[CANCER RESEARCH 59, 3581–3587, August 1, 1999]
`
`Advances in Brief
`
`Regulation of Cell Growth and Cyclin D1 Expression by the Constitutively Active
`FRAP-p70s6K Pathway in Human Pancreatic Cancer Cells1
`
`Martin Grewe, Frank Gansauge, Roland M. Schmid, Guido Adler, and Thomas Seufferlein2
`Departments of Internal Medicine I [M. G., R. M. S., G. A., T. S.] and Surgery [F. G.], University of Ulm, D-89081, Ulm, Germany
`
`include 59-terminal oligopyrimidine tract mRNA translation and pro-
`tein synthesis (11). The translation inhibitor 4E-BP1 is also phospho-
`rylated by the FRAP-p70s6K pathway. This leads to the dissociation of
`4E-BP1 from initiation factor eIF-4E, permitting increased protein
`translation and mitogenesis (12). In contrast, dephosphorylated 4E-
`BP1 interacts with eIF-4E and thereby inhibits cap structure-depen-
`dent protein synthesis and cell growth (13). Interestingly, rapamycin
`induces dephosphorylation of 4E-BP1,
`leading to inactivation of
`eIF-4E (6, 14). In contrast to previous studies, more recent reports
`suggest that 4E-BP1 is not a direct downstream target of p70s6K but
`is regulated by FRAP in a parallel manner (14). This view is supported
`by results obtained in p70s6K 2/2 cells, demonstrating that rapamy-
`cin can still prevent phosphorylation of 4E-BP1 and inhibit growth
`(15). Thus, rapamycin most likely inhibits growth via both p70s6K-
`dependent and -independent pathways. The role of these rapamycin-
`sensitive pathways in the growth of pancreatic cancer cells is un-
`known.
`In the present study, we demonstrate that the FRAP-p70s6K path-
`way is constitutively phosphorylated/active in MiaPaCa-2 and Panc-1
`human pancreatic cancer cell lines and a pancreatic cancer tissue
`sample. Rapamycin induced p70s6K dephosphorylation and inactiva-
`tion of constitutively active p70s6K in serum-starved MiaPaCa-2 and
`Panc-1 cells. Rapamycin also inhibited constitutive phosphorylation
`of the translation inhibitor 4E-BP1 in these cells. Furthermore, pro-
`liferation and colony formation of MiaPaCa-2 and Panc-1 human
`pancreatic cancer cells were markedly reduced in the presence of
`rapamycin. Finally, rapamycin profoundly inhibited expression of
`cyclin D1. Thus, our results suggest that the rapamycin-sensitive
`FRAP-p70s6K pathway could serve as a novel target for therapeutic
`intervention in pancreatic cancer.
`
`Materials and Methods
`
`Abstract
`
`The FRAP-p70s6K signaling pathway was found to be constitutively
`phosphorylated/active in MiaPaCa-2 and Panc-1 human pancreatic can-
`cer cells and a pancreatic cancer tissue sample as judged by the retarded
`electrophoretic mobility of the two major FRAP downstream targets,
`p70s6K and 4E-BP1. Treatment of cells with rapamycin, a selective FRAP
`inhibitor, inhibited basal p70s6K kinase activity and induced dephospho-
`rylation of p70s6K and 4E-BP1. Moreover, rapamycin inhibited DNA
`synthesis as well as anchorage-dependent and -independent proliferation
`in MiaPaCa-2 and Panc-1 cells. Finally, rapamycin strikingly inhibited
`cyclin D1 expression in pancreatic cancer cells. Thus, inhibitors of the
`constitutively active FRAP-p70s6K pathway may provide a novel thera-
`peutic approach for pancreatic cancer.
`
`Introduction
`
`Pancreatic cancer constitutes the fourth leading cause of cancer
`death in Western countries for both sexes and has a dismal prognosis
`(1). The growth of pancreatic cancer is driven by multiple factors such
`as activating mutations in the small GTPase Ki-ras and various
`growth factors acting in an autocrine or paracrine manner (2). How-
`ever, the downstream targets of these proteins and the arising signal
`transduction pathways involved in autocrine/paracrine human pancre-
`atic cancer cell growth are poorly understood. Defining these path-
`ways could give rise to novel therapeutic approaches that are urgently
`needed.
