Plasma Membrane-Associated py397fak Is a Marker of Cytotrophoblast Invasion in Vivo and in Vitro

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1 American Journal of Pathology, Vol. 159, No. 1, July 2001 Copyright American Society for Investigative Pathology Plasma Membrane-Associated py397fak Is a Marker of Cytotrophoblast Invasion in Vivo and in Vitro Duško Ilić,* Olga Genbačev,* Fang Jin,* Eduardo Caceres,* Eduardo A. C. Almeida,* Valérie Bellingard-Dubouchaud,* Erik M. Schaefer, Caroline H. Damsky,* and Susan J. Fisher* From the Departments of Stomatology,* Anatomy, Obstetrics, Gynecology and Reproductive Sciences, and Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California; and Quality Controlled Biochemicals, Division of BioSource International, Hopkington, Massachusetts During human pregnancy specialized placental cells of fetal origin, termed cytotrophoblasts, invade the uterus and its blood vessels. This tumor-like process anchors the conceptus to the mother and diverts the flow of uterine blood to the placenta. Previously, we showed that the expression of molecules with important functional roles, including a number of extracellular matrix integrin receptors, is precisely modulated during cytotrophoblast invasion in situ. Here we exploited this observation to study the role of the focal adhesion kinase (FAK), which transduces signals from the extracellular matrix and recruits additional signaling proteins to focal adhesions. Immunolocalization studies on tissue sections showed that FAK is expressed by cytotrophoblasts in all stages of differentiation. Because extracellular matrix-induced integrin clustering results in FAK (auto)phosphorylation on tyrosine 397 (Y397FAK), we also localized this form of the molecule. Immunolocalization experiments detected Y397FAK in a subset of cytotrophoblasts near the surface of the uterine wall. To assess the functional relevance of this observation, we used an adenovirus strategy to inhibit cytotrophoblast expression of FAK as the cells differentiated along the invasive pathway in vitro. Compared to control cells transduced with a wild-type virus, cytotrophoblasts that expressed antisense FAK exhibited a striking reduction in their ability to invade an extracellular matrix substrate. When cytotrophoblast differentiation was compromised (hypoxia in vitro, preeclampsia in vivo), Y397FAK levels associated with the plasma membrane were strikingly lower, although total FAK levels did not change. Together our results suggest that (auto)phosphorylation of Y397 on FAK is a critical component of the signaling pathway that mediates cytotrophoblast migration/invasion. Pathol 2001, 159:93 108) (Am J During tumorigenesis, precursor cells that presumably were once normal become cancerous. Clonal selection, which takes months to years, is an integral part of this process. In the end, only a subset of cells with favorable attributes, including unregulated proliferation, invasiveness, and the ability to acquire a blood supply, survive. In the interim it is difficult to define critical steps in the molecular transitions that select particular clones from heterogeneous mixtures of precursor cells. Identification of the individual changes that promote the metastatic phenotype in the context of the global nature of downstream consequences is a pivotal problem in modern tumor biology. During development of the human placenta, the organ s specialized epithelial cells, termed cytotrophoblasts (CTBs), encounter many of the same obstacles that tumor cells must overcome during clonal selection. Specifically, a subset of these CTBs must detach from the basement membrane, where they reside as a monolayer in the fetal component of the placenta, and invade the uterus, where they survive only if they access a supply of maternal blood. Because subsequent human development depends on the tumor-like properties of this subpopulation of CTBs, the molecular mechanisms that are involved have been carefully programmed. Consequently, the cells acquire the ability to invade as part of a tightly regulated, stepwise differentiation process (diagrammed in Figure 1A). 1 Furthermore, this subset of CTBs stops proliferating once it initiates the program leading to an invasive phenotype. 2 Therefore, studying the process of CTB differ- Supported by the University of California at San Francisco Academic Senate and the National Cancer Institute Howard Temin Award (KO1 CA ) (to D. I.); and by National Institutes of Health grants HD 30367, HD 26732, and HL D. I. and O. G. contributed equally to this work. Accepted for publication April 10, Present address of F. G.: SUGEN, Inc., Oncology Targets, 230 E. Grand Ave., South San Francisco, California Address reprint requests to Susan Fisher, University of California San Francisco, 513 Parnassus, HSW-604, San Francisco, CA sfisher@cgl.ucsf.edu. 93

2 94 Ilic et al Figure 1. Diagram (A) and histological section (B) of an anchoring chorionic villus at the fetal-maternal interface at the beginning of the second trimester of pregnancy (15 weeks of gestation). A: The anchoring villus (AV) functions as a bridge between the fetal and maternal compartments of the human placenta. Both AV and floating villi (FV) are covered with syncytiotrophoblasts (STB), contain fetal blood vessels (FBV), and are bathed by maternal blood in the intervillous space (IVS). CTBs in AV form cell columns (CC) that attach the fetal-placental unit to the uterine wall (UW). CTBs then invade decidua, myometrium, and uterine blood vessels (ictb). Long arrow denotes direction of CTB migration/invasion. B: A histological section of the fetal-maternal interface diagrammed in (A) was stained with anti-cytokeratin (CK) to image all of the trophoblast populations. vctb; villous CTB. entiation/invasion offers a unique opportunity to identify, by drawing functional analogies, molecules particularly likely to play important roles in promoting the invasive phenotype of tumor cells. Much of our previous work focused on the role of adhesion molecules in CTB invasion. Initially we immunolocalized a battery of adhesion receptors and their ligands on tissue sections that contained CTBs in all stages of differentiation/invasion. These studies allowed us to phenotype differentiating cells in situ. The results showed that CTBs intricately modulate their expression of a wide repertoire of antigens that function in adhesion. This modulation process begins when the cells leave their basement membrane in the placenta, and ends when they colonize maternal blood vessels in the uterus. The onset of CTB differentiation/invasion is signaled by reduced staining for receptors characteristic of polarized CTB epithelial stem cells integrin 6 4 and E-cadherin and the onset of expression of adhesion receptors expressed on endothelium VE-cadherin, Ig family members VCAM-1 and PECAM-1, and integrins v 3 and Cadherin-11 is also up-regulated on extravillus CTBs and on decidualizing endometrial stroma. 