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Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating Prostate Tumor Growth and Diminishing p27Kip1 Expression

Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 32, Issue of August 11, pp. 24500 –24505, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating Prostate Tumor Growth and Kip1 Expression* Diminishing p27 Received for publication, April 12, 2000, and in revised form, May 18, 2000 Published, JBC Papers in Press, May 25, 2000, DOI 10.1074/jbc.M003145200 Jeremy R. Graff‡§¶, Bruce W. Konicek‡§, Ann M. McNulty‡, Zejing Wangi, Keith Houck**, Sheryl Allen‡, Jonathan D. Paul‡, Ahed Hbaiu‡, Robin G. Goode‡, George E. Sandusky‡, Robert L. Vessellai, and Blake Lee Neubauer‡ From the ‡Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, the iDepartment of Urology, University of Washington, Seattle, Washington 98195, and **Sphinx Pharmaceuticals, Division of Eli Lilly and Company, Research Triangle Park, North Carolina 27707 The PTEN tumor suppressor gene is frequently inac- a release from the cell cycle arrest that follows androgen with- tivated in human prostate cancers, particularly in more drawal (4). Indeed, hPCA progression has been associated re- advanced cancers, suggesting that the AKT/protein ki- peatedly with decreased expression of the cell cycle regulator Kip1 nase B (PKB) kinase, which is negatively regulated by p27 (4 –9). PTEN, may be involved in human prostate cancer pro- The PTEN/MMAC tumor suppressor gene is frequently in- gression. We now show that AKT activation and activity activated in primary human prostate cancers, particularly in are markedly increased in androgen-independent, pros- the more advanced cancers (10), and in human prostate cancer tate-specific antigen-positive prostate cancer cells xenografts and cell lines including PC-3, Du145, and LNCaP (LNAI cells) established from xenograft tumors of the (11–13). These studies suggest that components of the phos- androgen-dependent LNCaP cell line. These LNAI cells phatidylinositol 3-kinase pathway that are negatively regu- show increased expression of integrin-linked kinase, lated by PTEN, such as the key cell survival kinase AKT which is putatively responsible for AKT activation/Ser- (14 –16), may be increasingly activated with prostate tumor 473 phosphorylation, as well as for increased phospho- progression. Indeed, activated AKT regulates a number of in- rylation of the AKT target protein, BAD. Furthermore, Kip1 cell cycle regulator was dimin- expression of the p27 tracellular targets implicated in prostate tumor progression ished in LNAI cells, consistent with the notion that AKT and androgen independence. For instance, AKT-dependent in- directly inhibits AFX/Forkhead-mediated transcription activation of pro-apoptotic proteins such as BAD and caspase-9 Kip1 . To assess directly the impact of increased of p27 (17, 18) may suppress the normal apoptotic response. Addition- AKT activity on prostate cancer progression, an acti- ally, AKT may enhance cell cycle progression by suppressing vated hAKT1 mutant was overexpressed in LNCaP cells, AFX/Forkhead transcription factor activity (19 –22), which resulting in a 6-fold increase in xenograft tumor growth. would result in diminished expression of AFX target genes Like LNAI cells, these transfectants showed dramati- Kip1 Kip1 such as the cell cycle inhibitor p27 (23). Furthermore, AKT expression. Together, these data cally reduced p27 can elicit enhanced translation of key growth-regulatory pro- implicate increased AKT activity in prostate tumor pro- teins, like cyclin D1 (24), by stimulating FRAP/mTOR kinase gression and androgen independence and suggest that Kip1 expression, which has been repeat- activity and de-repressing translation initiation (Refs. 25 and diminished p27 edly associated with prostate cancer progression, may 26; depicted in Fig. 1). be a consequence of increased AKT activity. Because AKT is a central regulator of many intracellular processes implicated in prostate tumor progression and be- cause PTEN, the negative regulator of AKT, is functionally The molecular alterations that facilitate human prostate inactivated in a significant proportion of advanced hPCa, we cancer (hPCa) progression and the emergence of androgen- explored whether increased AKT activity may be involved in independent tumor cells are unclear but may involve a progres- prostate tumor progression and androgen independence. To sive decrease in the normal apoptotic response (1–3) as well as better model androgen-independent hPCa, we established a 1 1 novel series of AR , PSA , androgen-independent hPCa cells (LNAI cells) derived from xenograft tumors of the androgen- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked sensitive/dependent LNCaP prostate cancer cell line. Our anal- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to yses of these cells reveals that AKT activity is increased with indicate this fact. androgen-independent progression corresponding to increased This paper is dedicated to the memory of Charles F. Konicek. expression of integrin-linked kinase, the kinase putatively re- § Denotes equal contributions. To whom correspondence should be addressed: Lilly Research Labs, sponsible for the activation of AKT by phosphorylation at Ser- Eli Lilly and Co., Lilly Corporate Ctr., DC 0546, Indianapolis, IN 46285. 473 (Refs. 27 and 28; Fig. 1), increased phosphorylation of the Tel.: 317-277-0220; Fax: 317-277-3652; E-mail: graff_jeremy@lilly. pro-apoptotic protein BAD and markedly reduced expression of com. Kip1 the cell cycle regulator p27 . In addition, overexpression of The abbreviations used are: hPCa, human prostate cancer; RT-PCR, reverse transcription-polymerase chain reaction; AR, androgen recep- an activated AKT-1 cDNA in LNCaP cells dramatically accel- tor; PSA, prostate-specific antigen; ILK, integrin-linked kinase; AFX, Kip1 erates xenograft tumor growth and suppresses p27 expres- AFX/Forkhead transcription factor; PTEN, PTEN/MMAC tumor sup- sion. These data implicate increased AKT activity in human pressor gene; LNAI, androgen-independent derivatives of the hPCa cell line, LNCaP; FRAP/mTOR, mammalian target of rapamycin kinase. prostate cancer progression and androgen independence and 24500 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. AKT Activity Increases with Prostate Tumor Progression 24501 FIG.1. AKT activation and signaling pathways. As a downstream consequence of phosphatidylinositol 3,4,5-triphosphate (PIP ) generation by phosphatidylinositol 3-kinase (PI-3 kinase), AKT is activated via phosphorylation at Thr-308 by phosphatidylinositol-dependent kinase-1 (PDK-1) and at Ser-473 by phosphatidylinositol-dependent kinase-2 (PDK-2), which may be ILK (27, 28). The PTEN lipid phosphatase degrades phosphatidylinositol 3,4,5-triphosphate thereby negatively regulating AKT activation. PTEN loss abrogates this negative regulation (15). Upon activation, AKT can suppress the pro-apoptotic functions of BAD, via Ser-136 phosphorylation, and caspase 9 (17, 18). AKT can also phosphorylate and inactivate the AFX/Forkhead (AFX/FKHR) family of transcription factors (19 –22) which in turn suppresses AFX-mediated transcription of Kip1 target genes such as the p27 cell cycle regulator (23). AKT can specifically enhance translation of key genes such as cyclin D1 (24) by activating the FRAP/mTOR kinase and de-repressing mRNA translation initiation (25, 26). Kip1 RT-PCR Analyses—RNA was isolated as described (31) or with the suggest that the reduction in p27 expression (4 –9), which RNEasy kit (Qiagen, Santa Clarita, CA) or RNA STAT-60 reagent™ has been routinely associated with prostate tumor progression, (TEL-TEST, Inc., Friendswood, TX). RT-PCR analyses with these RNAs may be a consequence of increased AKT activity. were performed with the Titan™ single-step RT-PCR kit (Roche Molec- ular Biochemicals) using the specific primer sequences and conditions EXPERIMENTAL PROCEDURES described for AKT-1, AKT-2, and AKT-3 (32). Restriction digestion with Cell Culture and Establishment of Cell Lines ex Vivo—Cells were HaeIII or NotI (Life Technologies, Inc.) was employed to verify RT-PCR cultured in phenol red-free RPMI 1640 media (Life Technologies, Inc.) product identity. HaeIII specifically cuts AKT-1, -2, and -3 PCR prod- supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). All ucts, whereas NotI cuts only the AKT-1 RT-PCR product. xenograft studies were performed by injecting 4 3 10 cells subcutane- Transfection Analyses—LNCaP cells were transfected using Lipo- ously in 100 ml of a 1:1 