`The serine/threonine kinase p70s6K is a highly conserved element in
`a wide array of cellular processes including the mitogenic response to
`growth factors (3). This enzyme is activated in vivo by phosphoryla-
`tion mediated in part by a phosphatidyl kinase-related kinase, FRAP3
`or mTOR (mammalian target of rapamycin; Ref. 4). The immunosup-
`pressant rapamycin has emerged as a useful tool to elucidate the
`cellular function of FRAP and its downstream target, p70s6K (3, 5, 6):
`rapamycin inhibits FRAP by forming a stable complex with the
`immunophilin FK506-binding protein, which binds to FRAP. As a
`result of this interaction, rapamycin induces dephosphorylation of
`several sites (Thr229, Thr389, and Ser404) on p70s6K, leading to its
`rapid inactivation (7). Interestingly, rapamycin blocks the prolifera-
`tion of a variety of cells that have not entered the cell cycle (8, 9).
`Furthermore, we have recently shown that rapamycin inhibits consti-
`tutive p70s6K phosphorylation and cell growth in classical small cell
`lung cancer cell lines (10). Consequently, there has been considerable
`interest in the downstream targets of rapamycin and p70s6K that
`
`Cell Culture. Human pancreatic cancer cell lines MiaPaCa-2 and Panc-1
`were purchased from the American Type Culture Collection (Manassas, VA).
`Stocks were maintained in DMEM supplemented with 10% (v/v) FBS in a
`humidified atmosphere of 5% CO2: 95% air at 37°C. The cells were passaged
`every 3 days.
`Tissue Samples. Pancreatic carcinoma tissue samples were obtained from
`a patient undergoing a surgical operation for pancreatic cancer at the Depart-
`ment of General Surgery, University of Ulm. Informed consent was obtained
`from the patient before surgery. The tissue was collected after surgical re-
`moval, snap-frozen immediately in liquid nitrogen, and stored at 280°C.
`Immunoprecipitations and Western Blotting. MiaPaCa-2 and Panc-1
`cells were washed twice in serum-free DMEM and incubated in fresh DMEM
`for an additional 24 h. Cells were then treated with rapamycin as indicated in
`the figure legends and lysed in 50 mM Tris-HCl, 5 mM EDTA, 100 mM NaCl,
`40 mM b-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 1
`mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupep-
`tin (pH 7.6; lysis buffer). For immunoprecipitations, lysates were incubated
`with a polyclonal anti-4E-BP1 antibody for 2 h at 4°C on a rotating wheel with
`protein A-Sepharose beads added for the second h. Beads were washed twice
`in lysis buffer and resuspended in 23 SDS-PAGE sample buffer. Proteins were
`further analyzed by SDS-PAGE, followed by Western blotting using a mono-
`3581
`
`Received 3/12/99; accepted 6/16/99.
`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 Supported by a Grant SFB 518/B3 from the DFG (to T. S.).
`2 To whom requests for reprints should be addressed, at Department of Internal
`Medicine I, Medizinische Universita¨tsklinik Ulm, Robert-Koch Strasse 8, D-89081
`Ulm/Germany. Phone: 49-731-502-4307; Fax: 49-731-502-4302; E-mail:
`thomas.
`seufferlein@medizin.uni-ulm.de.
`3 The abbreviations used are: FRAP, FK506-binding protein rapamycin-associated
`protein; FBS, fetal bovine serum; TGF, transforming growth factor; ERK, extracellular
`signal-regulated kinase; BrdUrd, bromodeoxyuridine.
`
`
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`REGULATION OF CELL GROWTH AND CYCLIN D1 EXPRESSION
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`Results
`
`from Upstate Biotechnology, Inc. The polyclonal anti-ERK2 antibody was a
`kind gift of Dr. Jo van Lint (Katholieke Universiteit, Leuven, Belgium). PD
`098059 was obtained from New England Biolabs. Protein A-Sepharose and the
`BrdUrd labeling and detection kit were from Boehringer Mannheim. Enhanced
`chemiluminescence reagents, [3H]thymidine, and [g-32P]ATP, were from Am-
`ersham. All other reagents were of the purest grade available.
