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ERK^MAPK Activity as a Determinant of Tumor Growth and Dormancy; Regulation by p38^SAPK1

Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029

	ABSTRACT

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After dissemination from a primary tumor, cancer cells may resume growth, leading to overt metastasis, or enter a state of protracted dormancy. However, mechanisms that determine their fate, or markers that predict it, are mostly unavailable. We previously showed that in HEp3 human head and neck carcinoma, the extracellular signal-regulated kinase (ERK)^MAPK/p38^SAPK activity ratio predicts whether the cells will proliferate or enter a state of dormancy in vivo. The proproliferative balance of high ERK/p38 ratio was induced by high urokinase (uPA) receptor (uPAR) expression, which activated 5ß1-integrin and epidermal growth factor receptor. This signaling pathway was additionally enhanced by uPA binding to uPAR and fibronectin binding to 5ß1-integrin. We tested whether the ERK/p38 balance is predictive of in vivo behavior in other cancer cell types and whether altering the balance will shift their phenotype between proliferation and dormancy. ERK and p38 activities were determined using either phospho-specific monoclonal antibodies or a trans-reporting system where GAL4-Elk and GAL4-CHOP trans-activation of luciferase gene served as reporters for ERK and p38 activities, respectively. We show that in breast, prostate, melanoma, and fibrosarcoma cell lines, the level of active phospho-ERK and the ERK/p38 activity ratio predict for the in vivo behavior in 90% of the cell lines tested. Modulation of ERK/p38 activity ratio by multiple pharmacological and genetic interventions confirms that high ERK/p38 ratio favors tumor growth, whereas high p38/ERK ratio induces tumor growth arrest (dormancy) in vivo and that ERK is negatively regulated by p38. A melanoma cell line appeared to have developed an escape mechanism to avoid the growth inhibitory effect of high p38 activity. Mechanistic analysis implicated high uPAR expression and its interaction with and activation of 5ß1-integrin as determinants of the in vivo growth promoting high ERK/p38 ratio in several cell lines. The small GTPase, Cdc42, was implicated in activation of p38 and growth arrest. These results suggest that even cells that originate in advanced cancers retain a degree of dependence on surface receptors and matrix for their proliferative signals in vivo and provide a therapeutic opportunity to change their phenotype from tumorigenic to dormant.

	INTRODUCTION

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Tumor latency, best defined as the time between the carcinogenic event and the onset of progressive growth, culminating in a clinically detectable tumor mass, has been documented in experimental cancer models and in people exposed to catastrophic radiation (1) . It is assumed that during this period, cells undergo genetic and epigenetic changes, and those with growth advantage are selected giving rise to progressively growing tumors (2) . A seemingly parallel phenomenon exists in cancer patients who, although cured of their primary disease, show no signs of metastasis but develop tumors in secondary sites, often after prolonged periods of dormancy. Although it is easy to envision that progression from a normal to a fully malignant cell may take time, the inability of a fully malignant cell such as a cell that separates from a growing primary cancer to resume growth upon arrival in a new organ is more difficult to understand. However, the fact that disseminated but undetected cancer cells eventually progress to overt metastases indicates that solitary cell, or small groups of cells, can survive away from the primary until, through as yet unknown mechanisms, they resume growth. Because of their scarcity and the absence of detection methods, dormant cells are difficult to isolate, and the mechanisms that maintain dormancy and regulate the transition from dormancy to progressive growth remain largely unknown. It is possible that metastases can grow to a clinically detectable size, only after they acquire the ability to induce new blood vessel (neoangiogenesis; Ref. 3 ), but other mechanisms must also exist (4) . It has been shown that large proportion of cancer cells in distant organs remains as single dormant cells (5, 6, 7) . Because these conclusions were drawn from experiments in which cells were injected directly into the circulation, thus bypassing the selection process that takes place during cancer progression, it remains to be seen if they will apply to induction of dormancy in spontaneous metastasis.

We propose that even cancer cells that may have accumulated multiple mutations may use the surface receptors and ECM³ components to regulate signaling pathways that control cell cycle progression and/or arrest. This implies that a shift between growth and dormancy may be anticipated upon cancer cell arrival in a new organ, either because of changed ECM or changed expression or function of their surface receptors.

In progressively growing tumors, constitutive activation of the ERK pathway allows for G₀-G₁-S-phase transition and cell division (8) . Although ERK is mostly involved in induction of proliferation (9) and in some cases differentiation (10) , a high level of p38 activity is believed to be a negative growth regulator (11) that, depending on the stimulating signal, may suppress cell proliferation by inhibiting ERK (12) , inducing G₀-G₁ arrest (13) or triggering senescence (14) or apoptosis (15) . There is evidence that activation of p38 can reverse Ras-induced fibroblast transformation (16) and that in epithelial cells, inhibition of p38 by Ras is required for transformation (17) . Moreover, published evidence shows that p53 phosphorylation by p38 is required for its tumor suppressing activity and that PPM1D, a specific p38 phosphatase, by dephosphorylating p38 and inhibiting its activity blocks the tumor suppressor function of p53 (18) . Thus, it seems that many effectors can alter the balance of ERK and p38 and that such change may have profound consequence for tumor growth and survival.

We previously found that a balance that favors p38 activation over ERK in highly malignant human carcinoma cells (HEp3) can induce persistent growth suppression (dormancy) in vivo (12) . Specifically, we found that the rapid growth of metastatic HEp3 carcinoma in vivo is regulated by high expression of uPAR that, by interacting and activating 5ß1-integrins, initiates a signaling cascade that culminates in very strong and persistent ERK activation (19) . This activation, which is mediated through ligand-independent activation of EGFR (20) , is additionally enhanced by uPA binding to uPAR and cell binding to FN (19 , 21) . Consequently, uPAR overexpression and activation of ERK generates a positive feedback loop that maintains uPAR mRNA and high protein levels, keeping the signaling cascade for cell cycle progression on (12) . Upon down-regulation of uPAR, ERK activity is lost, p38 becomes activated, and the balance is shifted in favor of p38 (12) . Increased p38 activity has been previously shown to inhibit activation of ERK (22 , 23) and to block cell proliferation in culture (14) . We found, however, that HEp3 cells with low ERK/p38 activity balance, while fully capable of proliferation in culture, when inoculated in vivo on the chick CAM, or s.c. in nude mice, rapidly arrest in G₀-G₁ and remain viable but dormant for a prolonged period of time (12 , 19 , 24) . This paradigm provides one of the first examples whereby down-regulation of a surface protease receptor, by altering the interaction between a cancer cell and the extracellular matrix, induces in vivo dormancy of an otherwise fully malignant cell. This additionally suggests that some cancer cells may not be fully growth autonomous and that, in the metastatic sites, either the necessary growth inducing stimuli may be absent or the solitary cells may transiently lack components necessary for the interpretation of the extracellular cues, giving rise to dormant metastases. We showed that in HEp3 cells, it was possible, by disrupting the expression or the mutual interactions between uPA, uPAR, 5ß1, FAK, EGFR, or FN proteins that constitute the ERK activating complex, to favor p38 activity and dormancy (12 , 19 , 21) . Here, we examine the generality of the above paradigm by characterizing the ERK/p38 ratios in a series of tumor cell lines representing cancers of different origin in the context of their in vivo behavior. Our results indicate that the majority of the cell lines studied conform to the paradigm established for HEp3 cells in that their ERK activity or ERK/p38 activity ratio is predictive of their in vivo behavior and that uPAR and 5ß1-integrin play a major role in that behavior.