mix of 13 phosphate-buffered saline and fectAMINE Plus™ reagent (Life Technologies, Inc.) with the empty Matrigel (Becton Dickinson, Bedford, MA) into castrated male nude vector pCDNA3.1 (CLONTECH) or with pCDNA3.1 carrying the cDNA mice (Harlan Sprague-Dawley nu/nu) or into intact male nude mice T308D/S473D for the activated mutant of human AKT-1 . LNCaP cells were supplemented with 90-day release testosterone pellets (Innovative Re- selected in culture media supplemented with 190 units/ml hygromycin search, Tampa, FL). First-generation LNAI cell lines, LNAI.T1 and (CalBiochem, San Diego, CA). LNAI.T2, were derived from a xenograft tumor of LNCaP established in an intact mouse that continued to grow following castration. Second RESULTS generation LNAI cells, designated T1.8, T1.16, T2.9, T2.10, and T2.11, 1 1 Establishment of AR , PSA Androgen-independent Human were established from individual xenograft tumors of LNAI.T1 and LNAI.T2, respectively, growing in castrated mice. To evaluate tumor Prostate Cancer Cell Lines—The majority of androgen-inde- formation and growth in the presence or absence of circulating andro- pendent hPCa express both the androgen receptor and pros- gens, these secondary LNAI cell lines were also then injected into intact tate-specific antigen (33). The most widely available and com- and castrated male nude mice. Tumor volumes were calculated by the 2 monly used androgen-independent human prostate cancer cell equation A 3 B 3 0.4, where A is the smallest tumor diameter, B is the lines, PC-3 and Du145, lack expression of both AR and PSA (34) largest tumor diameter, and 0.4 is a correction factor (29). Western Blot Analyses and AKT Kinase Activity Assays—Protein and therefore may not reflect the majority of advanced, andro- extracts were isolated as described (30) using radioimmune precipita- gen-independent hPCa. To better model androgen-independent tion cell lysis buffer for the xenograft tissues or 13 cell lysis buffer for prostate cancer, we developed a panel of cell lines from the the cell culture extracts (New England Biolabs, Cambridge, MA). For 1 1 AR , PSA , androgen-dependent/sensitive hPCa cell line, LN- Western blots, 20 –30 mg of protein extract/lane (as indicated in the CaP, following in vivo selection for xenograft tumor growth individual figure legends) was electrophoresed, transferred to polyvi- TM persisting after castration. These cell lines, designated LNAI nylidene membranes (Hybond-P , Amersham Pharmacia Biotech) us- TM ing the XCell II Mini-Cell apparatus (Novex, San Diego, CA), and cells, readily formed tumors both in intact, testosterone-sup- immunoblotted with the following antibodies: AKT (1:500), phospho- plemented male nude mice and in castrated male nude mice Ser-473 Kip1 AKT (1:500 dilution) (New England Biolabs); BAD (1:500), p27 (Table I), indicating that these cell lines are androgen-independ- (1:1000) (Transduction Laboratories, Lexington, KY); integrin-linked ent. Notably, the second-generation LNAI cells, T1.8, T1.16, Ser-136 kinase (1:2000) and phospho-BAD (1:250) (Upstate Biotechnology, T2.11, formed tumors more rapidly in both intact and castrated Inc., Lake Placid, NY); PSA (1:500) (M212091, Fitzgerald, Concord, hosts than the first-generation LNAI cells, LNAI.T1 and MA); and b-actin (1:2000) (Sigma). Anti-mouse IgG, anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-sheep IgG (Up- LNAI.T2. All of these LNAI cells expressed PSA (Fig. 2) and AR state Biotechnology) secondary antibodies were used at 1:1000 dilution. (data not shown). PSA was also evident in the serum from All Western blots were detected by chemiluminescence (Pierce) cap- LNAI xenograft-bearing intact and castrated mice (data not TM tured with the Lumi-imager and, where indicated, quantitated with shown). Therefore, we have successfully established androgen- TM the Lumi-analyst software (Roche Molecular Biochemicals). Immu- independent hPCa cells that, like the majority of androgen- noprecipitation/kinase assays were performed with the AKT activity kit independent hPCa, retain expression of both AR and PSA and according to the manufacturer’s instructions (New England Biolabs) using 200 mg of cell extract. share the same genetic background as the androgen-depend- 24502 AKT Activity Increases with Prostate Tumor Progression TABLE I Tumor formation: LNCaP versus LNAI cells Tumor cells (4 3 10 ) were injected subcutaneously into the left flank of intact, testosterone-supplemented or castrated male nude mice in a 1:1 mix of 13 phosphate-buffered saline and matrigel in 100 ml total volume. Tumor formation was monitored weekly. Latency period rep- resents the time (days post-injection) to a definable tumor (.100 3 3 mm ) 6 S.E. Time (days post-injection) to 1300 mm is indicated 6 S.E. Tumor incidence Latency period Time to 1300 mm Cell line Intact Castrate Intact Castrate Intact Castrate days days days LNCaP 7/7 0/5 51 6 6 N/A N/A N/A LNAI.T1 5/5 4/5 43 6048 6253 6386 6 6 LNAI.T1.8 5/5 4/5 34 6431 6453 6969 6 9 LNAI.T1.16 5/5 4/5 29 6431 6449 6545 6 6 LNAI.T2 4/5 2/5 51 6597 6 0 N/A N/A LNAI.T2.10 5/5 5/5 29 6440 6553 6588 6 8 LNAI.T2.11 4/5 5/5 26 6232 6662 6977 6 8 FIG.3. AKT activation and activity are increased in LNAI cells. Ser-473 A, activation of AKT was monitored by immunoblotting for AKT These data were pooled from two experiments. LNCaP tumors did phosphorylation in LNCaP and LNAI cell lysates (20 mg/lane). Immu- not grow to 1300 mm within 100 days-post injection. N/A, not appli- noblotting for total AKT protein was performed simultaneously on a cable. duplicate blot. To control for gel loading and transfer, blots were rou- tinely reprobed for b-actin. These immunoblots are representative of six independent experiments. B, AKT activity was assessed as described under “Experimental Procedures” following immunoprecipitation of AKT from 200 mg of cell lysate. Data are shown for LNCaP, AI.T1, T1.16, and cells derived from a xenograft tumor of T1.16 (T1.16X). These data are representative of four independent experiments. FIG.2. LNAI cells express prostate-specific antigen. Expression of PSA was evaluated in LNCaP or LNAI cell lysates by Western blot analyses (20 mg/lane). Recombinant human PSA (rPSA) was included as a positive control for PSA immunoblotting. These data are representa- tive of three independent experiments. ent/sensitive hPCa cell line, LNCaP. As such, differences be- tween LNAI and LNCaP cells can be related to androgen inde- pendence and tumor progression without being complicated by differing genetic backgrounds. AKT Activation and Activity Are Increased in the Androgen- independent LNAI Cells—Because AKT is a key regulator of many intracellular processes that have been implicated in pros- tate cancer progression and androgen independence (1, 25), we examined AKT expression and activation in androgen-inde- pendent LNAI cells relative to androgen-dependent LNCaP cells. Although AKT expression levels were unchanged (Fig. 3A), AKT activation (Ser-473 phosphorylation) was markedly increased in LNAI cells relative to LNCaP, particularly in the FIG.4. RT-PCR analyses for expression of AKT-1, AKT-2, and more aggressively growing second-generation LNAI cells, T1.8, AKT-3. RT-PCR analyses using primers specific for each AKT isoform T1.16, and T2.11, (Fig. 3A) and in xenografts (data not shown). (33) were run for 35 PCR cycles. The product sizes, in base pairs, are 330 for AKT-1, 335 for AKT-2, and 327 for AKT-3. A, analyses of LNCaP Phosphorylation of AKT at Thr-308, which is also required for and the second-generation LNAI cell lines T1.16 and T2.9. B, expres- activation, was not increased in LNAI cells (data not shown). sion of AKT-1 and AKT-3 in primary prostate cancer and normal pros- Moreover, AKT activity was also increased in LNAI cells rela- tate tissue. PC-3 is included as a positive control for AKT-3 expression, tive to LNCaP cells (Fig. 3B). Together, these data indicate that whereas LNCaP and T1.16 lack AKT-3 expression. The identity of the AKT-3 RT-PCR products was verified by restriction digestion with AKT activation (Ser-473 phosphorylation) and activity are in- HaeIII and NotI (data not shown). C, expression of AKT-1 and AKT-3 in creased in androgen-independent LNAI cells, particularly the tissue from normal prostate, prostate cancer, and prostate cancer me- more aggressively growing, second-generation lines (T1.8, tastases to bone and soft tissues. Note that each of these samples was T1.16, and T2.11), relative to androgen-dependent LNCaP positive for b -microglobulin by RT-PCR (data not shown). cells. Androgen-independent LNAI