`
`clonal anti-eIF-4E antibody or a polyclonal anti-4E-BP1 antibody with immu-
`noreactive bands visualized by enhanced chemiluminescence detection. For
`p70s6K2 and 4E-BP1 mobility shift assays and detection of cyclin D1, cyclin
`E, and p27kip1, cells were treated as indicated in the figure legends and lysed
`in SDS-PAGE sample buffer, and samples were further analyzed by SDS-
`PAGE and Western blotting with specific antisera to these proteins essentially
`as described above. Pancreatic tissues were lysed and dissociated by sonica-
`tion. The lysates were subsequently boiled in SDS-PAGE sample buffer for 5
`min and further analyzed by SDS-PAGE and Western blotting as described
`Rapamycin Inhibits Constitutive Phosphorylation and Activa-
`above.
`tion of p70s6k in Panc-1 and MiaPaCa-2 Cells. Activation of
`p70s6K and ERK2 Immune Complex Kinase Assays. Serum-starved
`p70s6K by mitogens can be determined by the appearance of slower-
`MiaPaCa-2 and Panc-1 cells were incubated with rapamycin, PD 098059, and
`migrating forms in SDS-PAGE due to the phosphorylation of p70s6K
`TGF-a as indicated in the figure legends. Controls received an equivalent
`amount of solvent. Cells were then lysed at 4°C in 1 ml of lysis buffer.
`on Thr229, Thr389, and Ser404, which are not basally phosphorylated in
`Immunoprecipitations were performed at 4°C using an anti-p70s6K antibody or
`quiescent cells (3). The phosphorylation of these sites can be pre-
`an anti-ERK2 antibody for 2 h, with protein A-agarose added for the second
`vented or reversed by treatment with rapamycin, which specifically
`hour. Immune complexes were washed three times in lysis buffer and once
`inhibits the p70s6K activator FRAP (3, 4). To examine the status of
`with p70s6K kinase buffer [20 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM DTT,
`p70s6K phosphorylation in human pancreatic cancer cells, cultures of
`and 10 mM b-glycerophosphate] or ERK kinase buffer [15 mM MgCl2, 15 mM
`Panc-1 and MiaPaCa-2 cells were incubated in the absence or pres-
`Tris-HCl (pH 7.4)], respectively. Kinase reactions were performed by re-
`ence of 20 ng/ml rapamycin. Cells were lysed, and the lysates were
`suspending the protein A-Sepharose pellets in 25 ml of kinase assay
`analyzed by immunoblotting. p70s6K exhibited a retarded electro-
`mixture containing the appropriate kinase buffer with 0.2 mM S6 peptide
`phoretic mobility characteristic of the phosphorylated form of this
`(RRRLSSLRA) or myelin basic protein, 20 mM ATP, 5 mCi/ml [g-32P]ATP, 2
`mM cyclic AMP-dependent protein kinase inhibitor peptide, and 100 nM
`enzyme in Panc-1 and MiaPaCa-2 cells. In control experiments, there
`was no retarded electrophoretic mobility of p70s6K in serum-starved
`microcystin LR. Incubations were performed under linear assay conditions at
`30°C for 20 min and terminated by spotting 25 ml of the supernatant onto
`mouse or human fibroblasts (data not shown), in accordance with
`Whatman p81 chromatography paper. Papers were washed four times for 5 min
`previous results (3). Treatment of cells with rapamycin induced a
`in 0.5% o-phosphoric acid, immersed in acetone, and dried before scintillation
`striking dephosphorylation of p70s6K, as demonstrated by the increase
`counting. The average radioactivity of two blank samples containing no
`in the electrophoretic mobility (Fig. 1A, top panels). The effect of
`immune complex was subtracted from the result of each sample.
`rapamycin on constitutive p70s6K phosphorylation was concentration
`DNA Synthesis Assays. MiaPaCa-2 and Panc-1 cells were serum-starved
`dependent; maximum effects were achieved at 6 and 20 ng/ml rapa-
`for 24 h and then incubated with various additions of rapamycin as described
`mycin in Panc-1 and MiaPaCa-2 cells, respectively (Fig. 1A, bottom
`in the figure legends. Control cells received solvent or DMEM/10% FBS to
`panels). Dephosphorylation of p70s6K by rapamycin in Panc-1 and
`determine maximum DNA synthesis. After 18 h of incubation at 37°C,
`MiaPaCa-2 cells was first visible after 5 min and reached a maximum
`[3H]thymidine (0.25 mCi/ml; 1 mM) was added to the cultures, and cells were
`10 min after the addition of rapamycin to the cells (data not shown).