	MATERIALS AND METHODS

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Reagents and Antibodies.
DMSO, Triton X-100, sodium orthovanadate, NaFl, protease inhibitors, DNase I, BSA, normal goat serum, collagenase type 1A, rhodamine-phalloidin conjugate, and human fibronectin were from Sigma Chemical Co. (St. Louis, MO). Aprotinin and trypsin were from ICN Biomedicals, Inc. (Aurora, OH). DMEM, OPTI-MEM medium, glutamine, antibiotics, and Lipofectin were from Life Technologies, Inc., Laboratories (Grand Island, NY). Fetal bovine serum was from JRH Biosciences (Lenexa, KS), COFAL-negative embryonated eggs were from Specific Pathogen-Free Avian Supply (North Franklin, CT); protein G-agarose beads were from Boehringer Mannheim (Indianapolis, IN). Polyvinylidene difluoride membranes and ECL detection reagents were from Amersham (Amersham Life Sciences, Little Chalfont, United Kingdom). PD98059, SB203580, and its inactive analogue SB202474 were from Calbiochem (San Diego, CA). The stock solutions were prepared in 100% DMSO. DAPI was from Hoechst Molecular Probes (Eugene, OR). Antibodies: antiphospho ERK 1/2 (antiphospho-Tyr 204; clone E4) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-ERK1/2 (Clone MK12) and anti-p38 (Clone 24) mAbs were from Transduction Laboratories (Lexington, KY). Antiphospho-p38 polyclonal antibodies were from Biosource (Camarillo, CA). Normal mouse IgG, fluorochrome-labeled secondary antibodies, and antihuman fibronectin polyclonal antibody (F3648) were from Sigma. Goat antimouse, Alexa-546-conjugated IgG, was from Molecular Probes (Eugene, OR). Antihuman uPAR mAb R2 was kindly provided by Dr. Michael Ploug (Finsen Laboratory, Copenhagen, Denmark). Anti-uPAR polyclonal rabbit antibody (399R) was from American Diagnostica, and polyclonal rabbit anti-ß1 antibody (Mab1952) and anti-5ß1 antibody (Clone HA5) were from Chemicon International, Inc. (Temecula, CA). Antimouse IgG HRP-conjugated mAb and mounting medium (Vectashield) were from Vector Laboratories (Burlingame, CA). Antirabbit IgG-HRP and anti-HA antibody (clone 12CA5) were from Boehringer Mannheim (Indianapolis, IN). All antibodies used in vivo or in culture were free of azide. The endotoxin content of antibodies used in culture or in vivo was tested using Pyrogen-Plus test from Biowhittaker (Walkersville, MD) and found to have <24 pg/ml.

Cell Lines, Stable cDNA Transfections, and Cell Culture Conditions.
Human epidermoid carcinoma HEp3 (T-HEp3), serially passaged on CAMs of chick embryos, was used as a source of tumorigenic cells (19 , 25) . The source of spontaneous dormant tumor cells (D-HEp3) were HEp3 cells passaged in culture 120–170 times (24) with uPAR level of only 20% of that in tumorigenic cells (19) . PC3, MDA-MB-231, MDA-MB-453, MDA-MB-468, MCF-7, and HT1080 cells were obtained from American Type Culture Collection. M24met cells were kindly provided by Dr. Barbara Muller from Scripps Institute (La Jolla, CA). T-HEp3, D-HEp3, PC3, MCF-7, MDA-MB-453, MDA-MB-468, and HT1080 cells were cultured in DMEM and MDA-MB-231, and M24met cells in RMPI with 10% heat-inactivated fetal bovine serum, penicillin (500 units/ml), and streptomycin (200 µg/ml). PC3 cells were transfected with an empty vector (pSV-Neo) or with a construct encoding an HA-tagged active mutant of Mek1, HA-R4F-Mek, kindly provided by Dr. Natalie Ahn (University of Colorado, Boulder, CO). G418-resistant clones were pooled to avoid clonal variation, and R4F-Mek expression was determined by Western blot. PC3-Neo and PC3-R4F-Mek cells were routinely cultured in the presence of 400 µg/ml G418. HT1080 cells were stably transfected with an MKK6b(E) active mutant in pCDNA3.1 (kindly provided by Dr. Jihuai Han (Scripps Institute) or with pCDNA3.1 only, and G418-resistant clones were pooled to avoid clonal variation, and MKK6b(E) expression was determined by Western blot.

Growth of Tumor Cells on CAMs.
Cells were detached with 2 mM EDTA in PBS, washed, and inoculated on the CAMs of 9–10-day-old chick embryos. At different times, postinoculation CAMs were excised, enzymatically dissociated, and single cell suspensions counted (19) . In addition, PC3 or MDA-MB-231 cells treated with 2 µM SB203580, PC3-Neo or PC3-R4F-Mek cells, or HT1080-vector, or HT1080-MKK6b(E) cells were also grown on the CAMs as indicated above. In addition M24met or HT1080 cells were pretreated in suspension at 37°C for 20 min, with 10 µg/ml of anti-uPAR antibody (R2) or left untreated, washed, and inoculated onto 10-day-old CAMs. After 2–4 days, tumor growth was evaluated as indicated above. Alternatively, for continuous monitoring of tumor growth, after measuring the nodules size, the tissue was minced and passaged onto new 9–10-day-old CAMs (25) .

IF Microscopy.
For IF analysis, cells grown on coverslips were fixed in 3% paraformaldehyde in PBS for 15 min. The coverslips were washed and either permeabilized with 0.1% Triton X-100 or left nonpermeabilized, washed, blocked with 3% normal goat serum in PBS (15 min), and incubated for 1 h at room temperature with antifibronectin (F3648, 1:400) antibodies in 0.1% BSA/PBS or with vehicle alone. After washing and blocking, the secondary antibodies in 0.1% BSA/PBS containing rhodamine-phalloidin conjugate (1:70), or DAPI were added. Coverslips were mounted in Vectashield and kept at -20°C. Standard epifluorescence was captured with a Nikon E-600 epifluorescence photomicroscope (Toyko, Japan) using Plan-Neofluar x40 and x100 (N.A. 1.5 Oil) lenses (Nikon) or Plan-Apochromat lens x63 and x100 (Nikon) through a Diagnostic Instruments, Inc. SPOT-RT digital camera using SPOT and Adobe Photoshop 6.0 software on a Macintosh G4 computer.

Detection of uPAR and Integrin Expression.
For uPAR, detection cell lysates of the different cell lines were prepared as previously described (19) , centrifuged, and equal amounts (50 µg) of the supernatant proteins were used in Western blots with R2 anti uPAR mAb. FACS analysis was performed as described previously (12) . Antibodies (HA5, anti-5ß1, or isotype-matched IgG) were added to 5 x 10⁵ cells at 10 µg/ml and incubated at 4°C for 30 min, followed by two washes and FITC-conjugated goat antimouse (1:1000 IgG) incubation. Cells were fixed in 5% formaldehyde in PBS and analyzed in FACS-SCAN equipped with laser 488 (Becton Dickinson, San Jose, CA). Data analysis was performed using Cell Quest software and a Macintosh G3 computer.

FN-Fibril Formation and Effect of Treatments.
Cells grown on glass coverslips in DMEM with 10% FBS or 5–10% FN-depleted FBS, with or without 5–10 µg/ml human serum FN were fixed and stained for FN, F-actin, or DAPI, as indicated above. To test the effect of disruption of uPAR/integrin interaction on FN-fibril formation, cells were plated on coverslips in 5% FN-depleted FBS/DMEM for 1 h and treated with anti-uPAR antibody to domain III (R2, 10–20 µg/ml) isotype-matched IgG (15 µg/ml) antibody or peptide 3–25 that inhibits uPAR/integrin interactions (26) for 20 min at 37°C, incubated overnight with 5–10 µg/ml human FN, fixed, and stained for FN as indicated above. FN-fibrils were counted on 200–350 cells/treatment in triplicate experiments and expressed as the percentage of DAPI-positive nuclei.