Cells Express AKT-1 and primers designed specifically for each AKT isoform (32) re- AKT-2 but not AKT-3—Three isoforms of human AKT have vealed that, like LNCaP cells, LNAI cells express only AKT-1 been isolated, AKT-1, AKT-2, and AKT-3 (25, 35). Expression of and AKT-2 (Fig. 4A), suggesting that the evolution of andro- AKT-3 has been specifically associated with hormone-indepen- gen-independent hPCa cells does not necessarily involve up- dent primary human breast cancers and breast cancer cell regulated AKT-3 expression. To evaluate further whether lines. Likewise, the androgen-independent human prostate AKT-3 expression is induced during hPCa development or pro- cancer cell lines PC-3 and Du145 express AKT-3, whereas the gression, we analyzed primary human prostate cancers, pros- androgen-dependent/sensitive LNCaP hPCa cell line expresses tate cancer metastases to bone and soft tissues, and normal only AKT-1 and AKT-2, suggesting that androgen-independent prostate tissue for expression of AKT-1 and AKT-3 (Fig. 4, B hPCA progression may involve AKT-3 induction (32). We there- and C). Our analyses reveal that, although not all tissues fore examined whether the increased AKT activity in LNAI express AKT-3, the majority of both normal and tumor tissue cells might reflect AKT-3 expression. RT-PCR analyses with shows AKT-3 expression, thereby indicating that AKT-3 ex- AKT Activity Increases with Prostate Tumor Progression 24503 Ser-136 FIG.6. BAD phosphorylation is increased in LNAI cells and xenograft tissue. Phosphorylation of BAD was detected by West- Ser-136 ern blotting with a BAD phospho-specific antibody (30 mg extract/ lane). Parallel Western blots were also run and immunoblotted for total Ser-136 BAD protein. Signal intensity for pBAD was normalized for BAD expression. The data presented are from a single experiment and are representative of six independent experiments with the cell culture lysates (upper panel) and three experiments with xenograft tissue ly- sates (lower panel). In the lower panel, each bar represents an individ- ual xenograft tumor. In both panels, hatched bars represent LNAI FIG.5. Integrin-linked kinase expression is increased in LNAI lysates, and the clear bars represent LNCaP lysates. cells. Expression of ILK was assessed by Western blotting of cell culture extracts (A) or extracts from individual xenografts (B) (20 mg/ lane). Blots were reprobed for expression of b-actin to control for loading and transfer. Specific bands for the 59-kDa ILK and the 46-kDa b-actin proteins are indicated. These blots are representative of three inde- pendent experiments each with the cell culture and xenograft lysates. Note that each of the LNAI xenograft tissues has high level expression of ILK, whereas only one of the four LNCaP xenograft tissues shows high level ILK expression. Kip1 FIG.7. p27 expression is diminished in LNAI cells. Expres- Kip1 sion of the cell cycle regulatory protein p27 was examined by West- pression is not induced in, or restricted to, malignancy. ern blotting (20 mg lysate/lane). Data presented are representative of LNAI Cells Overexpress Integrin-linked Kinase—Integrin- four independent experiments. linked kinase (ILK) has been putatively identified as responsi- Ser-473 ble for AKT phosphorylation (27, 28). Western blot anal- edly enhanced in androgen-independent LNAI cells compared yses revealed that ILK is overexpressed in LNAI cells and with androgen-dependent/sensitive LNCaP and suggest that xenografts (Fig. 5), particularly in the second-generation LNAI increased AKT activity may be intimately involved in the pro- cells in which AKT activation was highest (e.g. T1.16, Fig. 5), gression of hPCa and the emergence of androgen-independent Ser-473 suggesting that the enhanced AKT phosphorylation may hPCa cells. To determine directly how AKT may impact upon be a downstream consequence of up-regulated ILK expression. hPCa progression, we established stable overexpressors of the T308D/S473D Increased AKT Activity in LNAI Cells Corresponds to In- constitutively active AKT-1 mutant in LNCaP cells Ser-136 Kip1 creased BAD DD Phosphorylation and Decreased p27 Ex- (LNCaP:AKT cells) (Fig. 8, inset). Subcutaneous injection of DD pression—Because AKT activity was increased in androgen- these LNCaP:AKT cells into intact, testosterone-supple- independent LNAI cells, we addressed the question of whether mented male nude mice yielded rapidly growing tumors with molecules downstream from AKT were differentially affected in 100% incidence (4/4) in less than 30 days post-injection, LNAI cells compared with LNCaP. The pro-apoptotic protein whereas vector control cells formed tumors in only three of five BAD can be functionally inactivated by phosphorylation at mice that were not evident until day 37 post-injection (Fig. 8). Ser-136 by AKT (17, 36, 37). Xenograft tissue and cell culture DD Moreover, the LNCaP:AKT cells showed a dramatic increase extracts of LNAI showed increased phosphorylation of BAD at in tumor growth (Fig. 8), with a mean tumor volume more than the AKT target site, Ser-136, relative to LNCaP cell and xe- 6-fold that of the vector control transfectants at all time points nograft extracts (Fig. 6). (by Dunnett’s test on ranked tumor volumes, p , 0.05 for all AKT has recently been shown to phosphorylate and inacti- time points). Like the aggressively growing LNAI cells, LNCaP: DD Kip1 vate the Forkhead/AFX family of transcription factors (19 –22). AKT tumor cells showed a substantial decrease in p27 As AFX activates transcription of the cell cycle regulator expression (Fig. 8, inset). Tumors did not form in castrated Kip1 DD p27 (23), inhibition of AFX-mediated transcription by AKT mice following injection with either LNCaP:AKT or vector Kip1 may lead to decreased expression of p27 . In LNAI cells, in control LNCaP cells. A second, independent experiment con- Kip1 DD which AKT is hyperactivated, the protein expression of p27 firmed that LNCaP:AKT cells formed more rapidly growing was markedly reduced relative to that in LNCaP (Fig. 7). This tumors in intact male nude mice when compared with vector decrease was most pronounced in the most aggressively grow- control LNCaP cells but were unable to form tumors in cas- ing, second-generation LNAI cells with the highest AKT activ- trated male nude mice (data not shown). Therefore, although ity (e.g. T1.16). These data are consistent with the notion that insufficient to elicit growth in castrated male nude mice, over- AKT negatively regulates AFX-mediated transcription of DD expression of the constitutively active AKT dramatically en- Kip1 p27 but do not exclude the possibility that other mecha- hanced LNCaP xenograft tumor growth in intact male nude Kip1 nisms, such as decreased protein or RNA stability, may con- mice and resulted in markedly reduced expression of p27 . Kip1 tribute to p27 loss. Together, these data indicate that the DISCUSSION increased AKT activity in androgen-independent LNAI cells profoundly effects key downstream mediators involved in cell We have developed a series of androgen-independent hPCa cycle arrest and apoptotic signaling. cell lines (LNAI cells) from the androgen-dependent/sensitive Stable Overexpression of Constitutively Active AKT Dramat- hPCA cell line, LNCaP. Like the majority of androgen-independ- ically Enhances LNCaP Xenograft Tumor Growth in Intact ent hPCa (34), these LNAI cells retain expression of AR and Male Nude Mice—Our data indicate that AKT activity is mark- PSA. We have now shown that AKT activation (Ser-473 phos- 24504 AKT Activity Increases with Prostate Tumor Progression emergence of androgen-independent hPCa cells, such as a di- minished apoptotic response (1, 2, 39) as well as a release from the cell cycle control that follows androgen ablation (4). AKT can dampen the normal apoptotic response by suppressing the activity of numerous pro-apoptotic proteins including BAD, caspase 9, and the Forkhead family of transcription factors (Refs. 17–22; summarized in Fig. 1). AKT can also facilitate the release of cells from cell cycle control by inhibiting AFX/Fork- head-mediated transcription of the key cell cycle regulator, Kip1 p27 (23). Further, by activating FRAP/mTOR and de-re- pressing translation initiation, AKT may specifically enhance FIG.8. Xenograft growth of LNCaP:AKT transfectants in in- the translation of cyclin D1 (24) as well as other translationally DD tact male nude mice. LNCaP:AKT or LNCaP vector control cells controlled growth factors and growth regulatory proteins im- were injected (4 3 10 cells) into intact, testosterone-supplemented plicated