7,8 By using an in vitro model of CTB invasion that replicates the process of adhesion molecule switching in vivo, weas- sessed the contribution of individual adhesive interactions to the invasion process. In this regard, we found a particularly important role for integrin cell-extracellular matrix (ECM) receptors. A number of these molecules whose expression is up-regulated during differentiation promote invasion (eg, 1 1, v 3 ), whereas others are inhibitory (eg, 5 1 ). Thus, this highly regulated invasive program is governed by a system of checks and balances. Together, our previous work suggests that the intracellular mechanisms that CTBs use to translate the information they receive via integrin receptors into signals is crucial to their ability to invade the uterus. Integrin interactions with ECM components lead both to occupancy of their ligand-binding sites and to clustering, which assembles multimolecular signaling complexes in focal adhesions, sites of cell-ecm interactions. 9,10 Thus, integrin signals are connected to multiple intracellular signaling pathways. 11 One important outcome of integrin clustering by ECM is the (auto)phosphorylation of focal adhesion kinase (FAK) on tyrosine 397 (py397fak). py397fak, in turn, serves as the backbone of a scaffold that recruits additional signaling proteins to focal adhesions. 12 FAK has been implicated in the transduction of survival signals from ECM and in promoting cell migration Both processes are relevant to establishing a pool of cells with a migratory/invasive (metastatic) phenotype. Several studies have attempted to determine whether there is a correlation between the levels of FAK expression and the acquisition of metastatic potential. Although FAK was overexpressed in some types of highly invasive tumors, the degree of FAK expression was not a significant prognostic factor. 23 Interestingly, comparative studies of normal breast tissue and invasive breast carcinoma revealed that FAK levels can be elevated 24 or reduced 25 in transformed versus normal tissue. From these studies it seems that regulation of total FAK levels is not crucial for promoting an invasive phenotype. Thus, the key might be regulation of its activity. In the current study we have extended our work on the adhesion phenotype switching that occurs as CTBs differentiate/invade to address the putative role(s) of FAK in this unusual tumor-like behavior. Again, we have used a combination of in situ and in vitro approaches. Localization studies in placental tissues showed that CTBs in all stages of differentiation express FAK. However, only a subset of CTBs invade the uterine wall (Figure 1A). Therefore, we hypothesized that some of these cells had high levels of (auto)phosphorylated FAK. To test this idea, we immunolocalized py397fak on tissue sections of the fetal-maternal interface. We also tested the effect of re-

3 FAK and CTB Invasion 95 ducing CTB expression of FAK protein on the cells ability to invade a Matrigel substrate in vitro. Finally, we asked whether conditions that inhibit CTB invasion (hypoxia in vitro, preeclampsia in vivo) were associated with a reduction in FAK (auto)phosphorylation, as determined by measuring FAK kinase activity and by staining for py397fak. These results support the hypothesis that FAK (auto)phosphorylation on Y397 plays a key role in promoting the invasive behavior of normal CTBs and, by analogy, may also play an important role in tumor development. Materials and Methods Antibodies and Antibody Characterization The 7D3 rat anti-human cytokeratin monoclonal antibody (mab) was raised in this laboratory. 3 Several antibodies recognized both the phosphorylated and the nonphosphorylated forms of FAK: two mouse mabs (Transduction Laboratories, Inc., San Diego, CA, and Chemicon, Temecula, CA); rabbit polyclonal antibodies A-17, C-20 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and JF1; 17 and goat polyclonal antibodies A-17 and C-20 (Santa Cruz Biotechnology Inc.). Anti-paxillin mab was purchased from Zymed (South San Francisco, CA) and anti-phosphotyrosine 4G10 from UBI (Lake Placid, NY). An antibody that recognized only FAK (auto)phosphorylated on Tyr397 (py397fak) was obtained from Bio- Source International Inc. (Camarillo, CA). To validate its use in immunolocalization studies, the specificity of this antibody was rigorously tested before initiation of the experiments reported here. Briefly, tissue sections cut from several organs of fak knockout mice and the parental wild-type animals were analyzed along with cultured cells from both strains. 17 The samples from fak-null animals, which served as negative controls, also had enhanced levels of potentially cross-reactive antigens, including proteins phosphorylated on tyrosine and the homologous ( 50%) FAK family member Pyk2. 26 Initial experiments showed that anti-py397fak antibody at a concentration of 2.5 g/ml stained tissue sections and cells from wild-type, but not fak-null, animals. Staining was completely abolished when the antibody was preincubated with 100 g/ml of the phosphopeptide used to generate the antibody, whereas addition of 100 g/ml of the nonphosphorylated peptide had no effect. Preincubation with a phosphopeptide (100 g/ml) that corresponds to the homologous sequences in Pyk2 had also no effect on the staining specificity of anti-py397fak antibody. Tissue Sources for Immunolocalization Experiments Control samples of chorionic villi and the portion of the uterine wall to which they attached were obtained from pregnancy terminations for nonmedical reasons and from uncomplicated spontaneous deliveries. Because of their relatively small size, samples from each first trimester placenta were processed in toto. Second and third trimester samples were from three to five randomly chosen sites. None of the samples had abnormalities that could be detected either grossly or histologically. Tissues were obtained from women whose pregnancies were terminated during the first trimester (52 samples), second trimester (7 samples), or at the time of delivery (8 samples). Conclusions were based on analysis of all samples in each group. Samples of chorionic villi and the portion of the uterine wall to which they attached were also obtained by placental bed biopsy of women who received prenatal care at the University of California San Francisco (Moffitt/Long Hospital). Informed consent for the procedure was obtained during one of the late second/early third trimester clinic visits. The consent form and biopsy procedure were approved by the University of California San Francisco Committee on Human Research. Control samples were obtained from eight women who had no underlying medical conditions at 33 (n 1), 35 (n 4), and 36 (n 3) weeks of gestation. Eight samples were obtained from preeclamptic patients at 32 (n 1), 33 (n 4), and 35 (n 3) weeks of gestation. Preeclampsia was diagnosed according to the following criteria, recommended by Chesley 27 : nulliparity; no history of hypertension before pregnancy; increase in diastolic pressure of 15 mm Hg or systolic pressure of 30 mm Hg compared with blood pressure obtained before 20 weeks of gestation; proteinuria 0.3 g/24 hours (or 1 on urine dipstick) in a catheterized specimen; hyperuricemia 5.5 mg/dl (or 1 SD greater than the normal mean value before term); return to normal blood pressure and resolution of proteinuria by 12 weeks postpartum. Severe preeclampsia was diagnosed according to the following criteria recommended by the American College of Obstetricians and Gynecologists: systolic blood pressure 160 mm Hg and/or diastolic pressure 110 mm Hg; proteinuria of 5 gina 24-hour period or 3 on urine dipstick; presence of cerebral or visual disturbances. Two patients were diagnosed with severe preeclampsia and were delivered by Cesarean section (32 and 33 weeks); six patients with preeclampsia had vaginal deliveries [33 (n 3) and 35 (n 3) weeks]. Immunolocalization Placental tissues were processed for double indirect immunolocalization as previously described. 