Detection of ERK^MAPK and p38 ^SAPK Activities.
To monitor ERK and p38 activity levels, we used a reporter system (Pathdetect, Stratagene) based on the use of fusion proteins that comprise GAL4 DNA binding domain fused to the activation domain of specific transcription factors that, in turn, drive the expression of luciferase reporter gene. An expression plasmid encoding a chimeric protein GAL4-Elk1 (pFA-Elk, Stratagene; ERK phosphorylation target) or expression plasmids coding for GAL4-CHOP (pFA-CHOP, Stratagene) or GAL4-MEF2A (p38 phosphorylation targets) (in pCDNA3.1 kindly provided by Dr. Jihuai Han (Scripps Institute; were cotransfected with a Photynus pyralis luciferase reporter gene controlled by 5 GAL4-binding sites linked 5' to the herpes virus thymidine kinase (tk) promoter (pD700, kindly provided by Dr. Ari Melnick, Albert Einstein College of Medicine, New York, NY). All transient transfections were performed using Fugene reagent (3:1 Fugene/DNA ratio) and 1:5 ratio of the trans-activator and reporter plasmids. In some experiments, additional plasmids encoding a dominant negative mutant of p38 (12) , an active mutant of MKK6 [MKK6b(E)], active or inactive mutants of Cdc42 (Cdc42QL and Cdc42N17, kindly provided by Dr. Silvio Gutkind, NIH, Bethesda, MD) were cotransfected. In some experiments, after overnight transfection with the reporter system cells were treated for 12–24 h with 5–10 µM p38 inhibitor SB203580 and/or 50 µM arsenic trioxide as a stress inducer or with 25–50 µM PD98059 Mek inhibitor. In all transfections, a plasmid-encoding Renilla luciferase (Clontech) was cotransfected (100–500 ng), and firefly luciferase activity was normalized to Renilla luciferase activity. After 48–72 h after transfection, cells were lysed using the passive lysis buffer from Promega, and luciferase activity was detected with a luminometer using the Dual Luciferase Reporter Kit from Promega following the vendor’s instructions. Controls lacking the transactivator constructs (Elk-, CHOP-, or MEF2A-GAL4) but including the GAL4-tk-luciferase construct alone, or in combination with the SV40-renilla luciferase construct or untransfected cells, were included in every experiment and showed no or very low luciferase activity that was not affected by treatments. In addition, cell lysates of subconfluent monolayers of the different cell lines, prepared as previously described (19) , were centrifuged, and equal amounts (20–50 µg) of the supernatant proteins were used in Western blots to detect either active or total ERK and p38 levels using phospho-ERK (p42/p44) or ERK1 and phospho-p38 or p38 antibodies. The effect of p38-inhibition on ERK activation was tested by treating the cells with 1–10 µM SB203580 or its inactive analogue SB202474 or 0.05% DMSO for 5–20 min or 5–48 h in serum-free DMEM. The phospho-ERK and ERK levels were analyzed by Western blotting. In some experiments, cells pretreated with medium alone, control IgG (10 µg/ml), or R2 antibody (10 µg/ml) were plated on poly-L-lysine (5 µg/ml) or fibronectin (5 µg/ml), and after 20 min, the cells were lysed and assayed for ERK activation by Western blot. In all cases after SDS-PAGE and transfer, the membranes were blotted with the corresponding antibodies and the signal was developed using ECL.

Detection of Activated Rac and Cdc42 by PAK1-PBD-GST Pull-Down Assay.
Active (GTP-loaded) Rac or Cdc42 was detected using a Rac/Cdc42 activation kit from Upstate Biotechnology following the vendor’s instructions. Briefly, after removing the medium, cell monolayers were lysed directly with a 2% glycerol, 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, and 1 mM EDTA containing lysis buffer with protease and phosphatase inhibitors and snap frozen in liquid N₂. The lysates were cleared by centrifugation, and the supernatants (0.6–1.0 mg of protein) were incubated for 60 min with Sepharose beads conjugated with a GST-p21 binding domain of PAK-1 (10 µl of beads/10 µg of PAK1-PBD) fusion protein, which binds to GTP, but not GDP, loaded Rac or Cdc42. After incubation, the beads were spun down and washed twice with lysis buffer, resuspended in 2x Laemmli sample buffer with 100 mM ß-mercaptoethanol. The Rac or Cdc42 proteins in the precipitates or cell lysates were detected by Western blot using anti-Rac (clone 102) or anti-Cdc42 (clone 44) mAbs from BD PharMingen (San Diego, CA) as indicated above.

Surface Labeling with Sulfo-NHS-Biotin.
Subconfluent monolayers were washed three times with cold PBS, the cells were incubated on ice for 20 min with 5 ml of 0.5 mg/ml sulfo-NHS-biotin (Pierce, Rockford, IL), and the reaction was stopped by aspirating and washing the cells twice with 10 ml of ice-cold PBS. The cells were then scraped in 1 ml of PBS containing protease inhibitors, spun at 4°C, and the pellets were lysed with a buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and protease inhibitors as described for radioimmunoprecipitation assay buffer. Immunoprecipitation and biotinylated proteins detection were performed as indicated below.

Coimmunoprecipitation of 5ß1-Integrin and uPAR.
Untreated M24met and T-HEp3 cells or surface-biotinylated T-HEp3 and MDA-MB-231 cells were lysed and extracted for 1 h with a lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM orthovanadate, 1 mM NaFl, and protease inhibitors. Triton X-100 soluble fraction or insoluble fractions (400 µg protein) extracted for 30 min with radioimmunoprecipitation assay lysis buffer, after preclearing with protein-G beads and isotype-matched IgG for 30 min, were incubated with 4 µg of anti-5ß1 (HA5), R2 mAb, or isotype-matched IgG overnight at 4°C, precipitated with protein G-agarose beads and washed twice. The beads were resuspended in 2x Laemmli sample buffer, heated to 95°C for 10 min, and analyzed by Western blotting using anti-5ß1 integrin polyclonal antibodies or anti-uPAR 399R polyclonal antibodies. Alternatively, immunoprecipitates of surface-biotinylated proteins were separated on SDS-PAGE, transferred to a membrane, blotted with streptavidin-HRP, washed, and the signal developed with ECL reagent.

	RESULTS

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We previously showed that the functional assembly of a multimeric complex formed by uPA, uPAR, and 5ß1and EGFR and the formation of fibrillar FN on the surface of squamous carcinoma HEp3 cells are required to induce and maintain high ERK/p38 activity ratio. The high ERK/p38 ratio allows cell cycle progression and tumor growth. Disruption of this complex caused an inversion in the ERK/p38 ratio toward p38 signaling, a change that results in cell cycle arrest and dormancy in vivo (12 , 19) . Here, we set out to explore the generality of this paradigm by considering the role of ERK/p38 activity ratio as a predictor of in vivo behavior. We also tested whether direct targeting of ERK or p38 activities can shift the cancer cell phenotype between the states of dormancy and tumorigenicity.