`incubated for another 6 h at 37°C. Cells were then washed with PBS and
`incubated in 5% trichloroacetic acid at 4°C for 30 min, washed with ethanol,
`Next we performed immune complex kinase assays to directly assess
`and solubilized in 1 ml of 2% Na2CO3, 0.1 M NaOH, and 1% SDS. The
`p70s6K activity. Treatment of cells with rapamycin substantially re-
`acid-insoluble radioactivity was determined by Cerenkov counting in 6 ml of
`duced the basal kinase activity of p70s6K by 92% and 99% in serum-
`Ultima Gold (Packard). For detection of BrdUrd incorporation into cellular
`starved Panc-1 and MiaPaCa-2 cells, respectively (Fig. 1B, top pan-
`DNA, MiaPaCa-2 and Panc-1 cells were serum-starved in fresh DMEM for
`els). The effect of rapamycin on p70s6K activity was specific because
`24 h. Cells were then incubated with 20 ng/ml rapamycin or solvent for 24 h
`rapamycin did not affect basal and TGF-a-stimulated activation of the
`at 37°C, with 10 mM BrdUrd added for the last 6 h. Cultures were subsequently
`mitogen-activated protein kinase ERK2 in both cell lines. In contrast,
`washed with PBS, fixed in 70% ethanol for 20 min, and incubated with
`the mitogen-activated protein/ERK kinase 1 inhibitor PD 098059
`anti-BrdUrd monoclonal antibody, followed by labeling with an antimouse
`markedly inhibited basal and TGF-a-stimulated ERK2 activation
`IgG-fluorescein antibody. Cells were examined using a Zeiss Axiophot immu-
`(Fig. 1B, bottom panels).
`nofluorescence microscope. Data are expressed as the percentage of BrdUrd-
`labeled nuclei.
`Effect of Rapamycin on 4E-BP1 Phosphorylation in Panc-1 and
`Growth Assay. Three days after passage, MiaPaCa-2 and Panc-1 cells
`MiaPaCa-2 Cells. It has been suggested that phosphorylation and
`were washed in serum-free DMEM,
`trypsinized, and resuspended in
`thus inactivation of the translation inhibitor 4E-BP1 are also mediated
`DMEM/1% FBS. Cells were plated at a density of 1 3 104 cells in 1 ml of
`by a rapamycin-sensitive pathway (6, 14). Therefore, we examined the
`DMEM/1% FBS in the presence or absence of 20 ng/ml rapamycin in dupli-
`phosphorylation status of 4E-BP1 in serum-starved human pancreatic
`cate. At the times indicated in the figure legends, the cell number was
`cancer cells in the absence or presence of rapamycin. To determine
`determined using a cell counting chamber.
`4E-BP1 phosphorylation, a Western blot analysis was performed
`Clonogenic Assay. MiaPaCa-2 and Panc-1 cells were washed, trypsinized,
`using a polyclonal antibody against 4E-BP1. Three forms of 4E-BP1
`and resuspended in DMEM. The cell number was determined using a cell
`were present in serum-starved Panc-1 and MiaPaCa-2 cells, suggest-
`counting chamber. Cells (3 3 104 ) were mixed with DMEM/1% FBS
`ing constitutive phosphorylation of 4E-BP1 in these cells. Treatment
`containing 0.3% agarose in the presence or absence of rapamycin at the
`concentrations indicated and layered over a solid base of 0.5% agarose in
`with rapamycin leads to a decreased intensity of the upper two bands
`DMEM/1% FBS in the presence or absence of rapamycin at the same con-
`and to an increased intensity of the fastest migrating lower band
`centrations in 33-mm dishes. The cultures were incubated in humidified 5%
`(Fig.1C, left, top and bottom panels). According to previous studies,
`CO2: 95% air at 37°C for 14 days and then stained with the vital stain nitroblue
`the lowest band represents the unphosphorylated form of 4E-BP1 (16,
`tetrazolium. Colonies of .120 mm in diameter (20 cells) were counted using
`17). We next examined whether the constitutive phosphorylation of
`a microscope.