2,3 Briefly, tissues were fixed in 3% paraformaldehyde for 30 minutes, washed three times in phosphate-buffered saline (PBS), infiltrated with 5 to 15% sucrose followed by optimal cutting temperature (OCT) compound (Miles Scientific, Naperville, IL), and frozen in liquid nitrogen. Sections (6 m) were prepared using a cryostat (Slee International, Inc., Tiverton, RI). Nonspecific antibody reactivity was blocked by using a commercially available kit (Vector, South San Francisco, CA). Sections were then incubated for 2 days at 4 C with a mixture of the three antibodies: 1) 2.50 g/ml of ChromPure donkey whole IgG (Jackson ImmunoResearch, West Grove, PA); 2) 1 g/ml of 7D3

4 96 Ilic et al anti-human cytokeratin; and 3) 1 g/ml of either anti-fak (JF1) or anti-py397fak. Then, the sections were rinsed three times in PBS for 10 minutes and incubated for 30 to 60 minutes at room temperature with the appropriate species-specific secondary antibodies conjugated to rhodamine (donkey anti-rat IgG) or biotin (donkey antimouse or anti-rabbit IgG), and washed three times in PBS for 10 minutes. Sections were then incubated for 15 minutes at room temperature with streptavidin/fluorescein isothiocyanate conjugate (Vector) and 10 g/ml Hoechst (Molecular Probes, Eugene, OR), washed three times in PBS for 10 minutes, and mounted with Vectashield medium (Vector). Samples were examined with a Zeiss Axiophot Epifluorescence microscope (Thornwood, NY) equipped with filters to selectively view the rhodamine, fluorescein, and Hoechst fluorescence. When FAK was immunolocalized in cultured cells, fixation was followed by permeabilization (10 minutes in cold methanol). All other steps were performed using the protocol described for sections. Several types of control incubations were included. Sera (preimmune or isotype-matched nonimmune) and PBS were substituted for the primary antibodies. In addition, fluorescein isothiocyanate-donkey anti-mouse and fluorescein isothiocyanate-donkey anti-rabbit IgG were substituted for biotin-conjugated reagents to detect binding of the primary antibody. Finally, the effects on blocking and control peptides were assessed as described above. Staining of control tissue sections and cells was never observed. HAM/Dulbecco s modified Eagle s medium (1:1/v:v) culture medium (Sigma Chemical Co., St. Louis, MO) supplemented with an antibiotic/antimycotic mixture (100 U/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml amphotericin B; Sigma) and 10% fetal calf serum. Each group (control and experimental) consisted of five explants. In toto, 12 placentas were individually analyzed. Isolated CTBs, co-cultures and anchoring villi were maintained either under standard tissue conditions (5% CO 2 /95% air) or placed in a Bactron anaerobic incubator (Sheldon Manufacturing Inc., Cornelius, OR), where they were maintained in a 2%O 2 /93% N 2 /5% CO 2 environment. Dissolved O 2 at the cell-medium interface, measured using micro-oxygen electrodes (MI-730; Microelectrode Inc., Londonderry, NH), was 20% under standard tissue conditions and 2% in the Bactron anaerobic incubator. BrdU Incorporation Anchoring villi from 6- to 8-week-old placentas were cultured on Matrigel in either 20% O 2 or 2% O 2. After 48 hours, the original medium was replaced with fresh medium containing 1 mol/l 5-bromo-2 -deoxyuridine (BrdU, Roche Molecular Biochemicals). Twenty-four hours later tissue was washed, fixed, embedded in OCT, and frozen in liquid nitrogen. Sections cut from these blocks were stained with fluorescein isothiocyanate-labeled anti-brdu monoclonal antibody as recommended by the manufacturer (Roche Molecular Biochemicals). Cell and Tissue Culture CTBs were isolated from pools of multiple placentas as described. 28 The placentas were obtained immediately after first trimester terminations and third trimester deliveries. Remaining leukocytes were removed by using a mab to CD45 coupled to magnetic beads. The purified CTBs were cultured in DME H21 minimal essential medium containing 2% Nutridoma (Roche Molecular Biochemicals, Indianapolis, IN) and 50 g/ml of gentamicin on two substrates. Routinely the cells were plated on Matrigel-coated tissue culture wells (Collaborative Biomedical Products, Bedford, MA). Co-cultures were also established with decidua (n 4, 10 to 12 weeks of gestation). Anchoring villi were prepared for culture as previously described. 29,30 Briefly, small fragments of placental tissue from the fetal-maternal interface were teased apart until they had the characteristic tree-like appearance of chorionic villi as viewed in a stereomicroscope. Anchoring villi were identified by the attached remnants of cell columns. Light microscopic examination of hematoxylinstained sections of villus tissue preparations consistently showed the presence of floating and anchoring villi and the absence of endometrial contamination. Anchoring villi (wet weight 5 to 10 mg) were cultured on the surface of Matrigel-coated 12-mm Millicell-CM culture dish inserts (0.4 m pores; Millipore Corp., Bedford, MA). The inserts were then placed into 24-well dishes in a mixture of F12 Adenovirus Construction and Transduction Adenoviruses were generated by using published methods. 31 The antisense FAK vector (pcdna3 antisense FAK) was the kind gift of Drs. Y. Takeuchi and M. Suzawa from Kyoto University, Japan. 32 pegfp-c1 was purchased from Clontech (Palo Alto, CA). NdeI-BamHI fragments of pcdna3 antisense FAK and pegfp-c1 were inserted into the padlox shuttle vector. The viruses were established by transfecting the ligated adenoviral genome constructs with replication-defective 5 virus into CRE8 cells that stably overexpressed Cre-recombinase. Positive clones were expanded in HEK293 cells. Adenoviruses that expressed either a FAK antisense oligonucleotide (AdASFAK) or GFP (AdcGFP) were plaque purified ( pfu/ml) and isolated as described. 31 For transduction, explants or cells were infected with wildtype replication-defective 5 adenovirus, AdASFAK, or AdcGFP. Before plating, isolated CTBs and anchoring villus explants were incubated for 2 hours in medium containing 20 to 150 pfu/cell of virus. After one washing with PBS, the cells and villi were cultured as described above. At the end of the experiment FAK expression was monitored by immunostaining and immunoblotting. Invasion Assays Two assays were used. First, to quantify invasion, CTBs were cultured in Matrigel-coated Transwell inserts