in human prostate cancer progression (Refs. 40 – 43; male nude mice. Tumor volume (mm ) is depicted on the y axis and was calculated as described (see “Experimental Procedures”). Hatched bars see Fig. 1). DD represent the tumor volumes for individual LNCaP:AKT tumors, The data in this report clearly imply increased AKT activity whereas white bars represent individual LNCaP vector control tumors. in hPCa progression. The most aggressively growing, andro- DD LNCaP:AKT formed tumors in four of four mice injected, whereas the gen-independent LNAI cells had the greatest increase in AKT LNCaP vector control formed tumors in only three of five mice injected. The inset shows Western blot analyses (20 mg extract/lane) in LNCaP: activity. Moreover, overexpression of an activated AKT-1 mu- DD AKT transfectants and vector control LNCaP transfectants for ex- tant dramatically accelerated LNCaP xenograft tumor growth. pression of AKT (representative of three independent experiments) and However, our data also reveal that overexpression of an acti- Kip1 p27 (representative of six independent experiments). To control for vated AKT-1 was alone insufficient to drive xenograft tumor gel loading and transfer, these blots were reprobed for b-actin expres- growth in castrated mice, suggesting that factors in addition to sion. A second xenograft experiment showed a similar increase in LN- DD CaP:AKT xenograft growth relative to the LNCaP vector control increased AKT activity must contribute to the ability of LNAI (data not shown). cells to form and grow tumors in the absence of circulating androgens. For instance, the increased expression of ILK in phorylation) and activity are increased in these LNAI cells LNAI cells may influence alternate pathways in addition to the when compared with androgen-dependent LNCaP cells and AKT pathway, such as the Wnt pathway (27), that may con- may be a consequence of increased expression of the integrin- tribute to the androgen-independent phenotype. Moreover, Ser-473 linked kinase, the kinase putatively responsible for AKT other intracellular pathways may be activated in LNAI cells phosphorylation (27, 28). Like parental LNCaP cells, LNAI independent of, or upstream from, AKT that contribute to the cells express only AKT-1 and AKT-2, indicating that AKT-3 androgen-independent phenotype of LNAI cells. Indeed, acti- expression is not necessary for androgen independence, as has vation of the mitogen-activated protein kinase pathways, per- been suggested in studies with the androgen-independent PC-3 haps as a downstream consequence of HER2 activation, has and Du145 hPCa cells (32). Moreover, our data reveal that been implicated in hPCa progression and androgen independ- AKT-3 is expressed not only in prostate cancer tissues but also ence (44 – 46). in normal prostate tissue, indicating that AKT-3 expression is In summary, the data presented in this report indicate that not induced in, or restricted to, prostate malignancy. We have increased AKT activity, perhaps as a consequence of increased also shown that the increased AKT activity in these LNAI cells ILK expression, is involved in hPCa progression and suggest Kip1 corresponds to increased phosphorylation of the pro-apoptotic that the reduction in p27 expression, which has been re- protein, BAD, and markedly decreased expression of the peatedly associated with hPCa progression and androgen inde- Kip1 p27 cell cycle regulator, consistent with the notion that pendence (4 –9), may be a consequence of enhanced AKT activ- AKT inhibits AFX-mediated transcriptional activation of ity. Together with previously published reports that PTEN is Kip1 p27 (23, 38). Taken together, these data imply increased inactivated in a significant proportion of advanced primary AKT activity in hPCa progression and the emergence of andro- human prostate cancers (10) and that AKT regulates many of gen-independent human prostate cancer cells. the intracellular processes associated with hPCa progression To address whether AKT alone can drive prostate tumor (1, 25), the data in this report implicate AKT as a key mediator progression and/or the emergence of androgen-independent of prostate tumor progression and suggest that AKT may be a cells, we established LNCaP cells with stable overexpression of prime target for prostate cancer therapy. a constitutively active AKT-1 cDNA. Though alone insufficient Acknowledgments—We thank Drs. Mark Marshall, James G. to elicit xenograft tumor growth in castrated mice, overexpres- Herman, Jake Starling, and Steven M. Paul for critical review of this sion of activated AKT dramatically increased tumor growth in manuscript. We thank Zandra Lee for collecting some of the tissues intact mice relative to vector control transfectants. Like the used for RT-PCR analyses. We also thank Drs. Homer Pearce and most aggressively growing LNAI cells (e.g. T1.16), these LN- Steven M. Paul for supporting this work. DD CaP:AKT cells also showed a remarkable reduction in REFERENCES Kip1 Kip1 p27 expression, suggesting that p27 loss, which has 1. Isaacs, J. T. (1999) Urol. Clin. North Am. 26, 263–273 been repeatedly linked to hPCa progression (4 –9), may be a 2. Denmeade, S. R., Lin, X. S., and Isaacs, J. T. (1996) Prostate 28, 251–265 3. Berges, R. R., Vukanovic, J., Epstein, J. I., CarMichel, M., Cisek, L., Johnson, consequence of increased AKT activity. Taken together with D. E., Veltri, R. W., Walsh, P. C., and Isaacs, J. T. (1995) Clin. Cancer Res. the analyses of LNAI cells, these data reveal that increased 1, 473– 480 4. Agus, D. B., Cordon-Cardo, C., Fox, W., Drobnjak, M., Koff, A., Golde, D. W., AKT activity contributes to hPCa progression by dramatically and Scher, H. I. (1999) J. Natl. Cancer Inst. 91, 1869 –1876 accelerating prostate tumor growth, perhaps in relation to de- 5. Macri, E., and Loda, M. (1998 –99) Cancer Metastasis Rev. 17, 337–344 Kip1 creasing p27 expression, and is specifically associated with 6. Guo, Y., Sklar, G. N., Borkowski, A., and Kyprianou, N. (1997) Clin Cancer Res. 3, 2269 –2274 the more advanced, aggressively growing androgen-independ- 7. Yang, R. M., Naitoh, J., Murphy, M., Wang, H. J., Phillipson, J., deKernion, ent hPCa cells (e.g. T1.16). J. B., Loda, M., and Reiter, R. E. (1998) J. Urol. 159, 941–945 8. Tsihlias, J., Kapusta, L. R., DeBoer, G., Morava-Protzner, I., Zbieranowski, I., Increased AKT activity may play a profound role in the Bhattacharya, N., Catzavelos, G. C., Klotz, L. H., and Slingerland, J. M. progression of human prostate cancers. AKT regulates many of (1998) Cancer Res. 58, 542–548 the processes associated with metastatic progression and the 9. De Marzo, A. M., Meeker, A. K., Epstein, J. I., and Coffey, D. S. (1998) Am. J. AKT Activity Increases with Prostate Tumor Progression 24505 Pathol. 153, 911–919 (1998) Genes Dev. 12, 502–513 10. Ittmann, M. M. (1998) Oncol. Rep. 5, 1329 –1335 27. Delcommenne, M., Tan, C., Gray, V., Rue, L., Woodgett, J., and Dedhar, S. 11. Whang, Y. E., Wu, X., Suzuki, H., Reiter, R. E., Tran, C., Vessella, R. L., Said, (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11211–11216 J. W., Isaacs, W. B., and Sawyers, C. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 28. Lynch, D. K., Ellis, C. A., Edwards, P. A., and Hiles, I. D. (1999) Oncogene 18, 95, 5246 –5250 8024 – 8032 12. Vlietstra, R. J., van Alewijk, D. C., Hermans, K. G., van Steenbrugge, G. J., 29. Attia, M. A., and Weiss, D. W. (1966) Cancer Res. 26, 1787–1800 and Trapman, J. (1998) Cancer Res. 58, 2720 –2723 30. Graff, J. R., Boghaert, E. R., De Benedetti, A., Tudor, D. L., Zimmer, C. C., 13. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Chan, S. K., and Zimmer, S. G. (1995) Int. J. Cancer 60, 255–263 Miliaresis C, Rodgers, L., McCombie, R., Bigner, S. H., Giovanella, B. C., 31. Graff, J. R., De Benedetti, A., Olson, J. W., Tamez, P., Casero, R. A., Jr., and Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M. H., and Parsons, R. Zimmer, S. G. (1997) Biochem. Biophys. Res. Commun. 240, 15–20 (1997) Science 275, 1943–1947 32. Nakatani, K., Thompson, D. A., Barthel, A., Sakaue, H., Liu, W., Weigel, R. J., 14. Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C., and Roth, R. A. (1999) J. Biol. Chem. 274, 21528 –21532 Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., and Mak, T. W. 33. Craft, N., and Sawyers, C. L. (1998 –99) Cancer Metastasis Rev. 17: 421– 427 (1998) Cell 95, 29 –39 34. Webber, M. M., Bello, D., and Quader, S. (1997) Prostate 30, 58–64 15. Cantley, L. C., and Neel, B. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 35. Nakatani, K., Sakaue, H., Thompson, D. A., Weigel, R. J., and Roth, R. A. 4240 – 4245 (1999) Biochem. Biophys. Res. Commun. 257, 906 –910 16. Wu, X., Senechal, K., Neshat, M. S., Whang, Y. E., and Sawyers, C. L. (1998) 36. Craddock, B. L., Orchiston, E. A., Hinton, H. J., and Welham, M. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 95, 15587–15591 J. Biol. Chem. 274, 10633–10640 17. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, 37. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. Science M. E. (1997) Cell 91, 231–241 (1997) 278, 687– 689 18. Cardone, M. H., Roy, N., Stennicke, H. R., Salvesen, G. S., Franke, T. F., 38. Ramaswamy, S., Nakamura, N., Vazquez, F., Batt, D. B., Perera, S., Roberts, Stanbridge, E., Frisch, S., and Reed. J. C. (1998) Science 282, 1318 –1321 T. M., and Sellers, W. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 19. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, 2110 –2115 M. J., Arden, K. C., Blenis, J., and Greenberg, M. (1999) Cell 96, 857– 868 39. McConkey, D. J., Greene, G., and Pettaway, C. A. (1996) Cancer Res. 56, 20. Kops, G. J., de Ruiter, N. D., De Vries-Smits, A. M., Powell, D. R., Bos, J. L., 5594 –5599 and Burgering, B. M. (1999) Nature 398, 630 – 634 40. Russell, P. J., Bennett, S., and Stricker, P. (1998) Clin. Chem. 44, 705–723 21. Tang, E. D., Nunez, G., Barr, F. G., and Guan, K. L. (1999) J. Biol. Chem. 274, 41. Culig, Z., Hobisch, A., Cronauer, M. V., Radmayr, C., Hittmair, A., Zhang, J., 16741–16746 Thurnher, M., Bartsch, G., and Klocker, H. (1996) Prostate 28, 392– 405 22. Takaishi, H., Konishi, H., Matsuzaki, H., Ono, Y., Shirai, Y., Saito, N., 42. Raught, B., and Gingras, A. C. (1999) Int. J. Biochem. Cell Biol. 31, 43–57 Kitamura, T., Ogawa, W., Kasuga, M., Kikkawa, U., and Nishizuka, Y. 43. De Benedetti, A., and Harris, A. L. (1999) Int. J. Biochem. Cell Biol. 31, 59 –72 (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11836 –11841 44. Gioeli, D., Mandell, J. W., Petroni, G. R., Frierson, H. F., Jr., and Weber, M. J. 23. Medema, R. H., Kops, G. J., Bos, J. L., and Burgering, B. M. (2000) Nature 404, (1999) Cancer Res. 59, 279 –284 782–787 45. Craft, N., Shostak, Y., Carey, M., and Sawyers, C. L. (1999) Nat. Med. 5, 24. Muise-Helmericks, R. C., Grimes, H. L., Bellacosa, A., Malstrom, S. E., Tsichlis, 280 –285 P. N., and Rosen, N. (1998) J. Biol. Chem. 273, 29864 –29872 25. Coffer, P. J., Jin, J., and Woodgett, J. R. (1998) Biochem J. 335, 1–13 46. Yeh, S., Lin, H. K., Kang, H. Y., Thin, T. H., Lin, M. F., and Chang, C. (1999) 26. Gingras. A. C., Kennedy, S. G., O’Leary, M. A., Sonenberg, N., and Hay, N. Proc. Natl. Acad. Sci. U. S. A. 96, 5458 –5463 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating Prostate Tumor Growth and Diminishing p27Kip1 Expression

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 32, Issue of August 11, pp. 24500 –24505, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating Prostate Tumor Growth and Kip1 Expression* Diminishing p27 Received for publication, April 12, 2000, and in revised form, May 18, 2000 Published, JBC Papers in Press, May 25, 2000, DOI 10.1074/jbc.M003145200 Jeremy R. Graff‡§¶, Bruce W. Konicek‡§, Ann M. McNulty‡, Zejing Wangi, Keith Houck**, Sheryl Allen‡, Jonathan D. Paul‡, Ahed Hbaiu‡, Robin G. Goode‡, George E. Sandusky‡, Robert L. Vessellai, and Blake Lee Neubauer‡ From the ‡Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, the iDepartment of Urology, University of Washington, Seattle, Washington 98195, and **Sphinx Pharmaceuticals, Division of Eli Lilly and Company, Research Triangle Park, North Carolina 27707 The PTEN tumor suppressor gene is frequently inac- a release from the cell cycle arrest that follows androgen with- tivated in human prostate cancers, particularly in more drawal (4). Indeed, hPCA progression has been associated re- advanced cancers, suggesting that the AKT/protein ki- peatedly with decreased expression of the cell cycle regulator Kip1 nase B (PKB) kinase, which is negatively regulated by p27 (4 –9). PTEN, may be involved in human prostate cancer pro- The PTEN/MMAC tumor suppressor gene is frequently in- gression. We now show that AKT activation and activity activated in primary human prostate cancers, particularly in are markedly increased in androgen-independent, pros- the more advanced cancers (10), and in human prostate cancer tate-specific antigen-positive prostate cancer cells xenografts and cell lines including PC-3, Du145, and LNCaP (LNAI cells) established from xenograft tumors of the (11–13). These studies suggest that components of the phos- androgen-dependent LNCaP cell line. These LNAI cells phatidylinositol 3-kinase pathway that are negatively regu- show increased expression of integrin-linked kinase, lated by PTEN, such as the key cell survival kinase AKT which is putatively responsible for AKT activation/Ser- (14 –16), may be increasingly activated with prostate tumor 473 phosphorylation, as well as for increased phospho- progression. Indeed, activated AKT regulates a number of in- rylation of the AKT target protein, BAD. Furthermore, Kip1 cell cycle regulator was dimin- expression of the p27 tracellular targets implicated in prostate tumor progression ished in LNAI cells, consistent with the notion that AKT and androgen independence. For instance, AKT-dependent in- directly inhibits AFX/Forkhead-mediated transcription activation of pro-apoptotic proteins such as BAD and caspase-9 Kip1 . To assess directly the impact of increased of p27 (17, 18) may suppress the normal apoptotic response. Addition- AKT activity on prostate cancer progression, an acti- ally, AKT may enhance cell cycle progression by suppressing vated hAKT1 mutant was overexpressed in LNCaP cells, AFX/Forkhead transcription factor activity (19 –22), which resulting in a 6-fold increase in xenograft tumor growth. would result in diminished expression of AFX target genes Like LNAI cells, these transfectants showed dramati- Kip1 Kip1 such as the cell cycle inhibitor p27 (23). Furthermore, AKT expression. Together, these data cally reduced p27 can elicit enhanced translation of key growth-regulatory pro- implicate increased AKT activity in prostate tumor pro- teins, like cyclin D1 (24), by stimulating FRAP/mTOR kinase gression and androgen independence and suggest that Kip1 expression, which has been repeat- activity and de-repressing translation initiation (Refs. 25 and diminished p27 edly associated with prostate cancer progression, may 26; depicted in Fig. 1). be a consequence of increased AKT activity. Because AKT is a central regulator of many intracellular processes implicated in prostate tumor progression and be- cause PTEN, the negative regulator of AKT, is functionally The molecular alterations that facilitate human prostate inactivated in a significant proportion of advanced hPCa, we cancer (hPCa) progression and the emergence of androgen- explored whether increased AKT activity may be involved in independent tumor cells are unclear but may involve a progres- prostate tumor progression and androgen independence. To sive decrease in the normal apoptotic response (1–3) as well as better model androgen-independent hPCa, we established a 1 1 novel series of AR , PSA , androgen-independent hPCa cells (LNAI cells) derived from xenograft tumors of the androgen- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked sensitive/dependent LNCaP prostate cancer cell line. Our anal- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to yses of these cells reveals that AKT activity is increased with indicate this fact. androgen-independent progression corresponding to increased This paper is dedicated to the memory of Charles F. Konicek. expression of integrin-linked kinase, the kinase putatively re- § Denotes equal contributions. To whom correspondence should be addressed: Lilly Research Labs, sponsible for the activation of AKT by phosphorylation at Ser- Eli Lilly and Co., Lilly Corporate Ctr., DC 0546, Indianapolis, IN 46285. 