Tumor Growth and Expression Levels of uPAR and 5ß1-Integrin.
Human tumor cell lines representative of head and neck carcinoma (T-HEp3, tumorigenic, and D-HEp3 dormant), fibrosarcoma (HT1080), metastatic melanoma (M24met), prostate carcinoma (PC3), and breast carcinoma (MCF-7, MDA-MB-231, MDA-MB-453, and MDA-MB-468) reported to have varying malignant potential (27) were examined for their ability to form tumors on the CAM of chick embryo. The cells were inoculated onto 9–10-day-old CAMs (5 x 10⁵ cell/CAM), and their growth was monitored by weekly passage of similar amounts of tumor minces onto fresh CAMs for up to 8 weeks. Fig. 1A shows that M24met and HT1080 cells formed rapidly growing, large tumors, a behavior similar to that of T-HEp3 cells, in which tumorigenicity was shown to be uPAR and integrin dependent (12 , 19) . In contrast, PC3 cells and three breast cancer cell lines formed small tumor nodules, which, similarly to D-HEp3 cells (19 , 24) , did not increase in size for up to 8 weeks. An additional breast cancer cell line, MCF-7, and a prostate cancer cell line, LNCaP, behaved like the D-HEp3 cells (data not shown), even if the inoculum size was increased 4-fold (28) . Microscopic examination of trypan blue stained minces of the small CAM nodules showed that even after 8 weeks in vivo, they contained live cancer cells. We previously showed (19 , 25) that the in vivo dormancy of D-HEp3 cells results from reduced proliferation and not increased apoptosis. To distinguish between these two potential mechanisms of dormancy induction, sections of growing (T-HEp3) or dormant (MDA-MB-231, D-HEp3, and PC3) tumors were examined for apoptosis using TUNEL assay. These experiments revealed an apoptosis level of <5% that did not differ between the growing and the dormant tumors (data not shown), suggesting that as previously described (19 , 25) , dormancy most likely is the result of a failure to activate the proliferative signal.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. In vivo behavior (growth or dormancy) of cancer cell lines and their uPAR and 5ß1-integrin expression. A, cell lines [T-HEp3, tumorigenic head and neck carcinoma HEp3; HT1080, fibrosarcoma; M24met, melanoma, (group a) and MDA-MB-453, MDA-MB-468, and MDA-MB-231, breast carcinomas; PC3, prostate carcinoma; D-HEp3, dormant HEp3, (group b)] were inoculated on CAMs (T-HEp3 at 2 x 105, the rest of the cell lines at 5 x 105/CAM), and their growth was monitored by weekly measurements of the tumor diameters after which the tumors were excised, minced, and reinoculated onto new CAMs. Tumor volumes were calculated using the formula (Dxd2)/2, where D is the longest, and d is the shorter diameter. (Breast carcinoma MCF-7 and prostate carcinoma LNCaP behaved like the dormant, group b cell lines, results not shown). Increasing the inoculum of dormant cells to 2 x 106cell/CAM did not change the outcome (results not shown). B, uPAR in 50 µg of cell proteins detected by Western blot using anti-uPAR antibody, R2. C, 5ß1 integrin surface expression detected by FACS analysis using anti-5ß1 integrin antibodies (clone HA5, 10 µg/ml, open area) and isotype matched IgG (10 µg/ml, shaded area). The signal was developed using FITC-conjugated goat antimouse antibodies.

Is High uPAR Level Always Predictive of 5ß1-Integrin Activation, Increased ERK/p38 Activity Ratio, and Growth in Vivo?
We previously showed that when expressed at high levels, uPAR interacts with and activates 5ß1-integrin. This results in the integrin being able to organize FN into insoluble fibrils, suppression of p38 activity, tipping of the balance in favor of ERK activity, and tumor growth. Reduction of uPAR level reverses the balance in favor of p38 (12) . Should the paradigm be applicable to other cell lines, the prediction would be that HT1080, M24met, and MDA-MB-231 will form FN-fibrils and will have high ERK/p38 ratios. The prediction for cell lines with low or undetectable levels of uPAR and 5ß1 integrin (Fig. 1, B and C) would be that they will form no FN-fibrils and will have a ratio of ERK/p38 activities favoring p38. Examination of FN-fibrils by IF using anti-FN antibody revealed 100% conformity between FN-fibril presence and in vivo behavior (Fig. 2A and Table 1 ). Again, the MDA-MB-231cells, despite a robust uPAR and 5ß1-integrin expression, did not form FN-fibrils (Fig. 2A) , a finding that conformed to their dormancy on the CAM (Fig. 1A and Ref. 28 ).

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Surface detection of FN fibrils and uPAR-5ß1 complex. A, detection of FN fibrils by IF. Tumorigenic (T-HEp3, HT-1080, M24met) and dormant MDA-MB-231 cells grown in medium supplemented with 5 µg/ml human FN for 16 h were fixed and stained with antihuman FN antibody (see "Materials and Methods"). The arrows indicate FN-fibrils. The majority of fibrils were apical with some basolateral and many at cell-cell contacts. B, detection of the surface uPAR-5ß1 complex by surface biotinylation and immunoprecipitation. Surface-biotinylated cells were lysed and immunoprecipitated with anti-5ß1 (HA5), anti-uPAR (R2), or with isotype-matched IgG antibodies and analyzed by streptavidin-HRP binding to biotinylated proteins after SDS-PAGE and transfer to polyvinylidene difluoride membranes (bottom panel). The top part of the same blot was reacted with anti-5 integrin antibodies (top panel). uPAR runs as a smear because of its high degree of glycosylation. The three strong bands in MDA-MB-231 cells are nonspecific. C, detection of the surface uPAR in T-HEp3 and MDA-MB-231 cells. Surface biotinylation and lysis of T-HEp3 and MDA-MB-231 cells was performed as in B. Anti-uPAR R2 mAb or irrelevant IgG were used in the immunoprecipitate, and the signal was developed using streptavidin-HRP and ECL. The abbreviations: IP, immunoprecipitation; WB, Western blot. MW markers are indicated to the right of the streptavidin-HRP blot.