5 FAK and CTB Invasion 97 (6.5 mm; Costar Corp.) containing polycarbonate filters with 8- m pores. After 72 hours the cultures were rinsed in PBS, fixed, and stained with anticytokeratin antibodies as described below. To assay invasion, the filters were dissected from the inserts with a scalpel blade and mounted on poly-l-lysine-coated slides in a drop of mounting medium such that the underside of the filters faced upward. The number of cytokeratin-positive cell processes that penetrated the Matrigel and appeared on the underside of the filters was counted. In each of three independent experiments, three filters were used for each experimental condition. Second, CTBs were cultured with decidual explants. 33 Briefly, portions of the decidua parietalis, which does not contain CTBs before culture, were obtained at the time of pregnancy termination (10 to 12 weeks). The tissue was washed three times in ice-cold PBS and cut into 2-mm 3 pieces. After another three washes, five decidual pieces in 500 l of medium (DME/2% Nutridoma) were plated on each of several Millicell inserts (12-mm diameter) coated with 200 l of Matrigel. The inserts were transferred to 16-mm culture dishes containing 600 l of medium and incubated for 6 hours, the amount of time required for the explants to attach to the ECM substrate. Then the medium was aspirated from the upper chamber, and 10 6 CTBs in 500 l of medium was added. The cultures were maintained for 4 days in either a 2% or a 20% oxygen atmosphere, and medium was changed daily. Three co-cultures were set up for each experimental condition, and the experiments were repeated three times. The samples were fixed in 3% paraformaldehyde for 40 minutes, washed three times in PBS (4 C, 10 minutes), infiltrated with sucrose, embedded in OCT compound, and frozen in liquid nitrogen. Sections (6 m) were cut using a cryostat (Slee International Inc., Tiverton, RI) and collected on poly-l-lysinecoated microscope slides. The sections were stained as described below. Samples were examined with a Zeiss Axiophot epifluorescence microscope equipped with proper filters and photographed with Kodak T-Max 400 film (Kodak, Rochester, NY). Western Blots and FAK (Auto)phosphorylation Assay To examine the efficiency of FAK antisense in reducing levels of the endogenous FAK protein, cells were lysed in modified RIPA buffer [1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 150 mmol/l NaCl, 10 mmol/l Tris/HCl, ph 7.4, 1 mmol/l ethylenediaminetetraacetic acid] containing freshly added protease inhibitors (1 g/ml aprotinin, 1 g/ml leupeptin, 1 mmol/l phenylmethyl sulfonyl fluoride). Lysates were precleared with protein G-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) and separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 8% gels. Gels were transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) and blotted using monoclonal anti-fak antibody (Transduction Laboratories). For kinase assays FAK was immunoprecipitated from cells lysed in modified RIPA buffer using a combination of two goat polyclonal anti-fak antibodies, A-17 and C-20 (Santa Cruz Biotechnology). Immunoprecipitates were washed and then incubated in universal kinase buffer (10 mmol/l PIPES, ph.7.4, 10 mmol/l MnCl 2 ) in the presence of [ - 32 P]ATP for 30 minutes at 30 C. 34 Samples were separated using 8% SDS-PAGE and transferred to nitrocellulose. The membrane was blotted first with anti-fak mab (Transduction Laboratories). The bands were visualized using alkaline phosphatase-conjugated secondary antibodies and BCIP/NBT dye substrate (Roche Molecular Biochemicals). Dried membranes were exposed to BioMax MS film (Kodak) to detect incorporated radioactivity. To assess total protein tyrosine phosphorylation, whole cell/triton soluble lysates were prepared by solubilizing cells in a buffer containing 1% Triton X-100, 150 mmol/l NaCl, 10 mmol/l Tris/HCl, ph 7.4, 1 mmol/l ethylenediaminetetraacetic acid, and freshly added protease inhibitors (1 g/ml aprotinin, 1 g/ml leupeptin, 1 mmol/l phenylmethyl sulfonyl fluoride). Lysates were spun at maximum speed in a microfuge for 10 minutes at 4 C. Dissolving the pellets in modified RIPA buffer yielded a whole cell/triton insoluble/ripa soluble lysate. Lysates were spun at maximum speed in a microfuge for 10 minutes at 4 C. Dissolving the pellets in SDS-PAGE sample buffer yielded whole cell/triton-insoluble/ripa-insoluble lysates. Results Activated FAK Was Detected Only at Sites of CTB Invasion in Situ Initially, we used immunolocalization techniques to evaluate FAK expression during human CTB invasion in situ. These studies focused on the fetal-maternal interface, which includes floating villi together with anchoring villi, their sites of uterine attachment, and the portion of the uterine wall that CTBs invade. The histological organization of this region is diagrammed in Figure 1A. A photomicrograph of a tissue section stained with anti-cytokeratin (CK), which labels syncytiotrophoblasts (STBs) and all CTB subpopulations, is shown in Figure 1B. In both panels the long arrow denotes the direction of migration/invasion. CTBs in all locations showed high levels of FAK immunoreactivity (Figure 2). In mononuclear villus CTBs (vctb), FAK localized to the plasma membrane region (Figure 2A, short arrows). In multinucleate STBs, staining of the cytoplasm in a punctate pattern was often detected (Figure 2A, long arrows). However, the brightest immunoreactivity was found at the vctb-stb border, suggesting that FAK expression is associated with the apical surfaces of the vctb plasma membranes, the basal surfaces of STB plasma membranes, or both areas (Figure 2A, arrowheads). CK-positive invasive CTBs (ictb) within the uterine stroma also stained for FAK in a pattern that was primarily plasma membrane-associated (Figure 2C, short arrows). All of the antibodies against FAK protein that were tested, including those recognizing epitopes in

