associated with FAK in dormant cells, an effect exclusively dependent on integrin α5β1. To confirm this result, we analyzed the cells by immunfluorescence. Figure 6b demonstrates that GRAF became membrane localized in the dormant cells in a reciprocal relationship to the loss of RhoA membrane localization. As a control, Fig 6b demonstrates that the RhoA GAP p190 was not affected in dormant cells. To further confirm the activation by membrane localization of GRAF in dormancy, we carried out membrane fractionation experiments. Figure 6c demonstrates that GRAF was primarily cytoplasm localized in growing cells with a membrane to cytoplasm (m/c) ratio of 0.25. In dormant cells, GRAF membrane localization increased to an m/c ratio of 0.61. This effect once again was dependent on integrin α5β1, as blocking antibody to this integrin decreased the ratio to 0.15. With blocking antibody to integrin α2β1 used as a control, the GRAF m/c ratio was 0.80. These data support the hypothesis that the RhoA GAP GRAF becomes activated and membrane localized in dormant cells causing an inactivation of RhoA and that this effect depends on binding of integrin α5β1.
Activation of PI3K is Independent of Integrins α5β1 Binding in Dormant Cells
PI3K Activation is Necessary for Cortical Actin Redistribution in Dormant Cells
Membrane Localization of GRAF and Inactivation of RhoA Require PI3K Activity
We have previously demonstrated that estrogen sensitive breast cancer cells, analogous to ones likely to remain dormant as microscopic metastases in the bone marrow microenvironment of patients, can enter a state of relative dormancy in an in vitro model . The model encompasses some key components of the bone marrow niche, which include FGF-2 and fibronectin. Estrogen sensitive cells are induced by FGF-2 to go into G1 arrest through induction of cdk inhibitors , to re-express integrins lost with malignant progression  and to develop a distinct phenotype consisting of a large, spread out appearance, large cytoplasm to nucleus ratios  and to acquire resistance to chemotherapy with taxanes .
Here, we demonstrate that the spread appearance corresponds to cortically rearranged fibrillar actin and omnidirectionally activated FAK at the cell periphery. Circumferential actin bundle formation is another element of re-differentiation in these dormant cells. Cortical actin is observed exclusively in nontransformed mammary epithelial cells, disappears and is replaced by stress fibers during malignant transformation . These effects are similar to ones we have previously demonstrated to occur with re-differentiation of a highly malignant breast cancer cell line, MDA-MB-231, upon enforced expression of FGF-2 , a growth factor whose expression stops during the process of mammary epithelial cell progression to malignancy .
The activation of FAK, however, appears to be counterintuitive to the re-differentiation process when first encountered. FAK activation is associated with integrin-mediated adhesion and motility and is the mainstay of focal adhesion complexes initiating stress fibers. FAK levels are elevated and its activation plays a role in breast cancer progression [35, 36, 37, 38, 39]. However, our data showing that the activated FAK is complexed with GRAF in dormant breast cancer cells supports a role in a more differentiated state. GRAF is a protein with RhoA and dcdc42 GAP activity discovered in leukemic cells . GRAF binds to the C-terminal domain of FAK in an SH3 domain-dependent manner  and blocks Rho-mediated stress fiber formation . This can be regarded as contributing to partial cancer cell re-differentiation, since RhoA is the primary cause of stress fiber formation and increased motility of cancer cells, and trends to higher expression with tumor grade and nodal metastasis in breast cancer . This report is the first account for a putative role for GRAF in the inactivation of RhoA in dormant breast cancer cells in this in vitro model.
The inactivation of RhoA appears to be at steady state and Rhotekin pulldown assays for RhoA GTP did not demonstrate downregulation at earlier times (data not shown). It is most likely that actin polymerization took place before the steady state of dormancy was achieved, and F-actin was stabilized in the cortical distribution after inactivation of RhoA. We assayed for activation of both Rac and cdc42 to determine the effects of dormancy on other members of the small GTPase family. The GTP loading of cdc42 was diminished, but Rac GTP loading was unaffected (data not shown). The effect is likely due to family member specificity of the GRAF family [42, 44, 45] due to a favorable interaction of the GRAF 231-residue BH domain, which harbors its GAP activity  with Glu-95 of Cdc42 and Glu-97 of RhoA, and is absent in the corresponding Ala-95 of Rac1 . Rac and RhoA have a reciprocal relationship, and Rac activity remains unchecked with the inactivation of RhoA . This is one likely explanation for the distinct appearance of lamellopodia on dormant cells (Figs. 1a, 3b, 4a, 5a, 6b, 8a, 9a). However, without the ability to form stress fibers, the characteristic motility due to Rac activation does not occur .