The effect of uPAR-induced activation of integrins, or lack thereof, on the level of ERK activation was tested by Western blot analysis of cell line lysates using antibodies that recognize phosphorylated (active) and total ERK1/2 proteins (Fig. 3A) . To compensate for the differences in total ERK level between cell lines, ERK activity was determined by scanning the bands and calculating the ratios of phospho-ERK to ERK in each cell line. The ratios were expressed as a fraction of that in T-HEp3 cells, which was arbitrarily set as 1. ERK phosphorylation, which varied widely among cell lines (Fig. 3A) , provided an opportunity for testing their link to integrin activation and in vivo growth. The tumorigenic HT1080 and M24met cells, both with high uPAR and active integrins (as determined by their ability to form FN-fibrils; Fig. 2A and Table 1 ), had activity similar to that of T-HEp3 cells (0.63 and 1.04, respectively; Fig. 3A ). We previously showed that in T-HEp3 cells, the uPAR/5ß1-integrin complex activated EGFR in a ligand-independent fashion and that EGFR served as a mediator of the uPAR-induced signal to ERK (20) . Although, we did not specifically address this question here, it is likely that EGFR serves similar function in the HT1080 and M24met because both express EGFR (data not shown and Refs. 29, 30, 31 ). The phosphorylated ERK in the dormant cell lines, PC3, MCF-7, MDA-MB-453, and MDA-MB-468 ranged between 0.03 and 0.33 and was similar or lower than that of the dormant D-HEp3 cells, which was 0.27. Phosphorylated ERK in MDA-MB-231 cells was 0.49 (Fig. 3A) , a level that apparently was not sufficient to facilitate their in vivo growth. This fits our findings in HEp3 cells showing that a 2-fold reduction in ERK activity by experimental manipulation inhibits tumor cells growth in vivo (see below and Refs. 12 , 19 , 20 ). Overall, high basal ERK activity measured in vitro appears to be a good predictor of in vivo growth.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Analysis of MAPK, ERK and p38 activity and their down-stream targets. A, activation of ERK. Cells serum-starved for 24 h were lysed, and 50 µg of cell protein were analyzed by Western blotting for phospho-ERK (top panels) and for ERK1/2 (bottom panels). The bands were scanned by densitometry, the ratios of phopsho-ERK to ERK calculated, and the values expressed as a fraction of the ratio calculated for T-HEp3 cells which was arbitrarily set as 1. B, activation of p38. Activity of p38 and calculation of ratios was as in A, except that antiphospho-p38 and anti-p38 antibodies were used and the value for D-HEp3 cells was arbitrarily set as 1. C, activation of CHOP, a downstream target of p38. MCF-7 cells growing in medium with serum were transiently transfected with CHOP-GAL4/luciferase reporter plasmids (see "Materials and Methods") and either left untreated (control) or were treated with 5 µM of the p38 inhibitor (SB203580) or 50 µM arsenic trioxide (a strong inducer of p38 activity) or with a combination of the two; T-HEp3 cells were left untreated (control) or were treated with arsenic trioxide (50 µM) overnight; D-HEp3 cells were transfected with an empty vector or with a plasmid coding for an active mutant of MKK6, MKK6b(E). After overnight treatment or 48 h after transfection, CHOP-induced luciferase activity was determined and normalized to renilla luciferase activity as indicated in "Materials and Methods." D, activation of Elk, a downstream target of phospho-ERK, T-HEp3, D-HEp3, and MCF-7 cells were transfected with Elk-GAL4/luciferase reporter plasmids. Cells were either left untreated or were treated with a Mek1/2 inhibitor, PD98059 (50 µM). After overnight incubation, the cells were lysed, and luciferase activity was measured and normalized to renilla luciferase activity as indicated in "Materials and Methods." E, cell lines were transfected with the Elk-GAL4 or CHOP-GAL4 trans-reporting system, the Elk (ERK) and CHOP (p38) activities were determined as indicated in "Materials and Methods," and the Elk to CHOP activity ratios were calculated and plotted.

To show that the level of phosphorylated ERK or p38, measured by Western blots in lysates of cells, correspond to their in vivo activity, we measured their ability to phosphorylate and, thus activate, their downstream targets. Cells to be tested were transiently transfected with plasmids coding for GAL4-Elk1 (target of phosphorylation by ERK) or GAL4-CHOP/GADD153 [target of phosphorylation by p38 (32) ] fusion proteins. Controls lacking the transactivator constructs (Elk-, CHOP-, or MEF2A-GAL4) but including the GAL4-tk-luciferase construct alone or in combination with the SV40-renilla luciferase construct or untransfected cells were included in every experiment. The activation of ERK or p38 was measured by transactivation of a luciferase reporter. To establish the parameters of sensitivity and responsiveness of p38, the CHOP-dependent trans-activation of luciferase was examined in several cell lines under basal conditions, after treatment with 50 µM arsenic trioxide [a known inducer of p38 activity (11) ] with and without treatment with a specific inhibitor of p38 and ß-isoforms, SB203580 (5 µM), and after transfection with constitutively active MKK6b(E) (the immediate specific upstream activator of p38; Fig. 3C ). In MCF-7 cells, arsenic treatment increased CHOP activity by 2.5-fold. SB203580 treatment reduced the basal CHOP activity by >50% and eliminated arsenic-induced CHOP activity. In addition, treatment of T-HEp3 cells with arsenic also caused a strong induction of CHOP activity (Fig. 3C) . In D-HEp3 cells, MKK6b(E) expression caused an increase in CHOP activity similar to that produced by arsenic in MCF-7 cells. Taken together, these results suggest that CHOP activity reflects accurately p38 activation (Fig. 3C) . Similarly, the correlation between phospho-ERK levels and Elk activation was tested in T-HEp3, D-HEp3, and MCF-7 cells. As shown in Fig. 3D , T-HEp3 cells showed a 4-fold higher basal Elk activity than D-HEp3 and MCF-7 cells, a ratio similar to that determined by Western blot analysis (Fig. 3A) . The high basal ERK/Elk activity in T-HEp3 cells was eliminated almost completely by overnight incubation with 25–50 µM Mek1/2 inhibitor PD98059 (Fig. 3D) . These results show that Elk and CHOP trans-reporting system reproduces the basal levels of active ERK and p38 and that it reports with fidelity changes in their ratios.

To test whether the measure of functional activity ratios of ERK and p38 will improve the predictive value of P-ERK even further, we transfected the three tumorigenic and five dormant cells lines with Elk or CHOP trans-reporting system, measured their luciferase activity, and calculated the ratio of Elk to CHOP. As shown in Fig. 3E , two of three tumorigenic cells (T-HEp3 and HT1080) had a ratio of Elk/CHOP > 1, whereas the rest of the cell lines, including the tumorigenic M24met, had Elk/CHOP ratios < 1, with some as low as 0.01 (MCF-7 and PC3). Thus, the Elk/CHOP ratio predicts the in vivo behavior as well as the phospho-ERK level alone, except that it segregates the MDA-MB-231 cells in which P-ERK level is borderline for growth (Fig. 3A) , conclusively into the dormant group. In contrast, unlike the P-ERK level, the ELK/CHOP ratio in M24met wrongfully predicts their inability to grow in vivo.