6 98 Ilic et al Figure 2. FAK is ubiquitously expressed in placental trophoblasts. Sections of the fetal-maternal interface (18 weeks) were stained with antibodies against cytokeratin (CK) (B and D) to image all of the trophoblast populations and with several antibodies that recognized all forms of the FAK. A: An example of the staining pattern of one FAK antibody (JF1). FAK staining was detected in all CTB subpopulations, primarily in association with the plasma membrane (short arrows). The cytoplasm of STB stained in a punctate pattern (long arrows). The brightest immunoreactivity was found at the villous CTB (vctb)- STB border (arrowheads). C: Cytokeratin-positive CTBs that invaded the uterine wall (ictb) showed diffuse cytoplasmic and bright cell-membrane staining for FAK, denoted by the arrows. the C-terminal or N-terminal region, gave similar staining patterns. Data from numerous in vitro studies show that FAK (auto)phosphorylation on tyrosine 397 is involved in regulating this molecule s activity during migratory processes. Accordingly, we stained tissue sections of the fetal-maternal interface with an antibody that recognizes only py397fak (Figure 3A). vctbs and STBs showed little or no staining with anti-py397fak (not shown). Interestingly, this antibody distinguished two CK-positive ictb populations within the uterine wall. Strong staining for py397fak was seen in sites of initial invasion, CTBs near the surface of the uterine wall that borders the intervillous space, whereas CTBs that had penetrated more deeply did not stain (Figure 3B). In py397fak-positive cells, immunoreactivity was either diffuse throughout the cytoplasm (Figure 3, D F; arrowheads) or localized to a very specific region (Figure 3, D-F; short arrows). Antisense FAK Expression Restricted CTB Migration/Invasion from Anchoring Villus Explants in Vitro Next, we assessed the role of FAK in CTB migration/ invasion in vitro. In the absence of a specific inhibitor of FAK (auto)phosphorylation on Y397, we used an adenovirus approach to deliver antisense oligonucleotides that were designed to lower the cells expression of FAK protein (AdASFAK). Two in vitro models were used. The first was an organ culture system in which explanted anchoring villi were cultured on Matrigel-coated wells. 29,30 In this model, villi attach to the Matrigel via the remnants of severed CTB columns. Subsequently, the area of the column that is in contact with the Matrigel gives rise to invasive CTBs that migrate out of the explant. Initially, we used an adenovirus that was engineered to encode green fluorescent protein (AdcGFP) to determine which cells, among the many types found in the villus explant (see Figure 1), were infected. GFP was expressed primarily by CTBs in the cell columns (CC) (Figure 4A). This finding is consistent with our previous observation that CTBs in this location up-regulate expression of v 3 integrin, 6 which is an adenovirus receptor. 35 GFP was expressed throughout the column, including at the expanded tips that contain outgrowths of migrating/invading CTBs (Figure 4, B and C; arrow). In contrast, explants that were infected with AdASFAK (Figure 4D) showed a consistent reduction in CTB migration/ invasion from the cell columns (Figure 4D, arrowhead). At the end of the experiment, tissue sections were prepared

7 FAK and CTB Invasion 99 Figure 3. py397 FAK staining is detected on a subset of CTBs within the uterine wall (UW) in situ. A: Schematic diagram of FAK. The central kinase domain is flanked by large N- and C-terminal domains. The N-terminus contains a FERM (band 4.1, ezrin, radixin, moesin) domain and the C-terminus contains the focal adhesion targeting (FAT) domain. The major FAK (auto)phosphorylation site is on tyrosine Y397 (*Y397) near the junction of the FERM and kinase domains. Tissue sections included the superficial portion of the uterine wall that is adjacent to the intervillous space (IVS). C and E: Staining with anti-cytokeratin (CK) identified ictbs. B and D: Staining with an antibody that recognized only FAK (auto)phosphorylated on tyrosine 397 (py397fak) distinguished two CK-positive ictb populations within the uterine wall. Strong staining for py397fak was seen in CTBs near the uterine surface, adjacent to the intervillous space, whereas CTBs that penetrated more deeply did not stain. In py397fak-positive cells, immunoreactivity was either diffuse throughout the cytoplasm (arrowheads) or localized to a very specific region (short arrows). from control explants that expressed GFP and experimental explants that expressed antisense FAK. Immunolocalization showed that cell columns from the experimental explants stained only weakly for FAK as compared to cells in comparable regions of the control cultures (compare Figure 4E and 4F). Expression of Antisense FAK Reduced FAK Protein Levels in Isolated CTBs and Suppressed Their Migration/Invasion in Vitro We used a second tissue culture model to confirm and extend the results obtained in the villus explant system. When isolated vctbs are plated on a Matrigel substrate, they execute the differentiation pathway that gives rise to ictbs in vivo, including the adhesion molecule switching program. 1 Typically, this process takes 48 to 72 hours. Four different preparations of purified vctbs were transduced with AdASFAK or control virus. Effects on FAK protein levels, FAK localization, and CTB invasion were quantified for each preparation. An immunoblot from one of the preparations comparing FAK protein levels in lysates of CTBs transduced with AdASFAK or control virus is shown in Figure 5A. After 72 hours in culture, densitometric analyses showed that transduction with AdASFAK reduced FAK expression by 48% relative to control CTBs that were either transduced with the parental, wild-type virus (AdWT) or not exposed to virus. As an additional control, the lower molecular mass region of the same blot was probed with an antibody specific for paxillin ( 70

8 100 Ilic et al

9 FAK and CTB Invasion 101 Figure 5. FAK expression is associated with CTB invasion. A, top: Immunoblot comparing FAK protein levels in cell lysates of control CTBs that were not exposed to virus, CTBs transduced with an adenovirus that encoded antisense FAK (AdASFAK), and CTBs transduced with the wild-type virus. After 72 hours in culture, densitometric analyses showed that transduction with AdASFAK significantly reduced ( 50%) FAK expression as compared to controls. A, bottom: The lower molecular mass region of the same blot was probed with an antibody specific for paxillin ( 70 kd) as an independent confirmation that samples containing equal amounts of protein were loaded. B: Immunostaining also showed that transduction with AdASFAK reduced FAK expression. Staining with anti-cytokeratin was used to image all of the CTBs. C: Diagram of the invasion assay. Note that the size of the cells ( 15 to 20 m) is not proportional to the depth of the Matrigel (100 m). Isolated CTBs are plated on Matrigel-coated Transwell filter membranes (I). As the cells differentiate they invade the Matrigel substrate (II), pass through the filter pores and emerge on the opposite side of the membrane (II, III). Invasion is quantified after 48 to 72 hours by counting the number of cell processes on the underside of the filter. D: CTB invasion in cultures that were not exposed to virus was considered to be 100%. Transduction of CTBs with wild-type adenovirus and AdASFAK lowered invasion by 50 and 90%, respectively. Values are means of four experiments. Scale bars, SEM. kd). Bands of equal intensity visible in all of the lanes were an independent confirmation that samples containing equal amounts of protein were