The role of PI3K in GRAF activation is also novel. We demonstrated that the survival of these dormant cells depends, in part, on activation of the PI3K pathway. The data presented here demonstrate that parallel signaling induced by exogenous FGF-2 through PI3K and by integrin α5β1 is necessary for activation of this GAP. The levels of GRAF were not affected in dormant cells as demonstrated by western blot (data not shown). However, its membrane localization depended on both exogenous FGF-2 through PI3K and binding of integrin α5β1. The mechanism is not understood and will be studied in follow up investigations. However, an association with FAK has been demonstrated. Whether this association is direct or through elements of the well recognized large complex is yet to be determined and will be investigated.
PIK3CA, the gene coding for the catalytic subunit of PI3K, is mutated in 18–40% of breast cancers . The mutations are in “hotspots” in exons 9, corresponding to the helical domain and exon 20, corresponding to the kinase domain in 85–100% of cases [50, 51]. While the importance of the PI3K pathway in mammary tumorigenesis has been extensively investigated, opposing conclusions regarding mutations in the PIK3CA gene in primary breast tumors have been reached by different groups [50, 52]. A potential explanation for the conflicting reports came to light more recently when a more focused analysis reported that mutations in exon 9 are associated with a significantly worse prognosis for disease-free and overall survival while mutations in exon 20 are associated with prolonged survival . Also, while a mutation in Akt 1 has finally been identified in a number of malignancies, including breast cancer , the role of Akt activation in initiating malignant transformation is yet to be clarified . With respect to breast cancer dormancy, the significance of frequent mutations in the PI3K pathway is not at all understood. It is possible that activating mutations may render cells resistant to therapy and permit survival of metastatic cells in the bone marrow niche. We have previously shown that the activated PI3K pathway is necessary for survival of this dormancy model  but induction of the dormant, non-proliferative state depends on FGF-2-initiated signals that activate a variety of pathways in addition to PI3K.
In conclusion, we have shown that dormancy of estrogen sensitive breast cancer cells in a three component in vitro model encompasses a partial re-differentiation of breast cancer cells in close contact with elements of the microenvironement, which consist of inhibition of proliferation, re-expression of integrins lost with malignant transformation, cell spreading with omnidirectional FAK activation, activation of the RhoA GAP GRAF through exogenous FGF-2-mediated activation of PI3K and through integrin α5β1 ligation, with a resultant inactivation of RhoA and cortical actin rearrangement. Follow-up investigations will determine the mechanisms of achieving this steady state or dormancy and mechanisms for overcoming drug resistance in the dormant cells. Additional components will be added to the model, including a third dimension to validate the biological implications of our data prior to in vivo confirmation. In vivo effects of modulating RhoA activation in a murine metastasis model will confirm the functional role of RhoA inactivation in maintaining dormancy in micrometastases.
This model is one of several that have begun to generate data and hypotheses regarding this little understood but enormously significant biologic phenomenon. Our model fits with the concept of reversible growth/proliferation arrest or quiescence governed by a genetic program which ensures the suppression of terminal differentiation . The panel of genes comprising this state is activated regardless of the signal that initiates growth arrest. We have previously demonstrated that FGF-2 initiates reversible growth arrest in MCF-7 and T-47D cells  and that this effect is mediated through TGFβ . TGFβ and the BMP family are inhibitory to breast cancer cells that have not undergone epithelial mesenchymal transition  and can suppress micrometastases when administered in vivo .
A well-developed model of dormancy demonstrates a role for the urokinase receptor (u-PAR) activation in the exit from dormancy . The model describes the upregulation of integrin α5β1, and the ability of the latter to propagate signals from fibronectin through the EGF-receptor and ERK to cause single quiescent cells to enter the cell cycle . Similarly, a recent model of breast cancer dormancy demonstrated that the transition from quiescence to proliferation of breast cancer cells was dependent on fibronectin production and signaling through integrin β1, leading to cytoskeletal reorganization with F-actin stress fiber formation . These models are completely congruent with our hypothesis, despite first impressions. We have previously demonstrated that fibronectin increases the number of dormant MCF-7 and T-47D clones incubated with FGF-2, but nevertheless, the cells remain dormant . The likely explanation is that activation of proliferative signaling by ligation of integrin α5β1 by fibronectin and by FGF-2 mediated activation of the ERK pathway and upregulation of cyclin D  are trumped by FGF-2-induced parallel cell cycle inhibition and partial rediferrentiation. FGF-2-initiated signaling results in upregulation of p21WAF1/Cip1  and p27KIP1  and re-expression of integrins lost with de-differentiation , which collectively contribute to the dormant phenotype observed.
Supported by DAMD17-03-1-0524 (RW) and the Ruth Estrin Goldberg Memorial for Cancer Research (RW)
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