Decreasing the Balance of ERK/p38 Activity Reduces in Vivo Proliferation.
To examine the existence of a functional link between the ERK/p38 (Elk/CHOP) ratio and in vivo behavior, we used approaches previously verified in HEp3 cells (12 , 19 , 20) that shifted the balance of these two activities. HT1080 cells were chosen to examine the effect of ERK/p38 activity shift in favor of p38 on dormancy induction. We first established that a negative cross-talk between P-p38 and P-ERK, first discovered in HEp3 cells, also exists in HT1080 cells. Cells were transiently transfected with the Elk-luciferase reporter alone or cotransfected with a DNp38 expression vector, incubated with or without serum, lysed, and examined for luciferase activity. Fig. 4A shows that, as evidenced by a 2-fold increase in Elk-activated luciferase activity, it is possible, by inhibiting p38 activity, to additionally increase the level of active ERK. The effect was found to be serum independent (Fig. 4A) . This suggests that even in highly tumorigenic cells such as HT1080 with high ERK activity, p38 can still exert a negative regulatory effect. This conclusion was additionally supported by an experiment in which stable transfection of HT1080 cells with a constitutively active MKK6b(E) mutant, strongly reduced the P-ERK level (Fig. 4B) . We next examined whether inhibition of P-ERK and a resulting shift in balance in favor of p38 will affect the in vivo growth of HT1080. We used anti-uPAR antibody R2, which interferes with the uPAR/5ß1 complex and lowers P-ERK (19 , 20) or a direct inhibition of Mek activity with PD98059 inhibitor. To test the effect of R2 on ERK activity, cells were pretreated with preimmune IgG or R2 antibodies and plated on FN or PL, lysed 20 min later, and tested for ERK and P-ERK levels. As shown in Fig. 4C , adhesion to FN induced a 4-fold increase in P-ERK level that was reduced by 56% upon treatment with R2 antibody. Similar pretreatment of HT1080 cells with R2 antibody before inoculation on the CAM significantly reduced tumor volume (Fig. 4D) and tumor cell number (data not shown) after 4 days of in vivo growth. This indicates that inhibition of ERK and most likely a change in ERK/p38 ratio were responsible for a significant growth inhibition of these cells in vivo. We next tested whether inhibition of ERK activity by a specific pharmacological inhibitor of Mek1/2 (PD98059), an approach that completely bypasses the ERK inhibitory loop generated by interfering with the uPAR/5ß1 complex, will produce similar effect on growth. Pretreatment of HT1080 cells for 24 h with the 40-µM PD98059, although not affecting the viability of these cells in culture (data not shown), caused a significant growth inhibition after 4 days on CAMs (Fig. 4E) . Finally, we tested a direct effect of increased p38 activity on in vivo growth. HT1080 cells stably transfected with an active mutant of MKK6b(E) (Fig. 4B) , which grew in culture as well as vector-transfected cells (data not shown) upon inoculation on CAMs and after 7 days of incubation in vivo, showed significantly reduced growth (Fig. 4F) . Thus, regardless of the mechanism used, P-ERK inhibition that increases the relative contribution of p38 signaling reduces the in vivo proliferative ability of these cells.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. The in vivo growth of HT1080 fibrosarcoma is affected by changing the ERK/p38 activity through multiple interventions. A, inhibition of p38 induces ERK/Elk activation. HT1080 cells in medium with or without serum were transfected with Elk-GAL4 trans-reporting system and with a plasmid encoding dominant negative p38. Forty-eight h after transfection, the cells were lysed, and normalized luciferase activity was determined as indicated in methods. B, stable expression of active MKK6b(E) in HT1080 cells inhibits ERK activation. HT1080 cells were stably transfected with an empty pCDNA3.1 vector or with the same vector encoding an active mutant of MKK6 [MKK6b(E)]. The levels of active (P-ERK) and total ERK (ERK) as well as expression of MKK6b(E) (MKK6) were determined by Western blot (10 µg of protein/lane) as indicated in "Materials and Methods." C, disruption of uPAR/5ß1-integrin complex; effect on FN-dependent ERK activation. HT1080 cells were detached and incubated in suspension with medium alone (control), an irrelevant IgG (10 µg/ml) or with anti-uPAR mAb (R2, 10 µg/ml). Control cells were plated either on PL or FN, and antibody-treated cells were plated on FN. After 20 min, cells were lysed and the level of active phospho-ERK, or total ERK was determined by Western blot. D, cells were treated as in A with medium alone or with R2 mAb (10 µg/ml) and inoculated onto CAMs at 5 x 105 cells/CAM. Four days later, the tumors were excised and weighed. [Similar results were obtained when the excised tumors were dissociated and the cells counted (data not shown).] E, effect of Mek inhibition on the in vivo growth of HT1080 cells. Cells were pretreated for 36 h with 40 µM of the Mek inhibitor PD09589, inoculated at 5 x 105 cells/CAM and, after 4 days on CAM, excised and the number of cells/tumor counted. F, constitutive activation of p38 by MKK6b(E) and inhibition of ERK reduces tumor growth in vivo. Empty vector (HT-vector) or MKK6b(E) (HT-MKK6b(E))-expressing cells were inoculated at 5 x 105/CAM, and 7 days after inoculation, the tumor cells in each CAM were counted. (*, P < 0.001, Mann-Whitney test).

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Changing the ERK/p38 balance in PC3 cells in favor of ERK induces their in vivo growth. A, PC3 cells stably transfected with an active mutant of Mek1 (HA-R4F-Mek) or treated with (2 µM SB203580) were lysed, and the level of active (P-ERK) and total (ERK) ERK1/2 protein was determined by Western blot. In R4F-Mek1-transfected cells, the level of uPAR and the transgene expression were determined by Western blot using anti-uPAR (R2) and anti-HA-tag (12CA5, HA-R4F-Mek) antibodies, respectively. B, PC3 cells treated for 48 h with or without 5 µM SB203580 or PC3 cells expressing and empty vector or R4F-Mek were inoculated 5 x 105 cells/CAM, and after 6 days, the number of tumor cells/tumor nodule was determined and the population doublings calculated. (*, P < 0.001, Mann-Whitney test). C, vector or R4F-Mek-transfected cells were inoculated at 5 x 105 cells/CAM, and their growth was followed for 3 weeks as indicated in Fig. 1A . D, MDA-MB-231 cells treated for 48 h with or without 5 µM SB203580 were inoculated 5 x 105 cells/CAM, and after 1 week, the tumor volumes were determined as indicated in "Materials and Methods" (*, P < 0.02, Mann-Whitney test).

Can Tumor Cells with High p38 Activity Escape Dormancy?
The tumorigenic M24met cell line, with high uPAR, activated integrins and FN-fibrils (Figs. 1B and 2A ), was found to have high P-p38 levels (Fig. 3) , high CHOP activity (Fig. 6B) and a low Elk/CHOP ratio (Figs. 3E and 6B ). How could the high ERK activity be maintained in presence of very high p38 activity? Is it possible that the negative feedback between p38 and ERK is disrupted in these cells? To test this, M24met cells were incubated with 5 µM SB203580. Neither very short treatment (5–20 min), nor prolonged treatment (5–24 h), resulted in an increase in ERK phosphorylation (Fig. 6A) , indicating lack of negative regulation by p38. This was not attributable to a general loss of sensitivity to regulation because treatment with a combination of 4 µM SB203580 and 40 µM PD98059 (Mek inhibitor) reduced the level of ERK phosphorylation (Fig. 6A) , showing response expected of the classical Mek-ERK pathway. Was the lack of negative feedback indicative of other alterations in the p38 pathway? To examine this, M24met cells were transfected with either GAL4-Elk or GAL4-CHOP reporter constructs alone or in cotransfection with MKK6b(E) or DNp38 mutants (Fig. 6B) . The results show that the high CHOP activity was unaffected by inhibition of p38 with DNp38 mutant (Fig. 6B) , suggesting that the basal CHOP activity was p38 independent. The expression of MKK6b(E) produced a weak increase in CHOP activity (Fig. 6B) that did not correlate with a reduction in Elk, confirming the lack of cross-talk between p38 and ERK. To test whether some of P-p38 signaling capacity was retained, we examined the fate of another downstream target of p38, MEF2A (35) . Luciferase activity of M24met cells transfected with GAL4-MEF2A and luciferase reporter plasmid was only slightly increased when MKK6b(E) was expressed (probably because p38 was already maximally active) and strongly inhibited by cotransfection with DNp38 mutant (Fig. 6C) , suggesting that at least part of the p38 signaling pathway may have remained intact.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Negative cross-talk from p38 to ERK is altered in melanoma M24met cells. A, inhibition of p38 does not lead to ERK activation. Cells were treated for 5–20 min or 5–24 h with 5 µM SB203580 or with a combination of 5 µM SB203580 and 40 µM PD098059 or left untreated. The level of phospho-ERK and total ERK was determined by Western blot. B, increasing p38 activity activates CHOP, but p38 inhibition has no effect on CHOP and neither has an effect on Elk activity. Cells were transfected with the indicated plasmids, and 48 h after transfection, the cells were lysed and normalized luciferase activity determined. C, MEF2A (p38 reporter), another downstream target of p38 retains response to p38 inhibition. Cells were transfected with the indicated plasmids and normalized luciferase activity was determined 48 h after transfection.