473 (Refs. 27 and 28; Fig. 1), increased phosphorylation of the Tel.: 317-277-0220; Fax: 317-277-3652; E-mail: graff_jeremy@lilly. pro-apoptotic protein BAD and markedly reduced expression of com. Kip1 the cell cycle regulator p27 . In addition, overexpression of The abbreviations used are: hPCa, human prostate cancer; RT-PCR, reverse transcription-polymerase chain reaction; AR, androgen recep- an activated AKT-1 cDNA in LNCaP cells dramatically accel- tor; PSA, prostate-specific antigen; ILK, integrin-linked kinase; AFX, Kip1 erates xenograft tumor growth and suppresses p27 expres- AFX/Forkhead transcription factor; PTEN, PTEN/MMAC tumor sup- sion. These data implicate increased AKT activity in human pressor gene; LNAI, androgen-independent derivatives of the hPCa cell line, LNCaP; FRAP/mTOR, mammalian target of rapamycin kinase. prostate cancer progression and androgen independence and 24500 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. AKT Activity Increases with Prostate Tumor Progression 24501 FIG.1. AKT activation and signaling pathways. As a downstream consequence of phosphatidylinositol 3,4,5-triphosphate (PIP ) generation by phosphatidylinositol 3-kinase (PI-3 kinase), AKT is activated via phosphorylation at Thr-308 by phosphatidylinositol-dependent kinase-1 (PDK-1) and at Ser-473 by phosphatidylinositol-dependent kinase-2 (PDK-2), which may be ILK (27, 28). The PTEN lipid phosphatase degrades phosphatidylinositol 3,4,5-triphosphate thereby negatively regulating AKT activation. PTEN loss abrogates this negative regulation (15). Upon activation, AKT can suppress the pro-apoptotic functions of BAD, via Ser-136 phosphorylation, and caspase 9 (17, 18). AKT can also phosphorylate and inactivate the AFX/Forkhead (AFX/FKHR) family of transcription factors (19 –22) which in turn suppresses AFX-mediated transcription of Kip1 target genes such as the p27 cell cycle regulator (23). AKT can specifically enhance translation of key genes such as cyclin D1 (24) by activating the FRAP/mTOR kinase and de-repressing mRNA translation initiation (25, 26). Kip1 RT-PCR Analyses—RNA was isolated as described (31) or with the suggest that the reduction in p27 expression (4 –9), which RNEasy kit (Qiagen, Santa Clarita, CA) or RNA STAT-60 reagent™ has been routinely associated with prostate tumor progression, (TEL-TEST, Inc., Friendswood, TX). RT-PCR analyses with these RNAs may be a consequence of increased AKT activity. were performed with the Titan™ single-step RT-PCR kit (Roche Molec- ular Biochemicals) using the specific primer sequences and conditions EXPERIMENTAL PROCEDURES described for AKT-1, AKT-2, and AKT-3 (32). Restriction digestion with Cell Culture and Establishment of Cell Lines ex Vivo—Cells were HaeIII or NotI (Life Technologies, Inc.) was employed to verify RT-PCR cultured in phenol red-free RPMI 1640 media (Life Technologies, Inc.) product identity. HaeIII specifically cuts AKT-1, -2, and -3 PCR prod- supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). All ucts, whereas NotI cuts only the AKT-1 RT-PCR product. xenograft studies were performed by injecting 4 3 10 cells subcutane- Transfection Analyses—LNCaP cells were transfected using Lipo- ously in 100 ml of a 1:1 mix of 13 phosphate-buffered saline and fectAMINE Plus™ reagent (Life Technologies, Inc.) with the empty Matrigel (Becton Dickinson, Bedford, MA) into castrated male nude vector pCDNA3.1 (CLONTECH) or with pCDNA3.1 carrying the cDNA mice (Harlan Sprague-Dawley nu/nu) or into intact male nude mice T308D/S473D for the activated mutant of human AKT-1 . LNCaP cells were supplemented with 90-day release testosterone pellets (Innovative Re- selected in culture media supplemented with 190 units/ml hygromycin search, Tampa, FL). First-generation LNAI cell lines, LNAI.T1 and (CalBiochem, San Diego, CA). LNAI.T2, were derived from a xenograft tumor of LNCaP established in an intact mouse that continued to grow following castration. Second RESULTS generation LNAI cells, designated T1.8, T1.16, T2.9, T2.10, and T2.11, 1 1 Establishment of AR , PSA Androgen-independent Human were established from individual xenograft tumors of LNAI.T1 and LNAI.T2, respectively, growing in castrated mice. To evaluate tumor Prostate Cancer Cell Lines—The majority of androgen-inde- formation and growth in the presence or absence of circulating andro- pendent hPCa express both the androgen receptor and pros- gens, these secondary LNAI cell lines were also then injected into intact tate-specific antigen (33). The most widely available and com- and castrated male nude mice. Tumor volumes were calculated by the 2 monly used androgen-independent human prostate cancer cell equation A 3 B 3 0.4, where A is the smallest tumor diameter, B is the lines, PC-3 and Du145, lack expression of both AR and PSA (34) largest tumor diameter, and 0.4 is a correction factor (29). Western Blot Analyses and AKT Kinase Activity Assays—Protein and therefore may not reflect the majority of advanced, andro- extracts were isolated as described (30) using radioimmune precipita- gen-independent hPCa. To better model androgen-independent tion cell lysis buffer for the xenograft tissues or 13 cell lysis buffer for prostate cancer, we developed a panel of cell lines from the the cell culture extracts (New England Biolabs, Cambridge, MA). For 1 1 AR , PSA , androgen-dependent/sensitive hPCa cell line, LN- Western blots, 20 –30 mg of protein extract/lane (as indicated in the CaP, following in vivo selection for xenograft tumor growth individual figure legends) was electrophoresed, transferred to polyvi- TM persisting after castration. These cell lines, designated LNAI nylidene membranes (Hybond-P , Amersham Pharmacia Biotech) us- TM ing the XCell II Mini-Cell apparatus (Novex, San Diego, CA), and cells, readily formed tumors both in intact, testosterone-sup- immunoblotted with the following antibodies: AKT (1:500), phospho- plemented male nude mice and in castrated male nude mice Ser-473 Kip1 AKT (1:500 dilution) (New England Biolabs); BAD (1:500), p27 (Table I), indicating that these cell lines are androgen-independ- (1:1000) (Transduction Laboratories, Lexington, KY); integrin-linked ent. Notably, the second-generation LNAI cells, T1.8, T1.16, Ser-136 kinase (1:2000) and phospho-BAD (1:250) (Upstate Biotechnology, T2.11, formed tumors more rapidly in both intact and castrated Inc., Lake Placid, NY); PSA (1:500) (M212091, Fitzgerald, Concord, hosts than the first-generation LNAI cells, LNAI.T1 and MA); and b-actin (1:2000) (Sigma). Anti-mouse IgG, anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-sheep IgG (Up- LNAI.T2. All of these LNAI cells expressed PSA (Fig. 2) and AR state Biotechnology) secondary antibodies were used at 1:1000 dilution. (data not shown). PSA was also evident in the serum from All Western blots were detected by chemiluminescence (Pierce) cap- LNAI xenograft-bearing intact and castrated mice (data not TM tured with the Lumi-imager and, where indicated, quantitated with shown). Therefore, we have successfully established androgen- TM the Lumi-analyst software (Roche Molecular Biochemicals). Immu- independent hPCa cells that, like the majority of androgen- noprecipitation/kinase assays were performed with the AKT activity kit independent hPCa, retain expression of both AR and PSA and according to the manufacturer’s instructions (New England Biolabs) using 200 mg of cell extract. share the same genetic background as the androgen-depend- 24502 AKT Activity Increases with Prostate Tumor Progression TABLE I Tumor formation: LNCaP versus LNAI cells Tumor cells (4 3 10 ) were injected subcutaneously into the left flank of intact, testosterone-supplemented or castrated male nude mice in a 1:1 mix of 13 phosphate-buffered saline and matrigel in 100 ml total volume. Tumor formation was monitored weekly. Latency period rep- resents the time (days post-injection) to a definable tumor (.100 3 3 mm ) 6 S.E. Time (days post-injection) to 1300 mm is indicated 6 S.E. Tumor incidence Latency period Time to 1300 mm Cell line Intact Castrate Intact Castrate Intact Castrate days days days LNCaP 7/7 0/5 51 6 6 N/A N/A N/A LNAI.T1 5/5 4/5 43 6048 6253 6386 6 6 LNAI.T1.8 5/5 4/5 34 6431 6453 6969 6 9 LNAI.T1.16 5/5 4/5 29 6431 6449 6545 6 6 LNAI.T2 4/5 2/5 51 6597 6 0 N/A N/A LNAI.T2.10 5/5 5/5 29 6440 6553 6588 6 8 LNAI.T2.11 4/5 5/5 26 6232 6662 6977 6 8 FIG.3. AKT activation and activity are increased in LNAI cells. Ser-473 A, activation of AKT was monitored by immunoblotting for AKT These data were pooled from two experiments. LNCaP tumors did phosphorylation in LNCaP and LNAI cell lysates (20 mg/lane). Immu- not grow to 1300 mm within 100 days-post injection. N/A, not appli- noblotting for total AKT protein was performed simultaneously on a cable. duplicate blot. To control for gel loading and transfer, blots were rou- tinely reprobed for b-actin. These immunoblots are representative of six independent experiments. B, AKT activity was assessed as described under “Experimental Procedures” following immunoprecipitation of AKT from 200 mg of cell lysate. Data are shown for LNCaP, AI.T1, T1.16, and cells derived from a xenograft tumor of T1.16 (T1.16X). These data are representative of four