loaded. Immunoblot analysis of two of the other preparations transduced with AdASFAK showed a reduction in FAK expression of 42 and 54%, respectively, relative to FAK levels in the control cells. In the fourth experiment, FAK protein was not detected after transduction with AdASFAK, whereas strong signals were observed in the control cell lysates. A portion of the CTBs from each of the four experiments described above was used for immunocytochemistry to detect FAK in individual cells. Figure 5B shows the results of the experiment in which transduction with AdASFAK reduced FAK expression by 48% as assessed by immunoblotting. Staining with anti-ck was used to image all of the CTBs. Control cells on the Matrigel substrate were well spread and extended numerous protrusions. In comparison, CTBs that were transduced with AdASFAK had a more rounded appearance. When control cells were stained with an antibody that recognizes all forms of FAK, high levels of immunoreactivity were detected throughout the cytoplasm and in association with the plasma membrane. In the AdASFAK-transduced samples, FAK staining was weak or undetectable in most CTBs. The remaining CTBs from the four preparations were used to assess the effects of reducing FAK expression on their ability to invade. The assay is diagrammed in Figure 5C. Isolated CTBs are plated on Matrigel-coated Transwell filter membranes (Figure 5C, I). As the cells differentiate, they invade the Matrigel substrate (Figure 5C, II), pass through the filter pores and emerge on the opposite side of the membrane (Figure 5C, II and III). Invasion is quantified after 48 to 72 hours by counting the number of cell processes on the underside of the filter. The data from four experiments are summarized in Figure 5D. In this analysis, the level of CTB invasion in cultures that were not exposed to virus was considered to be 100%. Transduction of the cells with wild-type adenovirus lowered invasion by 50% without affecting the Figure 4. Anti-sense FAK inhibited the migration/invasion of CTBs from anchoring villi. FAK function was assessed using explanted anchoring villi (AV) cultured on Matrigel-coated wells. In this system, villi attach to the Matrigel and CTBs migrate from cell columns (CC) to invade the substrate. A and B: An adenovirus that was engineered to encode green fluorescent protein (GFP) was used to determine which cells in the villus explant were infected. GFP was primarily expressed by CTBs in the cell columns, including at the expanded tips that consist of migrating/invading CTBs (B, arrow). As compared to the control explants that were not exposed to virus (C), explants that were infected with an adenovirus that encoded anti-sense FAK showed a consistent reduction in CTB migration/invasion from the cell columns (D, arrowhead). At the end of the experiment tissue sections were prepared from control explants that expressed GFP and experimental explants that expressed anti-sense FAK. Immunolocalization showed that cell columns from the experimental explants stained only weakly for FAK (E) as compared to cells in comparable regions of the control cultures (F).

10 102 Ilic et al Figure 6. Hypoxia suppresses generation of py397fak in column CTBs of anchoring villi. A and B: In standard culture conditions (20% oxygen), explanted anchoring villi (AV) give rise to CTB cell columns (CC). There is very little BrdU incorporation under these conditions (C). D: Cytokeratin staining of adjacent section. A portion of the border between the explant and the cell column is denoted by the dotted line. py397fak staining of the area in the pink box is shown in (E). Intense immunoreactivity was detected in association with CTBs at the ECM interface. F: Cytokeratin staining of the same cells. G and H: Hypoxia (2% oxygen) stimulates CTB mitosis. Therefore, anchoring villus explants maintained in hypoxic conditions have larger cell columns than those cultured in 20% oxygen 37 and BrdU incorporation dramatically increases (I). J: Cytokeratin staining of adjacent section. K: Staining for py397fak was greatly reduced in CTBs cultured in hypoxia as compared to 20% oxygen (compare with C). L: Cytokeratin staining of the same cells. number of cells that attached, the expression of several differentiation markers (eg, 5 1, HLA-G), or the rate of apoptosis (data not shown). Thus, transduction with adenovirus affected invasion by an unexplained mechanism, possibly by interfering with normal function of the V 3 integrin in adhesion. Nevertheless, transduction of CTBs with AdASFAK resulted in an additional four-fold reduction in the cells ability to invade, again without affecting attachment, differentiation, or apoptosis. Finally, we also assayed CTB migration on fibronectin-coated Transwell filters using methods we described previously. 36 The results showed that migration was inhibited to the same extent as invasion (data not shown). Conditions that Restricted CTB Invasion in Vitro and in Vivo also Reduced py397fak Levels Because CTBs that penetrate the uterine wall in vivo must assume an invasive phenotype, we also examined FAK expression in situations in which their invasion is impaired. First, we investigated the effects of hypoxia, an important regulator of CTB invasion, on py397fak immunoreactivity. The morphology of control explants that were cultured for 72 hours under standard conditions (20% oxygen) is illustrated in Figure 6, A and B. As we showed previously, under these conditions CTBs in column remnants exit the mitotic cycle (Figure 6, C and D) and switch on the expression of stage-specific antigens that mediate migration and invasion (data not shown). 37 Here we found that CTBs at the edges of these explants stained brightly with antibodies that recognize all forms of FAK (data not shown) and py397fak. Particularly strong immunoreactivity for py397fak was noted in association with protrusions at the leading edge of cells at the periphery of explants (Figure 6, E and F). The morphology of experimental explants that were cultured under hypoxic conditions (2% oxygen) is illustrated in Figure 6, G and H. As we showed previously, under these conditions CTBs proliferate as indicated by BrdU incorporation (Figure 6, I and J). 37 Thus, the columns are larger than in control explants (compare Figure 6, A D, with Figure 6, G J). Also in hypoxia, CTB invasion, assayed as diagrammed in Figure 5C, is impaired. 33,37,38 Here we found strong FAK staining in CTBs that were cultured in both 2% and 20% oxygen (data not shown). However, staining for py397fak was greatly reduced in CTBs cultured in hypoxia. In addition, the staining pattern changed. Much of the immunoreactivity was diffuse and cytoplasmic rather than plasma membrane-associated (compare Figure 6K to 6E). To determine whether conditions that impaired CTB invasion have an impact on levels of total tyrosine phosphorylation, we cultured CTBs for 12 and 24 hours in 20% or 2% O 2. Overall, tyrosine phosphorylation in either the cytosolic fraction (Triton soluble) or the cytoskeleton-associated fraction (Triton insoluble, RIPA insoluble) did not