View larger version (72K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Analysis of uPAR/5ß1 function in M24met cells. A, association of 5ß1-integrin and uPAR. Cells were lysed, immunoprecipitated with anti-5ß1 integrin antibody, and Western blotted for ß1-integrin (top panel) and uPAR (bottom panel). CL, cell lysate of HEp3 cells. B, disruption of FN-fibrils. Cells grown overnight in FN-free serum supplemented with 5 µg/ml human FN (control, top and bottom left panels) or with 10 µg/ml R2 (anti-uPAR) mAb (top right panel), or 20 µM 3-25 peptide (bottom right panel). FN-fibrils were detected with rabbit antihuman FN antibody followed by fluorescein-coupled goat antirabbit IgG. (Preimmune IgG did not affect FN-fibrils, data not shown). The two top panels, cells stained with DAPI for nuclei detection. C, Quantitation of M24met cells positive for FN fibrils at 7 and 18 h. For each treatment (control, preimmune IgG and R2 antibody), 300 cells were scored. The FN-positive cells are expressed as percentage of total cells, determined by DAPI-positive nuclei. D, effect of pretreatment with anti-uPAR mAbs on the in vivo growth of M24met cells. Cells, untreated, or treated with 15 µg/ml anti-uPAR R2 antibody for 30 min at 37°C were resuspended in PBS and inoculated at 5 x 105 cells/CAM. After 48 h, the number of tumor cells/CAM was determined.

Cdc42 Contributes to uPAR/5ß1 Regulation of p38 Activity.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Cdc42 regulation of p38 signaling. A, T-HEp3 cells have higher level of active Rac than D-HEp3 cells. Cell lysates were tested by a pull-down assay using a p21-binding domain of PAK-GST fusion protein and Western blotting using anti-Rac antibody (see "Materials and Methods"). B, dormant cells lines have high level of active Cdc42. Pull-down assays were performed on lysates of cells as in A, except that membranes were blotted with an anti-Cdc42 antibody; active and total Cdc42 are shown in the top and bottom panels, respectively. The numbers under panels A and B show the active/total Rac and Cdc42 ratio, respectively. C, regulation of Cdc42 activity affects the activity of CHOP, the p38-donwstream target in T-HEp3 cells. Cells were transiently transfected with empty vector or Cdc42QL active or Cdc42N17 dominant negative mutant of Cdc42, and CHOP activation was measured using the CHOP trans-activating system (see "Materials and Methods"). Normalized luciferase activity was determined 48 h after transfection. The effects were serum independent. D, expression of dominant negative Cdc42 mutant inhibits basal CHOP activity. The CHOP trans-activating system was used to measure CHOP activity after transfection with an active MKK6 mutant [MKK6b(E)], a p38-activator, or with a dominant negative mutant of Cdc42 (Cdc42N17) on CHOP activity in D-HEp3, MDA-MB-231, and MCF-7 cells. Normalized luciferase activity is expressed as percentage of control.

Taken together, our results support the notion that the regulation of ERK and p38 activities by the uPAR-integrin complex is important for the growth of several tumor types and that targeting this complex may be clinically beneficial. Moreover, even in cells that do not form uPAR/integrin complex, either because of missing components or because of changes in molecular interactions, altering the ERK/p38 ratio by direct targeting of either one of these kinases, shifts their in vivo behavior between proliferation and dormancy.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The results of our experiments led us to conclude that ERK activity level and ERK/p38 activity balance are valid general predictors of tumorigenicity and dormancy in vivo. On the basis of our previous work, we functionally defined dormancy as a state in which cancer cells inoculated in vivo persist in a viable state without leading to an increase in tumor volume. In our experimental model of human head and neck carcinoma HEp3 grown on CAM, dormancy was achieved by proliferation arrest and not through increased apoptosis, and it was reversible (19 , 25) . We now find that many cancer cell lines, and especially the ones maintained in culture for prolonged periods, are dormant on the CAM. The fact that cells such as PC3 or MDA-MB-231, known to produce tumors in nude mice, enter dormancy when inoculated on the CAM, may seem paradoxical. This discrepancy can, however, be reconciled by the observation that in the nude mice, inocula as large as 10⁷ cell often require >6 weeks to produce palpable tumors (40, 41, 42) . The reason for this delay is believed to be scarcity of tumorigenic cells within the inoculum capable of in vivo proliferation. Our studies of the CAM tumors, where the cells are accessible for continuous examination, indicate that the entire tumor cell population enters dormancy and, after a period of time in vivo, reemerges to initiate progressive tumor growth (25) . A similar mechanism of dormancy may exist in the nude mouse and may be responsible for the latency of many transplantable tumors.

We confirmed our finding that uPAR/integrin complex and FN fibrils are important regulators of ERK activation by showing that in 4 of the 10 cell lines tested that expressed high uPAR and 5ß1-integrin, 3 had active ERK and were tumorigenic when inoculated on the CAM. In contrast, in 6 cell lines, which had low or no uPAR and/or low integrin level, the signal to ERK was greatly reduced. In these cells, the p38 activity was elevated, resulting in an ERK/p38 ratio that favored p38 and dormancy on CAMs. Over a period of several months, they consistently produced only small nodules that, despite the continuous presence of live tumor cells, did not increase in volume (Fig. 1A and Ref. 12 ), a behavior conforming to our definition of dormancy.

As previously shown for T-HEp3 cells (12) , we showed that changing the balance of ERK/p38 in HT1080 cells by inhibiting Mek or activating p38 by an active MKK6 mutant or disruption of uPAR/integrin complex by anti-uPAR antibody, resulted in reduction of ERK activity in HT1080 cells and inhibition of their in vivo growth. (These experiments were not of sufficient duration to conclude that persistent dormancy was established.) In a reverse approach, changing the ERK/p38 ratio in favor of ERK in dormant PC3 cells, using an inhibitor of p38, SB203580, or transfection with an active Mek-R4F mutant, led to resumption of in vivo growth. Thus, cancer cells remain sensitive to changes in their ERK/p38 activities and they respond by a profound shift in their in vivo behavior.

Although the molecular interactions leading to uPAR-induced positive signals to ERK are at least partially understood (19, 20, 21) , the mechanism of p38 activation induced by loss of uPAR is unknown. Because uPAR appears to regulate integrin activation, we tested whether classical downstream transducers of integrin signaling such as Rac and Cdc42 GTPases are involved in the differential regulation of ERK and p38. We found that in high uPAR-HEp3 cells, activation of integrins corresponds with strongly activated Rac. These results are in agreement with the recently published data showing that uPAR transfection of fibroblasts caused Rac-dependent membrane ruffling (43) . Rac has also been shown to be required for facilitating the Ras-ERK signaling and cell cycle progression (44) . In contrast, we found that cells with low uPAR and inactive 5ß1-integrins (D-HEp3, AS24), or cells with low uPAR and integrin levels (MCF-7) or even cells with high uPAR but inactive 5ß1-integrin (MDA-MB-231, see below), had active Cdc42 and high p38 activity levels. Moreover, we showed that Cdc42 activation was functionally linked to p38 activation in all these cells. These results are in agreement with reports showing that Cdc42, by activating p38, induces p21/p27 and represses cyclin D1 expression, thus causing cell-cycle arrest (13, 14, 15 , 45) . Our attempt at identifying molecules that may be responsible for regulation of Cdc42 included a 53-kDa Cdc42GAP (46) . However, testing its association with 5ß1 or FAK did not provide clues of its role in the differential activation of Rac and Cdc42 in the uPAR-rich and uPAR-deficient cells (data not shown). Recent findings (47) suggest that analysis of spatial and temporal regulation of effectors of Rac or Cdc42 activity by GDIs may have to be undertaken to understand the differential regulation of p38.