independent experiments. FIG.2. LNAI cells express prostate-specific antigen. Expression of PSA was evaluated in LNCaP or LNAI cell lysates by Western blot analyses (20 mg/lane). Recombinant human PSA (rPSA) was included as a positive control for PSA immunoblotting. These data are representa- tive of three independent experiments. ent/sensitive hPCa cell line, LNCaP. As such, differences be- tween LNAI and LNCaP cells can be related to androgen inde- pendence and tumor progression without being complicated by differing genetic backgrounds. AKT Activation and Activity Are Increased in the Androgen- independent LNAI Cells—Because AKT is a key regulator of many intracellular processes that have been implicated in pros- tate cancer progression and androgen independence (1, 25), we examined AKT expression and activation in androgen-inde- pendent LNAI cells relative to androgen-dependent LNCaP cells. Although AKT expression levels were unchanged (Fig. 3A), AKT activation (Ser-473 phosphorylation) was markedly increased in LNAI cells relative to LNCaP, particularly in the FIG.4. RT-PCR analyses for expression of AKT-1, AKT-2, and more aggressively growing second-generation LNAI cells, T1.8, AKT-3. RT-PCR analyses using primers specific for each AKT isoform T1.16, and T2.11, (Fig. 3A) and in xenografts (data not shown). (33) were run for 35 PCR cycles. The product sizes, in base pairs, are 330 for AKT-1, 335 for AKT-2, and 327 for AKT-3. A, analyses of LNCaP Phosphorylation of AKT at Thr-308, which is also required for and the second-generation LNAI cell lines T1.16 and T2.9. B, expres- activation, was not increased in LNAI cells (data not shown). sion of AKT-1 and AKT-3 in primary prostate cancer and normal pros- Moreover, AKT activity was also increased in LNAI cells rela- tate tissue. PC-3 is included as a positive control for AKT-3 expression, tive to LNCaP cells (Fig. 3B). Together, these data indicate that whereas LNCaP and T1.16 lack AKT-3 expression. The identity of the AKT-3 RT-PCR products was verified by restriction digestion with AKT activation (Ser-473 phosphorylation) and activity are in- HaeIII and NotI (data not shown). C, expression of AKT-1 and AKT-3 in creased in androgen-independent LNAI cells, particularly the tissue from normal prostate, prostate cancer, and prostate cancer me- more aggressively growing, second-generation lines (T1.8, tastases to bone and soft tissues. Note that each of these samples was T1.16, and T2.11), relative to androgen-dependent LNCaP positive for b -microglobulin by RT-PCR (data not shown). cells. Androgen-independent LNAI Cells Express AKT-1 and primers designed specifically for each AKT isoform (32) re- AKT-2 but not AKT-3—Three isoforms of human AKT have vealed that, like LNCaP cells, LNAI cells express only AKT-1 been isolated, AKT-1, AKT-2, and AKT-3 (25, 35). Expression of and AKT-2 (Fig. 4A), suggesting that the evolution of andro- AKT-3 has been specifically associated with hormone-indepen- gen-independent hPCa cells does not necessarily involve up- dent primary human breast cancers and breast cancer cell regulated AKT-3 expression. To evaluate further whether lines. Likewise, the androgen-independent human prostate AKT-3 expression is induced during hPCa development or pro- cancer cell lines PC-3 and Du145 express AKT-3, whereas the gression, we analyzed primary human prostate cancers, pros- androgen-dependent/sensitive LNCaP hPCa cell line expresses tate cancer metastases to bone and soft tissues, and normal only AKT-1 and AKT-2, suggesting that androgen-independent prostate tissue for expression of AKT-1 and AKT-3 (Fig. 4, B hPCA progression may involve AKT-3 induction (32). We there- and C). Our analyses reveal that, although not all tissues fore examined whether the increased AKT activity in LNAI express AKT-3, the majority of both normal and tumor tissue cells might reflect AKT-3 expression. RT-PCR analyses with shows AKT-3 expression, thereby indicating that AKT-3 ex- AKT Activity Increases with Prostate Tumor Progression 24503 Ser-136 FIG.6. BAD phosphorylation is increased in LNAI cells and xenograft tissue. Phosphorylation of BAD was detected by West- Ser-136 ern blotting with a BAD phospho-specific antibody (30 mg extract/ lane). Parallel Western blots were also run and immunoblotted for total Ser-136 BAD protein. Signal intensity for pBAD was normalized for BAD expression. The data presented are from a single experiment and are representative of six independent experiments with the cell culture lysates (upper panel) and three experiments with xenograft tissue ly- sates (lower panel). In the lower panel, each bar represents an individ- ual xenograft tumor. In both panels, hatched bars represent LNAI FIG.5. Integrin-linked kinase expression is increased in LNAI lysates, and the clear bars represent LNCaP lysates. cells. Expression of ILK was assessed by Western blotting of cell culture extracts (A) or extracts from individual xenografts (B) (20 mg/ lane). Blots were reprobed for expression of b-actin to control for loading and transfer. Specific bands for the 59-kDa ILK and the 46-kDa b-actin proteins are indicated. These blots are representative of three inde- pendent experiments each with the cell culture and xenograft lysates. Note that each of the LNAI xenograft tissues has high level expression of ILK, whereas only one of the four LNCaP xenograft tissues shows high level ILK expression. Kip1 FIG.7. p27 expression is diminished in LNAI cells. Expres- Kip1 sion of the cell cycle regulatory protein p27 was examined by West- pression is not induced in, or restricted to, malignancy. ern blotting (20 mg lysate/lane). Data presented are representative of LNAI Cells Overexpress Integrin-linked Kinase—Integrin- four independent experiments. linked kinase (ILK) has been putatively identified as responsi- Ser-473 ble for AKT phosphorylation (27, 28). Western blot anal- edly enhanced in androgen-independent LNAI cells compared yses revealed that ILK is overexpressed in LNAI cells and with androgen-dependent/sensitive LNCaP and suggest that xenografts (Fig. 5), particularly in the second-generation LNAI increased AKT activity may be intimately involved in the pro- cells in which AKT activation was highest (e.g. T1.16, Fig. 5), gression of hPCa and the emergence of androgen-independent Ser-473 suggesting that the enhanced AKT phosphorylation may hPCa cells. To determine directly how AKT may impact upon be a downstream consequence of up-regulated ILK expression. hPCa progression, we established stable overexpressors of the T308D/S473D Increased AKT Activity in LNAI Cells Corresponds to In- constitutively active AKT-1 mutant in LNCaP cells Ser-136 Kip1 creased BAD DD Phosphorylation and Decreased p27 Ex- (LNCaP:AKT cells) (Fig. 8, inset). Subcutaneous injection of DD pression—Because AKT activity was increased in androgen- these LNCaP:AKT cells into intact, testosterone-supple- independent LNAI cells, we addressed the question of whether mented male nude mice yielded rapidly growing tumors with molecules downstream from AKT were differentially affected in 100% incidence (4/4) in less than 30 days post-injection, LNAI cells compared with LNCaP. The pro-apoptotic protein whereas vector control cells formed tumors in only three of five BAD can be functionally inactivated by phosphorylation at mice that were not evident until day 37 post-injection (Fig. 8). Ser-136 by AKT (17, 36, 37). Xenograft tissue and cell culture DD Moreover, the LNCaP:AKT cells showed a dramatic increase extracts of LNAI showed increased phosphorylation of BAD at in tumor growth (Fig. 8), with a mean tumor volume more than the AKT target site, Ser-136, relative to LNCaP cell and xe- 6-fold that of the vector control transfectants at all time points nograft extracts (Fig. 6). (by Dunnett’s test on ranked tumor volumes, p , 0.05 for all AKT has recently been shown to phosphorylate and inacti- time points). Like the aggressively growing LNAI cells, LNCaP: DD Kip1 vate the Forkhead/AFX family of transcription factors (19 –22). AKT tumor cells showed a substantial decrease in p27 As AFX activates transcription of the cell cycle regulator expression (Fig. 8, inset). Tumors did not form in castrated Kip1 DD p27 (23), inhibition of AFX-mediated transcription by AKT mice following injection with either LNCaP:AKT or vector Kip1 may lead to decreased expression of p27 . In LNAI cells, in control LNCaP cells. A second, independent experiment con- Kip1 DD which AKT is hyperactivated, the protein expression of p27 firmed that LNCaP:AKT cells formed more rapidly growing was markedly reduced