11 FAK and CTB Invasion 103 Figure 7. py397fak and paxillin tyrosine phosphorylation decrease in hypoxia. A: Tyrosine phosphorylation in whole lysates of CTBs cultured at 20% O 2 and 2% O 2 for 0, 12, or 24 hours. B: Total tyrosine phosphorylation (top) and (auto)phosphorylation of FAK (bottom) in cells cultured under the same conditions. Immunoblotting with an antibody that recognizes total FAK demonstrates comparable levels of FAK in immunoprecipitates (middle). C: Tyrosine phosphorylation of paxillin decreased in CTBs cultured in 2% O 2 for 24 hours. However, similar levels of FAK were detected in paxillin immunoprecipitates regardless of oxygen concentration and levels of paxillin phosphorylation. However, either the amount or the phosphorylation of an unknown protein that associated with paxillin (asterisk) increased in hypoxia. change under the conditions tested. However, 100- to 130-kd proteins in the membrane-associated fraction (Triton insoluble, RIPA soluble) showed a decrease in tyrosine phosphorylation (Figure 7A). To determine whether this decrease included FAK (125 kd) phosphorylation, we examined levels of total tyrosine phosphorylation and (auto)phosphorylation in FAK immunoprecipitates. Interestingly, there was very little difference in overall tyrosine phosphorylation of FAK, whereas (auto) phosphorylation as shown by [ 32 P] incorporation was lower (Figure 7B). Consequently, we examined tyrosine phosphorylation of paxillin, a putative FAK substrate. Lower levels of paxillin phosphorylation were observed in conditions associated with lower levels of FAK (auto) phosphorylation. Finally, this did not change the amount of FAK that co-immunoprecipitated with paxillin. However, either the amount or the phosphorylation of an unknown protein that associated with paxillin (asterisk) increased in hypoxia (Figure 7C). We also studied the effects of hypoxia on py397fak expression by isolated CTBs. To simulate more closely conditions in vivo, the cells were co-cultured with decidua, a likely source of important paracrine signals that influence CTB invasion. In accord with our published work, 33 we found rapid invasion of CTB aggregates beneath the decidual surface in co-cultures that were maintained for 48 hours under standard tissue culture conditions (20% oxygen) (Figure 8A). ictbs in all locations showed strong staining for py397fak, primarily in association with the plasma membrane (Figure 8C). Also in accord with our published work, we found that in hypoxia (2% oxygen) the CTB aggregates, which attached to the decidua, failed to invade below the surface (Figure 8D). Staining with an antibody specific for py397fak showed only weak immunoreactivity in the cytoplasm of CTBs throughout the aggregates (Figure 8F). This difference was noted despite the fact that hypoxia did not affect the staining patterns of antibodies that recognized all forms of FAK (data not shown). Finally, we investigated whether placental hypoxia in vivo would have similar effects on CTB expression of FAK. As an example of placental hypoxia we studied tissue

12 104 Ilic et al Figure 8. Hypoxia down-regulates (auto)phosphorylation of Y397FAK in isolated CTBs co-cultured with human decidual explants. Sections of first trimester CTBs co-cultured with decidual explants in either standard conditions (20%; A C) or in hypoxia (2% oxygen; D F) were stained with hematoxylin (A and D), anti-cytokeratin (CK: B and E), or anti-py397fak (C and F). A: In 20% oxygen, CTBs deeply invaded the decidua. py397fak staining of the area in the yellow box is shown in (C). Strong staining for py397fak was detected on CTBs particularly in the area of the plasma membrane. D: In hypoxia (2% oxygen), CTB invasion of decidua was inhibited and staining for py397fak was faint and diffuse (F). samples of the fetal-maternal interface that were obtained from women with preeclampsia a pregnancy complication that is associated with shallow CTB invasion and, consequently, a reduction in blood flow to the placenta. 39,40 Control samples, matched for gestational age, were obtained from women with clinically normal pregnancies. The results are shown in Figure 9. In the control samples, staining for all forms of FAK was detected in association with the plasma membrane of CTBs found near the surface of the uterine wall (Figure 9A). A very similar staining pattern was observed in preeclampsia, except that the immunoreactivity was often more intense than in control samples (Figure 9B). In contrast, we noted dramatic differences between the two groups in the immunolocalization of py397fak. Whereas bright staining was detected in association with the plasma membranes of CTBs in the control samples (Figure 9E), little or greatly reduced immunoreactivity was detected in CTBs when the pregnancy was complicated by preeclampsia (Figure 9F). We confirmed the results of the immunolocalization experiments by examining FAK (auto)phosphorylation and py397fak expression in vitro (Figure 10). Control CTBs were isolated from the placenta of a patient who had a clinically normal pregnancy, and experimental CTBs were isolated from the placenta of a woman with preeclampsia. Then the cells were cultured under standard conditions for up to 48 hours. To assess (auto)phosphorylation, cell lysates were prepared at three time points: before plating, and after either 24 or 48 hours in culture. Then we assayed for incorporation of [ ] 32 Pin FAK immunoprecipitates (see Material and Methods). Afterward the products were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblotting with an antibody that recognized all forms of FAK showed that each of the lysates contained very similar levels of FAK protein (Figure 10A). However, as compared to control cells, FAK (auto)phosphorylation on Y397 was not detected in CTBs isolated from the placenta of a patient diagnosed with preeclampsia (Figure 10A). In four separate experiments, we also monitored the cells expression of py397fak as they invaded a Matrigel substrate, using the assay diagrammed in Figure 5C. To image the cell processes that make the initial tracks through the Matrigel, photomicrographs were taken from the underside of the filter. In control cultures, numerous cytokeratin-positive cell processes were visible. These stained brightly for py397fak (Figure 10B). When the pregnancy was complicated by preeclampsia, no cell processes had emerged on the underside of the filter by 72 hours. Instead, anti-cytokeratin staining revealed only halos of CTBs that remained on the upper, Matrigelcoated surface (Figure 10B). Similar halos of antipy397fak immunoreactivity were not observed in association with these cells. Discussion Human placental development entails a novel differentiation process in which CTB stem cells that are attached to a basement membrane in floating chorionic villi form aggregates of nonpolarized cells that subsequently lose their association with one another and invade the uterine wall (Figure 1). Our previous work showed that molecules whose expression is up-regulated coincident with uterine invasion in situ play important functional roles in culture models that replicate this process in vitro. 