Of the cell lines studied, two, the MDA-MB-231 and the M24met, warrant discussion. The MDA-MB-231 cell line, despite having high levels of uPAR and 5ß1-integrin, does not form FN-fibrils and has a level of active ERK that makes its assignment to the tumorigenic or dormant group uncertain. The lack of fibrils suggests that despite high uPAR level, its 5ß1-integrin is inactive. The finding of relatively high level of active Cdc42 and p38 supports this conclusion. We showed that a prerequisite for uPAR-activation of integrin is the association of the two proteins (19) , yet whereas anti-5ß1-integrin antibody coimmunoprecipitated uPAR from lysates of T-HEp3 cells, no such coimmunoprecipitation was found in MDA-MB-231 cells. It is possible that in some cells, uPAR partitions with other membrane receptors and becomes unavailable to partner with 5ß1-integrin. Chapman et al. (37) showed that in 293 cells in which uPAR was overexpressed by transfection, 90% of uPAR was found in a complex with 3ß1 integrin. Because they found that the 5ß1-integrin content exceeded that of 3ß1 by 4–5-fold, yet uPAR interacted almost exclusively with 3ß1, they suggested that association of uPAR with 3 was preferred. However, we find that both in T-HEp3 cells and in MDA-MB-231 cells uPAR may associate with the predominantly expressed integrin. In T-HEp3 cells, uPAR associates with 5ß1, which is at 6-fold excess over 3 (results not shown), whereas it seems to associate with 3 in MDA-MB-231 cells, which have a 2-fold excess of this integrin (37) . If, however, a preference for association with 3 exists, then it is possible that 3ß1-integrin has to be sequestered by other membrane proteins to allow uPAR to interact with 5ß1 or, as suggested by published evidence, to prevent it from exerting a trans-dominant effect on other integrins (48) . Hemler et al. (49 , 50) have shown that a tetraspanin CD151 (PETA) enters into specific, lateral interactions with the 3ß1 integrin. Testa et al. (51) has shown that CD151 is overexpressed in metastatic HEp3 cells in which we showed the 5ß1-integrin to be activated by frequent association with uPAR (19 , 20) . CD151 could thus be a regulator of 3ß1 availability. We are in the process of identifying the mechanism that dictates the preferential association of uPAR with individual integrins.

The second exception to the rule that high uPAR/integrin content and their proper interaction lead to high ERK and low p38 activities is the melanoma M24met. This cell line forms extensive FN-fibrils and has very high ERK activity but also very high p38 activity. This creates an ERK/p38 ratio predictive of dormant cells, yet the cells are highly tumorigenic and metastatic. This finding poses a dilemma because we showed that in all other cell lines studied, p38, through a negative feedback, exerts an inhibitory effect on ERK. How is it then possible that in M24met cells high ERK activity can coexist with high p38? The mechanism responsible for the negative feedback from p38 to ERK remains unknown. One study (52) shows that ERK1/2 coimmunoprecipitates p38 and that this interaction with p38 prevents ERK phosphorylation. Regardless of the mechanism, published reports suggest that the p38 pathway may be altered or dysfunctional in melanomas (53, 54, 55, 56) . In some melanoma cell lines, the downstream targets appear to be selectively uncoupled from p38 (57) . An aberrant function of the p38 pathway was also identified in a rhabdomyosarcoma (58) , which despite high myoD expression, known to induce differentiation in normal myoblasts, continue to proliferate without undergoing differentiation and, only upon hyperactivation of the p38 pathway by MKK6b(E), proceed to differentiation. In some of these tumors, p38 does not signal properly to the downstream targets (58) , suggesting an escape mechanism from the high p38 activity. In confirmation of these results, we found no evidence that the high p38 activity in M24met cells is engaged in a negative cross-talk to ERK (Fig. 6A) . Moreover, we found CHOP activity, a transcription factor known to be specifically activated by p38 phosphorylation, to be largely independent of p38 signaling (Fig. 6B) , yet another downstream target, MEF2A, to be regulated by p38 (Fig. 6C) . Interestingly, a p38-independent, phosphorylated form of CHOP that functions in interleukin 1-induced interleukin 6 gene transactivation has been identified (56) . These results and published data (56 , 57) suggest that the network of cross-talk and the downstream targets may be altered in melanoma and that, despite high levels of p38 activity, these cancer cells continue to proliferate in vivo. Moreover, because p38 activity has been shown to play a role in mRNA stability, including genes important for cancer invasion (59) , M24met cell line may define a subgroup of cancers that take advantage of p38 activity while at the same time maintaining and exploiting the proliferative advantage of high ERK.

Overall, based on the study of 10 different cancer cell lines, our results show that uPAR and 5ß1 expression and activation, most likely in presence of EGFR (20) , generate a high level of ERK activity and, with the exception of melanoma, low p38 activity, which is necessary for the in vivo growth of cancer cells. The high ERK activity feeds into a positive feedback loop that transactivates uPAR (and uPA) expression (12 , 34 , 60 , 61) . In turn, the high uPAR, by activating 5ß1 maintains high ERK activity (19 , 20) . The signaling loop required for cancer cell proliferation can be interrupted by a reduction in uPAR level by cleavage of its domain 1, important for uPAR interaction and activation of 5ß1 integrin (19 , 20 , 62) , and by loss of uPA and/or FN. It is plausible that these changes take place during cancer cell spread and establishment of metastases causing their initial dormancy. Because we showed that multiple experimental interventions decrease the ERK/p38 ratio, they should be considered in induction of dormancy. Although p38 inducers must be used with caution because some tumor properties may be dependent on this activity (63) , direct inhibition of the ERK pathway may be safe. Mek inhibitors have been tested in preclinical studies and showed tumor growth inhibition without toxicity (33) . This approach as well as therapies aimed at disruption of the uPAR/integrin interactions to persistently lower the ERK/p38 ratio may induce a state of prolonged dormancy of cells that have already disseminated but not yet entered progressive growth.

	ACKNOWLEDGMENTS

 
We thank rotating PhD students Luciana Giono and Felix Lohmann for help with some of the experiments and Dr. Samuel Waxman for continuous encouragement and support. We also thank the following individuals for donating reagents: monoclonal anti-uPAR antibody R2 from Dr. Michael Ploug, (Finsen Laboratory); P-p38 antibody from Erik Schaefer (Biosource); pD700 plasmid from Dr. Ari Melnick, (Albert Einstein College of Medicine); Cdc42 constructs from Dr. Silvio Gutkind (NIH); HA-R4F-Mek from Dr. Natalie Ahn (University of Colorado); MKK6b(E) and MEF2A constructs from Dr. Jihuai Han (Scripps Institute).

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by USPHS Research Grant CA-40578 (to L. O.), The Charles H. Revson Foundation (to J. A. A-G.), NIH/Mount Sinai School of Medicine Medical Scientist Training Program and National Cancer Institute Predoctoral Training Grant CA78207 (to D. L.), the Samuel Waxman Cancer Research Foundation, and The Peter J. Sharp Foundation.

² To whom requests for reprints should be addressed, at Department of Medicine, Box 1178, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-3194, (212) 241-5899, or (212) 241-6771; Fax: (212) 996-5787; E-mail: Liliana.Ossowski{at}mssm.edu

³ The abbreviations used are: ERK, extracellular signal-regulated kinase; uPA, urokinase; uPAR, uPA receptor; EGFR, epidermal growth factor receptor; ECL, enhanced chemiluminescence; DAPI, 4',6-diamidino-2-phenylindole; HRP, horseradish peroxidase; HA, hemagglutinin; IF, immunofluorescence; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; GST, glutathione S-transferase; ECM, extracellular matrix; FN, fibronectin; CAM, chorioallantoic membrane; FAK, focal adhesion kinase; GDI, GDP dissociation inhibitor; PETA, platelet-endothelial tetraspan antigen.

Received 10/14/02. Accepted 2/ 3/03.

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