`4E-BP1 was sufficient to decrease the interaction of eIF-4E with
`Materials. Rapamycin was obtained from Calbiochem-Novabiochem. An-
`4E-BP1. Serum-starved Panc-1 cells were incubated with solvent or
`tibodies against p70s6K, 4E-BP1, p27KIP1, cyclin D1, and cyclin E were
`20 ng/ml rapamycin. Cell lysates were immunoprecipitated with a
`obtained from Santa Cruz Biotechnology. The monoclonal anti-eIF-4E anti-
`polyclonal anti-4E-BP1 antibody and analyzed further by Western
`body was from Transduction Laboratories. The NH2-terminally directed anti-
`blotting with a monoclonal anti-eIF-4E antibody. As shown in Fig.1C
`p70s6K polyclonal antibody used to determine p70s6K activity was obtained
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`REGULATION OF CELL GROWTH AND CYCLIN D1 EXPRESSION
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`Fig. 1. Constitutive phosphorylation/activation of p70s6K and 4E-BP1 in Panc-1 and MiaPaCa-2 cells. A, top panels, serum-starved cultures of Panc-1 (left) and MiaPaCa-2 cells
`(right) were treated with 20 ng/ml rapamycin (Rapa) or received an equivalent amount of solvent (—). p70s6K mobility shift assays were performed as described in “Materials and
`Methods.” The results shown in each case are representative of three independent experiments. The positions of hypophosphorylated p70s6K (p70s6K) and the slower-migrating
`phosphorylated p70s6K (pp70s6K) are indicated by arrows. Bottom panels, serum-starved cultures of Panc-1 (left) and MiaPaCa-2 cells (right) were treated with various concentrations
`of rapamycin as indicated for 20 min or received an equivalent amount of solvent (—). p70s6K mobility shift assays were performed as described in “Materials and Methods.” The results
`shown in each case are representative of three independent experiments. The positions of hypophosphorylated p70s6K (p70s6K) and the slower-migrating phosphorylated p70s6K
`(pp70s6K) are indicated by arrows. B, top panels, serum-starved cultures of Panc-1 (left) and MiaPaCa-2 cells (right) were treated with 20 ng/ml rapamycin for 20 min (Rapa) or received
`an equivalent amount of solvent (—). Cells were subsequently lysed, and immune complex kinase assays for p70s6K activity were performed as described in “Materials and Methods.”
`Bottom panels, serum-starved cultures of Panc-1 (left) and MiaPaCa-2 cells (right) were treated with 20 ng/ml rapamycin (Rapa 1) for 20 min or 20 mM PD 098059 (PD 1) for 40
`min or received an equivalent amount of solvent (—). Cells were then incubated with 50 ng/ml TGF-a for 5 min (TGF-a 1) and lysed, and immune complex kinase assays for ERK2
`activity were performed as described in “Materials and Methods.” C, left panels, serum-starved cultures of Panc-1 (top) and MiaPaCa-2 cells (bottom) were treated with 20 ng/ml
`rapamycin (Rapa) as indicated for 20 min. Control cells received an equivalent amount of solvent (—). 4E-BP1 mobility shift assays were performed as described in “Materials and
`Methods.” The results shown in each case are representative of three independent experiments. The positions of hypophosphorylated 4E-BP1 (4E-BP1) and the slower-migrating
`phosphorylated forms of 4E-BP1 (p4E-BP1) are indicated by arrows. Middle panels, cells were treated as described above and lysed, and lysates were immunoprecipitated using a
`specific anti-4E-BP1 antibody as described in “Materials and Methods.” Western blotting was performed using an anti-eIF-4E antibody (top) or an anti-4E-BP1 antibody (bottom) as
`described in “Materials and Methods.” Right panels, human pancreatic cancer tissue samples were lysed and subsequently analyzed by SDS-PAGE with an anti-p70s6K (top) or an
`anti-4E-BP1 antibody (bottom) as described in “Materials and Methods.” The positions of hypophosphorylated and hyperphosphorylated p70s6K (p70s6K and pp70s6K) and 4E-BP1
`(4E-BP1 and p4E-BP1) are indicated by arrows.
`
`anti-4E-BP1 antibody, similar amounts of 4E-BP1 protein could be
`(middle, top panel), a small amount of eIF4E coimmunoprecipitated
`detected in rapamycin-treated and untreated cells (Fig. 1C, middle,
`with 4E-BP1 in nontreated cells. Upon treatment with rapamycin, the
`bottom panel). In addition, using 7-methyl-GTP-Sepharose that spe-
`amount of eIF-4E that could be detected in 4E-BP1 immunoprecipi-
`cifically binds eIF-4E, an increased amount of 4E-BP1 was found in
`tates increased strikingly. When blots were stripped and reprobed with
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`REGULATION OF CELL GROWTH AND CYCLIN D1 EXPRESSION
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`Fig. 2. Rapamycin inhibits DNA synthesis in
`Panc-1 and MiaPaCa-2 cells. A, serum-starved cul-
`tures of Panc-1 (left) or MiaPaCa-2 cells (right)
`were washed and incubated at 37°C for 24 h in
`serum-free DMEM containing either 20 ng/ml ra-
`pamycin or an equivalent amount of solvent.