28,41,42 Subsequent gene deletion studies have confirmed that a number of these molecules play important roles in vivo during murine placentation. 43,44 Here we exploited the combination of approaches we have developed to study human placentation to understand the role of FAK during CTB invasion. In situ, FAK was highly expressed, primarily in a plasma membraneassociated pattern, at all stages of CTB differentiation. This observation was somewhat unexpected given our previous work that strongly suggested the importance of integrin function to the cells acquisition of a migratory and invasive phenotype. 41 This result prompted us to assess the staining pattern of an antibody that recognized only the form of the molecule that is phosphorylated on tyrosine 397, py397fak, the primary (auto)phosphorylation site that initiates further FAK activation. Although vctbs and STBs showed little or no reactivity with this

13 FAK and CTB Invasion 105 Figure 9. Preeclampsia is associated with reduced staining for py397fak. A H: Frozen sections of biopsies from the fetal-maternal interface were stained with anti-ck (C, D, G, and H) and either anti-fak (A and B) or anti-py397fak (E and F). A: At week 35 of normal pregnancy, control CTBs stained for FAK. B: In preeclampsia, CTBs of the same gestational age also stained for FAK, usually with greater intensity than in control samples. E: Immunostaining of the control sample with anti-py397fak revealed groups of CTBs within the uterine wall that stained strongly, primarily at the cell surface. F: In comparison, CTB staining for py397fak was very weak when the pregnancy was complicated by preeclampsia.

14 106 Ilic et al Figure 10. In preeclampsia, reduced FAK (auto)phosphorylation and staining correlated with impaired invasion. To assess (auto)phosphorylation, lysates of purified CTBs were prepared at the time points indicated. [ - 32 P] incorporation into FAK was assayed as described in Materials and Methods. A, top: Immunoblotting with an antibody that recognized all forms of FAK showed that each of the lysates contained very similar levels of FAK protein. A, bottom: As compared to control cells, FAK (auto)phosphorylation on Y397, determined by [ - 32 P] incorporation, was significantly reduced in CTBs isolated from the placenta of a patient with preeclampsia. CTBs isolated from the same placentas as those used in the FAK (auto)phosphorylation kinase assay were cultured on Matrigel-coated Transwell inserts, fixed, and stained with anti-cytokeratin (CK) and anti-py397fak. B, top: CTBs from the control placenta invaded extensively, as shown by the CK-positive cell processes on the underside of the filter. The tips of these processes stained brightly for py397fak. B, bottom: In preeclampsia CTB invasion was impaired, as shown by the absence of CK-positive processes on the underside of the filter (arrows). A bright halo of out-of-focus CK fluorescence (see lined markings) revealed the outlines of CTBs that remained on the upper Matrigel-coated surface. No reactivity with anti-py397fak was observed. antibody, strong staining was observed in association with a subset of CTBs near the surface of the uterine wall. In vitro function-perturbation experiments showed that reducing FAK expression diminished CTB invasion. The inverse correlation also held: situations in which CTB invasion was down-regulated, such as hypoxia in vitro and preeclampsia in vivo, were associated with a dramatic reduction of py397fak levels in the cells especially in membrane-associated areas. Together our results suggest that (auto)phosphorylation of Y397 on FAK, rather than absolute FAK levels, is a critical component of the signaling pathway that mediates migration/invasion. Our findings have interesting implications that are particularly relevant to current theories regarding tumor cell invasion, a prerequisite for metastatic spread, the most common cause of morbidity and mortality in various neoplastic diseases. 45 Invasion, the movement of cells beyond their normally circumscribed boundaries, is a complex process that requires coordination of many extracellular signals, including those generated by soluble factors, as well as contact with the ECM and other cells. Likewise, intracellular signals, such as those that lead to assembly and disassembly of the actin cytoskeleton, are also crucial. 46 The strongest evidence that FAK is a key point of convergence of both ECM- and growth factor-regulated signals that influence cell migration comes from studies of FAK-null mouse embryos. Specifically, the cells without FAK exhibit a reduced migration rate because of impaired focal adhesion turnover. 17,18,22 Conversely, overexpression of FAK leads to an increase in cell migration. 19 In light of the results of both types of experiments, it is not surprising that a number of groups report that FAK expression is up-regulated in cancer cells as compared to their normal counterparts. 24,47 51 Our results strongly suggest that (auto)phosphorylation of Y397FAK could be a critical determinant of the metastatic potential of tumor cells. This is supported by our analysis of a rare malignant ictb tumor (placental site tumor) that we fixed according to the protocol that allowed immunolocalization of FAK and py397fak (Ilić and Genbačev, unpublished data). Recent work from three laboratories shows that FAK also plays a role in cell proliferation, suggesting additional tie-ins to other cellular processes that are critical to tumor progression Prompted by these findings, we studied the relationship of FAK expression to migration versus proliferation in several cell lines at the population level. Immunoblotting showed that the cells, including the JAR choriocarcinoma (malignant vctb) cell line, with the highest invasive potential and lowest proliferative rate had relatively lower levels of FAK in toto, but higher levels of py397fak. In comparison, other lines, including JEG (malignant vctb) cells, with the lowest invasive potential and highest proliferation rate had higher levels of total FAK but less FAK (auto)phosphorylated on Y397 (Ilić and Genbačev, in preparation). In the tissue sections and culture models analyzed here, we failed to detect immunostaining for py397fak in proliferating cells villus and column CTBs in situ and CTBs in explanted anchoring villi cultured under hypoxic conditions in vitro. Instead, expression was detected in migrating CTBs that had withdrawn from the cell cycle. 2 Together, our results suggest that it is possible to separate FAK signals that lead to proliferation from those that promote migration/invasion. In conclusion, our studies of human placental CTB invasion both in situ and in vitro suggest that regulation of FAK (auto)phosphorylation, rather than FAK levels, is