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• W2074531516 abstract "PAK physically interacts with CDC42 by pull down (View interaction) Formation of protrusions into the extracellular matrix (ECM) and subsequent migration through the ECM are the initial steps required for dissemination of cancer cells into the surrounding tissues, leading to metastasis [1, 2]. In order to observe cancer cell-specific protrusions in a 3-D ECM, we have previously developed a 3-D gel culture system where a native type-I collagen matrix containing epidermal growth factor (EGF) is overlaid with the Matrigel, a substance that mimics a thin basement membrane [3]. Plating of highly invasive MDA-MB-231 breast cancer cells or HT1080 fibrosarcoma cells onto the Matrigel leads to migration into the collagen and the formation of forming long protrusions morphologically reminiscent of invadopodia, actin-cytoskeletal protrusions formed in the ventral cell membrane that mediate characteristic focal degradation of the ECM ([4, 5] for recent reviews). Protrusion formation and invasion into the collagen are dependent upon matrix metalloproteinases (MMPs) activity, Wiskott-Aldrich Syndrome protein (WASP) family verprolin-homologous protein 2 (WAVE2) that regulates rearrangement of the actin cytoskeleton, and microtubule dynamics [3]. At present however, a requirement for EGFR signaling in the invasion of 3-D ECM by MDA-MB-231 cells has only been inferred from previous observations showing that 2-D migration of these cells is stimulated by EGF [6-8]. Additionally, participation of Rac or Cdc42, upstream regulators of WAVE2 and/or invadopodia formation, respectively [9-11] to the invasion of MDA-MB-231 cells into a 3-D ECM is unknown. In the present study, we characterized the requirement for EGFR signaling and Rho-family GTPases in invasion of a 3-D collagen matrix by MDA-MB-231 cells. MDA-MB-231 human breast cancer cell line was obtained from the European Collection of Cell Cultures (Porton Down, Wiltshire, UK). The cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). Recombinant human TGF-α was purchased from PeproTech (Rocky Hill, NJ). 3-D collagen gels were generated as previously described [3]. Briefly, 100 μL of 2 mg/mL neutralized type-I collagen gel (Nitta Gelatin Co., Osaka, Japan) containing final 1xRPMI1640 medium and 1% bovine serum albumin (BSA) was placed a 2 cm × 2 cm well on a glass slide (AR Brown Co., Tokyo, Japan) and solidified in CO2 incubator containing 5% CO2. A total of 5 × 104 serum-starved cells in 500 mL of RPMI1640 medium containing 0.05% BSA and 10 mg/mL transferrin were plated onto the 2 cm × 2 cm 3-D collagen gel. After 20–24 h of incubation, cells were fixed and stained with AlexaFluor 488-conjugated phalloidin (Molecular Probes, Carlsbad, CA). Stained cells were visualized using a laser scanning confocal microscope (Carl Zeiss, model LSM 5 Pascal; Jena, Germany). Numbers of the cells with long protrusions that projected into the collagen or that migrated into the collagen were counted as previously described [3]. To analyze migration within the collagen gel, 5 × 105 cells were mixed with 1000 μL of neutralized collagen, and 100 μL of the mixture was plated onto the glass slide. After the collagen-cell mixture hardened, it was overlaid with medium. Serum-starved cells were plated on PLL-coated two-well chamber slides (BD biosciences, Franklin Lakes, NJ) or on the 3-D collagen gels formed in the chamber slides as describe above. The cells were cultured for the indicated times prior to the onset of video recording. Image capture and time-lapse recording of the cultures were performed using the 20× objective of a Keyence BZ-9000 microscope (Keyence Co., Osaka, Japan) equipped with a CO2 incubation chamber unit kept maintained at 37 °C. Motion Analyzer software (Keyence Co.) was used to track the x–y coordinates of the cells in time-lapse images and to calculate the velocity and displacement of the cells from their respective start points. Cell motion paths were plotted using Deltagraph