`[3H]Thymidine was added during the last 6 h of the
`incubation, and [3H]thymidine incorporation was
`determined as described in “Materials and Meth-
`ods.” Each point represents the mean of three de-
`terminations and is representative of at least two
`independent experiments. B, serum-starved cultures
`of Panc-1 (left panels) or MiaPaCa-2 cells (right
`panels) were washed and incubated at 37°C for 24 h
`in DMEM containing either 20 ng/ml rapamycin
`(Rapa; bottom panels) or an equivalent amount of
`solvent (—; top panels). BrdUrd was added during
`the last 6 h of theincubation. After 24 h, BrdUrd
`incorporation into cell nuclei was determined as
`described in “Materials and Methods.” Labeled nu-
`clei were visualized by fluorescence microscopy.
`Typical fields are presented. C, BrdUrd incorpora-
`tion was performed as described above for rapamy-
`cin (Rapa 1)-treated or vehicle-treated (—) Panc-1
`(left) or MiaPaCa-2 cells (right). Results are pre-
`sented as the percentage of labeled nuclei and are
`the means of three distinct fields from two separate
`experiments.
`
`active FRAP-p70s6K pathway for pancreatic cancer cell growth. Basal
`7-methyl-GTP-Sepharose immunoprecipitates of lysates of Panc-1
`DNA synthesis in serum-free DMEM as assessed by [3H]thymidine
`cells treated with rapamycin as compared to untreated cells (data not
`shown). Thus, the level of constitutive phosphorylation of 4E-BP1 in
`incorporation was 35% of maximum stimulation in response to 10%
`serum-starved pancreatic cancer cells is sufficient to prevent interac-
`FBS in Panc-1 and MiaPaCa-2 cells (Fig. 2A). Upon treatment with
`rapamycin, basal [3H]thymidine incorporation decreased substantially
`tion with eIF-4E.
`Constitutive p70s6K- and 4E-BP1 phosphorylation was not re-
`in both cell lines. The effect of rapamycin was concentration depend-
`ent, with half-maximal effects at 1 and 3 ng/ml
`in Panc-1 and
`stricted to pancreatic cancer cells exhibiting activating Ki-ras muta-
`MiaPaCa-2 cells, respectively, and maximal effects at 20 ng/ml in
`tions such as MiaPaCa-2 and Panc-1 cells but could also be detected
`both cell
`lines. At
`this concentration, basal DNA synthesis was
`in the human pancreatic cancer cell lines SW 850 and SW 979 that
`reduced by 77% and 66% in Panc-1 and MiaPaCa-2 cells, respectively
`exhibit wild-type Ki-ras (data not shown). Thus, constitutive phos-
`phorylation of p70s6K and 4E-BP1 is not due to the activating Ki-ras
`(Fig. 2A). These results are in good agreement with the data obtained
`on rapamycin-induced dephosphorylation of p70s6K (Fig. 1A). To
`mutation in these cells. Interestingly, constitutive phosphorylation of
`p70s6K and 4E-BP1 could also be observed in a human pancreatic
`further substantiate our observations, we applied a distinct technique
`in which DNA synthesis was determined using an immunofluores-
`carcinoma tissue sample (Fig. 1C, right panels).
`Rapamycin Inhibits DNA Synthesis in Panc-1 and MiaPaCa-2
`cence assay to detect BrdUrd incorporation into cell nuclei. As shown
`Cells. We first examined the effect of rapamycin on basal DNA
`in Fig. 2B, BrdUrd incorporation into cell nuclei was markedly
`inhibited in the presence of rapamycin. At a concentration of 20 ng/ml
`synthesis in both cell lines to determine the role of the constitutively
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`solid medium in the presence or absence of rapamycin. As shown in
`Fig. 3B, rapamycin markedly inhibited the formation of colonies by
`MiaPaCa-2 and Panc-1 cells in a concentration-dependent manner.
`Half-maximal and maximal effects were achieved at 6 and 20 ng/ml
`rapamycin in both cell lines.
`Rapamycin Inhibits Expression of Cyclin D1 but not Cyclin E
`and p27KIP1. Progression from G1 to the S phase of the cell cycle is
`regulated by the expression of cyclins D and E, which modulate the
`activities of the cyclin-dependent kinases. Rapamycin can block G1 to
`S-phase cell cycle progression in a number of cell types by blocking
`growth factor-stimulated elimination of the cyclin-dependent kinase
`inhibitor p27KIP1 (20, 21). We have recently shown that rapamycin
`strikingly reduces bombesin-induced expression of cyclins D1, D3,
`and E in Swiss 3T3 fibroblasts (22). These effects of rapamycin may
`be a consequence of the inhibition of p70s6K or may be due to a
`distinct pathway(s) regulated by the FRAP-p70s6K pathway. To fur-
`ther explore the nature of the rapamycin-sensitive and -insensitive
`mechanisms regulating the basal growth of human pancreatic cancer
`cells, we determined the expression of cyclin D1, cyclin E, and
`p27KIP1 and correlated these with the activation of p70s6K. As shown
`in Fig. 4, treatment of cells with rapamycin leads to a sustained
`dephosphorylation of p70s6K for up to 36 h. Similar results were
`obtained for 4E-BP1 phosphorylation (data not shown). Rapamycin
`treatment of MiaPaCa-2 cells at a concentration that
`inhibited
`p70s6K2 and 4E-BP1 phosphorylation as well as basal cell growth
`
`Fig. 3. Rapamycin inhibits anchorage-dependent and -independent proliferation of
`Panc-1 and MiaPaCa-2 cells. A, cultures of Panc-1 (left) and MiaPaCa-2 cells (right) were
`incubated at a density of 1 3 104 cells in 1 ml of DMEM containing 1% FBS and 20 ng/ml
`rapamycin (E) or vehicle (F), and cells were counted at day 0, 1, 4, 5, 6, and 7 as
`indicated. Each point represents the mean of two determinations and is representative of
`at least two independent experiments. B, single cell suspensions of Panc-1 (left) or
`MiaPaCa-2 cells (right) were plated at a density of 3 3 104 cells/dish in agarose medium
`containing DMEM/1% FBS and various concentrations of rapamycin as indicated. Col-
`onies were counted after 2 weeks of incubation. In all cases, a representative of two
`independent experiments, each performed in triplicates, is shown.
`
`rapamycin, a maximum 62% reduction in BrdUrd incorporation could
`be detected in both cell lines (Fig. 2C).
`Effect of Rapamycin on Anchorage-dependent and -independ-
`ent Proliferation of Panc-1 and MiaPaCa-2 Cells. Next we exam-
`ined the effect of
`rapamycin on actual cellular proliferation.
`MiaPaCa-2 and Panc-1 cells were incubated in the absence or pres-
`ence of 20 ng/ml rapamycin, and cell numbers were determined over
`a period of up to 7 days. In the presence of rapamycin, cell numbers
`were reduced by a maximum of 66% and 68% at day 7 in Panc-1 and
`MiaPaCa-2 cells, respectively (Fig. 3A). Thus, the constitutively ac-
`tive, rapamycin-sensitive FRAP-p70s6K signaling pathway is likely to
`participate in sustaining anchorage-dependent growth of human
`Panc-1 and MiaPaCa-2 cells. In contrast to recent results obtained in
`rhabdomyosarcoma cells (18), rapamycin did not induce apoptosis in
`pancreatic cancer cells, as judged by an in situ assay to detect DNA
`fragmentation (data not shown).
`Tumors and transformed cells, including human pancreatic cancer
`cells, are able to grow in an anchorage-independent manner by form-
`Fig. 4. Inhibition of cyclin D1 expression by rapamycin in MiaPaCa-2 cells. Subcon-
`fluent cultures of MiaPaCa-2 cells were treated with 20 ng/ml rapamycin for various
`ing colonies in agarose medium. There is even a positive correlation
`times, as indicated. Control cells received an equivalent amount of solvent (—). Cells
`between the cloning efficiency of tumor cells in soft agar and the
`were lysed and further analyzed by Western blotting with either anti-p70s6K antibody,
`anti-cyclin D1 antibody, anti-cyclin E antibody, or anti-p27KIP1 antibody as indicated by
`histological involvement and invasiveness of the tumor in specimens
`an arrow. The results shown in each case are representative of at least three independent
`taken from different carcinomas (19). Consequently, we determined
`experiments. The positions of hypophosphorylated p70s6K (p70s6K) and the slower-
`the ability of MiaPaCa-2 and Panc-1 cells to form colonies in semi-
`migrating phosphorylated p70s6K (pp70s6K) are indicated by arrows.