software (Red Rock Software Inc., Salt Lake City, UT). Rac1-targeted siRNAs, Cdc42-targeted siRNAs and corresponding control siRNAs were obtained from Dharmacon (Smart siRNA pools; Thermo Scientific, Lafayette, CO). The cells were transfected with siRNAs using LipofectamineRNAiMAX reagent (Invitrogen) for 24 h as previously described [3]. Proteins were extracted and blotted onto a polyvinylidene difluoride membranes as previously described [3]. Membranes were incubated with anti-Rac1 monoclonal antibodies (Upstate Biotechnology clone 23A8, Charlottesville, VA), anti-Cdc42 monoclonal antibodies (Santa Cruz Biotechnology clone B-8, Santa Cruz, CA) or anti-β-actin monoclonal antibodies (Sigma, St. Louis, MO). Bound antibodies were detected using an enhanced chemiluminescence system (GE Healthcare; Buckinghamshire, UK). MDA-MB-231 cells were serum-starved for 18 h. The cells either stimulated with 50 ng/ml TGF-α for 3 h or unstimulated cells were lysed in a buffer containing 25 mM Hepes–NaOH (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol and 1% NP40. Cell lysates containing 1800 μg protein were incubated with 10 μg PAK-protein binding domain–glutathione S-transferase fusion protein (Cytoskeleton Inc., Denver, CO.) for 1 h at 4 °C. Then activated Rac1 or Cdc42 bound by PAK-PBD were separated using Glutathione-Sepharose beads (GE Healthcare) and detected by Western blot analysis as described above. Activation of Rac1 or Cdc42 was evaluated by the amount of PAK-PBD-bound Rac1 or Cdc42 normalized to the amount of Rac1 or Cdc42 in whole cell lysates, respectively. We first analyzed whether invasion of MDA-MB-231 cells into 3-D collagen gels was TGF-α-dependent. TGF-α was selected as an EGFR ligand because it is abundantly expressed in breast cancer tissues [12, 13]. Cells were cultured on a type-I collagen matrix for 22 h either in the presence or absence of TGF-α and cellular morphology and localization were examined by confocal microscopy (Fig. 1 ). In the absence of TGF-α, approximately 99% of the cells remained on the surface of the collagen (Fig. 1A, left panels and Fig. 1B). In the presence of TGF-α, approximately 5% of the cells on the collagen formed long protrusions into the collagen (Fig. 1A, arrows in right panels and Fig. 1B) and approximately 8% of the cells invaded into the collagen (Fig. 1A, arrowheads in right panels and Fig. 1B). We then explored whether TGF-α stimulated invasion by inducing the motility of MDA-MB-231 cells. MDA-MB-231 cells were cultured on a PLL-coated glass surface and stimulated with media containing TGF-α or collagen-I, then their movements were monitored by videomicroscopy (Fig. 2 ). TGF-α did not induce significant cell motility (Fig. 2A, middle panel), while collagen-I stimulated cell migration (Fig. 2A, right panel) evident as an increase in the speed of migration (Fig. 2B, left panel). An increase in the directionality of cell movement, evaluated by dividing the displacement of the cells from start point to end point by the path [14], was not evident in collagen-stimulated cells (Fig. 2B, right panel). The effects of TGF-α on the migration of cells either plated onto a 3-D collagen gel or embedded within the collagen were also analyzed (Fig. 3 ). Cells actively migrated on the surface of the collagen in the absence of TGF-α (Fig. 3A). Addition of TGF-α further increased the speed of migration by 50% (Fig. 3A). Migration of the cells embedded in the collagen was evaluated by tracing cell movements projected into the x–y plane (Fig. 3B, left panels showing tracks). The cells actively migrated through the collagen matrix in the absence of TGF-α, although the speed was decreased. Addition of TGF-α did not have a substantial effect on migration, with respect to either speed or directionality (Fig. 3B, right panels), in agreement with the previous report that MDA-MB-231 cells migrate within 3-D collagen in the absence of growth factors [15]. Although we cannot exclude the possibility that TGF-α induces invasion of the cells by increasing migration on collagen surface, because TGF-α induced invasion rather in an on–off manner, we may conclude that TGF-α primarily promotes movement of the cells from the surface to the inside of the collagen matrix, likely by initiating the formation of long protrusions into the collagen. We next investigated the potential roles of the Rho-family proteins, Rac1 and Cdc42, activities of which are stimulated in TGF-α -treated MDA-MB-231 cells (Fig. 4 A), in invasion and collagen-stimulated motility of the cells. When we examined the effect of siRNA-mediated downregulation of Rac1 or Cdc42 (Fig. 4B) on invasion, we found that depletion of Cdc42 strongly suppressed protrusion formation and invasion into 3-D collagen gels, while depletion of Rac1 had no inhibitory effects (Fig. 4C). To determine whether inhibition of invasion was a direct consequence of the suppression of cell motility, we examined the motility of Cdc42-depleted cells by videomicroscopy (Fig. 5 A). Cells deprived of Cdc42 exhibited minimal defects in cell motility, although a slight but significant decrease in migration speed was observed (Fig. 5B). This result opposes the findings a previous study showing that Cdc42 is required for EGF-stimulated directional motility in fibroblasts [16]. However, recent studies have indicated that the contributions of Cdc42 to cell migration and protrusion formation differ in different cell types, likely due to compensatory effects of other Rho-family GTPases [17], expression of which vary across different cell types [18, 19]. The results of this study and those from a recent report demonstrating a crucial role for Cdc42 in invasion of 3-D collagen gels by HT1080 human fibrosarcoma cells [20] suggest that the role of Cdc42 in invasion of 3-D collagen matrices cannot be compensated by other Rho-family GTPases, differing from the roles of Cdc42 in directed migration on 2-D surfaces. Unlike Cdc42, depletion of Rac1 did not affect the invasiveness of MDA-MB-231 cells. Rac1 plays an important role in the activation of WAVE2 [9]. Therefore, one would hypothesize that knockdown of Rac1 expression should suppress invasion, similar to depletion of WAVE2 [3]. Currently, the reasons underlying failure of Rac1 knockdown in MDA-MB-231 cells to suppress invasion remain unknown. It is possible that residual levels of Rac1 activity after knockdown were sufficient for cell migration [14, 21]. Alternatively, Rac3 can compensate for Rac1 in the activation of WAVE2 in cancer cell invasion [22, 23]. In conclusion, we have characterized the role of TGF-α in the invasion of 3-D collagen gels by MDA-MB-231 cells. TGF-α was shown to promote the formation of protrusions into collagen, the initial step of invasion, and may be dispensable for cell migration, which is effectively stimulated by collagen. Cdc42 is crucial for the TGF-α-induced invasion, but dispensable for collagen-stimulated migration. Recently, Cdc42 in HT1080 fibrosarcoma cells has been shown to regulate invasion of 3-D collagen gels by forming complexes with Membrane Type-I (MT1)-MMP, IQGAP1 that regulates reorganization of both the actin and microtubule cytoskeletons, and several other proteins involved in signal transduction [20]. It remains to be determined whether similar “Cdc42/MT1-MMP invasion signaling complexes” [20] also direct the formation of long protrusions in MDA-MB-231 cells in response to TGF-α. We thank Dr. Eiju Tsuchiya (Kanagawa Cancer Center Research Institute) for helpful discussions and encouragement, Dr. Kazuhide Takahashi (Kanagawa Cancer Center Research Institute) for critical review of the manuscript and Nobuyuki Endo (Keyence Co.) for use of the videomicroscope and associated software. This work was supported in part by a Grant-in-Aid for the Encouragement of Basic Science and Technology from the ." @default.
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• W2074531516 title "Invasion of breast cancer cells into collagen matrix requires TGF-α and Cdc42 signaling" @default.
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