`3585
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`Research.
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`West-Ward Pharm.
`Exhibit 1014
`Page 005
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`REGULATION OF CELL GROWTH AND CYCLIN D1 EXPRESSION
`
`resulted in a marked and sustained reduction in the expression of
`cyclin D1, compared to the levels observed in cells treated with
`vehicle alone. Reduced cyclin D1 expression was first visible after 6 h
`of incubation and was still detectable after 36 h of incubation with
`rapamycin. In contrast, expression of cyclin E and levels of p27KIP1
`expression did not change in response to rapamycin treatment (Fig. 4).
`Similar data were obtained in Panc-1 cells (data not shown). These
`results suggest that the inhibitory effect of rapamycin on basal pan-
`creatic cancer cell growth is mediated by inhibition of cyclin D1
`expression rather than by inhibition of p27KIP1 elimination.
`
`Discussion
`
`This is the first report demonstrating that the FRAP-p70s6K signal-
`ing pathway is constitutively active in serum-starved human pancre-
`atic cancer cells. The two major downstream targets of FRAP kinase,
`p70s6K and 4E-BP1, were found to be constitutively phosphorylated in
`pancreatic cancer cell lines as well as a human pancreatic cancer
`tissue sample. Activation of both could be prevented by treating cells
`with the selective FRAP inhibitor rapamycin. The effect of rapamycin
`on constitutive p70s6K2 and 4E-BP1 phosphorylation was indeed
`selective, because rapamycin did not prevent basal or TGF-a-induced
`ERK2 activation and hence tyrosine kinase activity of the epidermal
`growth factor receptor, which is required for the activation of ERK2
`by TGF-a. Interestingly, rapamycin substantially inhibited anchorage-
`dependent and -independent growth of MiaPaCa-2 and Panc-1 cells.
`p70s6K mediates 59-terminal oligopyrimidine tract mRNA translation
`(11), and 4E-BP1 is a major regulator of eIF-4E mediated 59 cap
`mRNA translation (12). Thus, these mechanisms are likely to sustain
`protein synthesis and growth in human pancreatic cancer cells. The
`precise contribution of the two FRAP targets to pancreatic cancer cell
`growth is difficult to determine. Recent data suggest that p70s6K and
`4EB-P1 are regulated by FRAP in a parallel rather than sequential
`manner (14). Furthermore, 4E-BP1 obviously plays an independent
`role in the rapamycin-sensitive regulation of cell growth: rapamycin
`can still prevent 4E-BP1 phosphorylation and cell growth in
`p70s6K2/2 cells (15). The antiproliferative effect of rapamycin in
`p70s6K2/2 cells could possibly be due to the inhibition of a novel,
`rapamycin-sensitive p70s6K isoform, which has recently been de-
`scribed in these cells (23). However, dephosphorylated 4E-BP1 has
`clearly been demonstrated to inhibit cell growth (13). Thus, constitu-
`tively phosphorylated p70s6K and 4EB-P1 are likely to contribute to
`both anchorage-dependent and -independent growth of human pan-
`creatic cancer cells. Rapamycin also blocks constitutive p70s6K phos-
`phorylation and proliferation in classical small cell lung cancer cells
`(10). Therefore,
`if cancer cells exhibit constitutive activation of
`FRAP/p70s6K, this signaling pathway is likely to play a role in the
`autonomous growth of these tumors.
`Because p70s6K regulates progression from G1 to the S phase of the
`cell cycle, we reasoned that the molecular mechanism underlying
`growth inhibition by rapamycin could be related, at least in part, to the
`cell cycle machinery.
`Indeed, the inhibitory effect of rapamycin on pancreatic cancer
`cell growth was associated with a reduction in cyclin D1 expres-
`sion. This was not due to a general inhibition of protein synthesis
`by rapamycin because we could not detect any change in the
`expression of cyclin E or p27KIP1. It
`is presently not known
`whether the effects of rapamycin on p70s6K phosphorylation/acti-
`vation, 4E-BP1 phosphorylation, and cyclin D1 expression in
`human pancreatic cancer cells are mediated by a linear, split, or
`parallel pathway(s). However, because cyclin D1 expression is
`rapamycin-sensitive in human pancreatic cancer ce