relative to that in LNCaP (Fig. 7). This tumors in intact male nude mice when compared with vector decrease was most pronounced in the most aggressively grow- control LNCaP cells but were unable to form tumors in cas- ing, second-generation LNAI cells with the highest AKT activ- trated male nude mice (data not shown). Therefore, although ity (e.g. T1.16). These data are consistent with the notion that insufficient to elicit growth in castrated male nude mice, over- AKT negatively regulates AFX-mediated transcription of DD expression of the constitutively active AKT dramatically en- Kip1 p27 but do not exclude the possibility that other mecha- hanced LNCaP xenograft tumor growth in intact male nude Kip1 nisms, such as decreased protein or RNA stability, may con- mice and resulted in markedly reduced expression of p27 . Kip1 tribute to p27 loss. Together, these data indicate that the DISCUSSION increased AKT activity in androgen-independent LNAI cells profoundly effects key downstream mediators involved in cell We have developed a series of androgen-independent hPCa cycle arrest and apoptotic signaling. cell lines (LNAI cells) from the androgen-dependent/sensitive Stable Overexpression of Constitutively Active AKT Dramat- hPCA cell line, LNCaP. Like the majority of androgen-independ- ically Enhances LNCaP Xenograft Tumor Growth in Intact ent hPCa (34), these LNAI cells retain expression of AR and Male Nude Mice—Our data indicate that AKT activity is mark- PSA. We have now shown that AKT activation (Ser-473 phos- 24504 AKT Activity Increases with Prostate Tumor Progression emergence of androgen-independent hPCa cells, such as a di- minished apoptotic response (1, 2, 39) as well as a release from the cell cycle control that follows androgen ablation (4). AKT can dampen the normal apoptotic response by suppressing the activity of numerous pro-apoptotic proteins including BAD, caspase 9, and the Forkhead family of transcription factors (Refs. 17–22; summarized in Fig. 1). AKT can also facilitate the release of cells from cell cycle control by inhibiting AFX/Fork- head-mediated transcription of the key cell cycle regulator, Kip1 p27 (23). Further, by activating FRAP/mTOR and de-re- pressing translation initiation, AKT may specifically enhance FIG.8. Xenograft growth of LNCaP:AKT transfectants in in- the translation of cyclin D1 (24) as well as other translationally DD tact male nude mice. LNCaP:AKT or LNCaP vector control cells controlled growth factors and growth regulatory proteins im- were injected (4 3 10 cells) into intact, testosterone-supplemented plicated in human prostate cancer progression (Refs. 40 – 43; male nude mice. Tumor volume (mm ) is depicted on the y axis and was calculated as described (see “Experimental Procedures”). Hatched bars see Fig. 1). DD represent the tumor volumes for individual LNCaP:AKT tumors, The data in this report clearly imply increased AKT activity whereas white bars represent individual LNCaP vector control tumors. in hPCa progression. The most aggressively growing, andro- DD LNCaP:AKT formed tumors in four of four mice injected, whereas the gen-independent LNAI cells had the greatest increase in AKT LNCaP vector control formed tumors in only three of five mice injected. The inset shows Western blot analyses (20 mg extract/lane) in LNCaP: activity. Moreover, overexpression of an activated AKT-1 mu- DD AKT transfectants and vector control LNCaP transfectants for ex- tant dramatically accelerated LNCaP xenograft tumor growth. pression of AKT (representative of three independent experiments) and However, our data also reveal that overexpression of an acti- Kip1 p27 (representative of six independent experiments). To control for vated AKT-1 was alone insufficient to drive xenograft tumor gel loading and transfer, these blots were reprobed for b-actin expres- growth in castrated mice, suggesting that factors in addition to sion. A second xenograft experiment showed a similar increase in LN- DD CaP:AKT xenograft growth relative to the LNCaP vector control increased AKT activity must contribute to the ability of LNAI (data not shown). cells to form and grow tumors in the absence of circulating androgens. For instance, the increased expression of ILK in phorylation) and activity are increased in these LNAI cells LNAI cells may influence alternate pathways in addition to the when compared with androgen-dependent LNCaP cells and AKT pathway, such as the Wnt pathway (27), that may con- may be a consequence of increased expression of the integrin- tribute to the androgen-independent phenotype. Moreover, Ser-473 linked kinase, the kinase putatively responsible for AKT other intracellular pathways may be activated in LNAI cells phosphorylation (27, 28). Like parental LNCaP cells, LNAI independent of, or upstream from, AKT that contribute to the cells express only AKT-1 and AKT-2, indicating that AKT-3 androgen-independent phenotype of LNAI cells. Indeed, acti- expression is not necessary for androgen independence, as has vation of the mitogen-activated protein kinase pathways, per- been suggested in studies with the androgen-independent PC-3 haps as a downstream consequence of HER2 activation, has and Du145 hPCa cells (32). Moreover, our data reveal that been implicated in hPCa progression and androgen independ- AKT-3 is expressed not only in prostate cancer tissues but also ence (44 – 46). in normal prostate tissue, indicating that AKT-3 expression is In summary, the data presented in this report indicate that not induced in, or restricted to, prostate malignancy. We have increased AKT activity, perhaps as a consequence of increased also shown that the increased AKT activity in these LNAI cells ILK expression, is involved in hPCa progression and suggest Kip1 corresponds to increased phosphorylation of the pro-apoptotic that the reduction in p27 expression, which has been re- protein, BAD, and markedly decreased expression of the peatedly associated with hPCa progression and androgen inde- Kip1 p27 cell cycle regulator, consistent with the notion that pendence (4 –9), may be a consequence of enhanced AKT activ- AKT inhibits AFX-mediated transcriptional activation of ity. Together with previously published reports that PTEN is Kip1 p27 (23, 38). Taken together, these data imply increased inactivated in a significant proportion of advanced primary AKT activity in hPCa progression and the emergence of andro- human prostate cancers (10) and that AKT regulates many of gen-independent human prostate cancer cells. the intracellular processes associated with hPCa progression To address whether AKT alone can drive prostate tumor (1, 25), the data in this report implicate AKT as a key mediator progression and/or the emergence of androgen-independent of prostate tumor progression and suggest that AKT may be a cells, we established LNCaP cells with stable overexpression of prime target for prostate cancer therapy. a constitutively active AKT-1 cDNA. Though alone insufficient Acknowledgments—We thank Drs. Mark Marshall, James G. to elicit xenograft tumor growth in castrated mice, overexpres- Herman, Jake Starling, and Steven M. Paul for critical review of this sion of activated AKT dramatically increased tumor growth in manuscript. We thank Zandra Lee for collecting some of the tissues intact mice relative to vector control transfectants. Like the used for RT-PCR analyses. We also thank Drs. Homer Pearce and most aggressively growing LNAI cells (e.g. T1.16), these LN- Steven M. Paul for supporting this work. DD CaP:AKT cells also showed a remarkable reduction in REFERENCES Kip1 Kip1 p27 expression, suggesting that p27 loss, which has 1. Isaacs, J. T. (1999) Urol. Clin. North Am. 26, 263–273 been repeatedly linked to hPCa progression (4 –9), may be a 2. Denmeade, S. R., Lin, X. S., and Isaacs, J. T. (1996) Prostate 28, 251–265 3. Berges, R. R., Vukanovic, J., Epstein, J. I., CarMichel, M., Cisek, L., Johnson, consequence of increased AKT activity. Taken together with D. E., Veltri, R. W., Walsh, P. C., and Isaacs, J. T. (1995) Clin. Cancer Res. the analyses of LNAI cells, these data reveal that increased 1, 473– 480 4. Agus, D. B., Cordon-Cardo, C., Fox, W., Drobnjak, M., Koff, A., Golde, D. W., AKT activity contributes to hPCa progression by dramatically and Scher, H. I. (1999) J. Natl. Cancer Inst. 91, 1869 –1876 accelerating prostate tumor growth, perhaps in relation to de- 5. Macri, E., and Loda, M. (1998 –99) Cancer Metastasis Rev. 17, 337–344 Kip1 creasing p27 expression, and is specifically associated with 6. Guo, Y., Sklar, G. N., Borkowski, A., and Kyprianou, N. (1997) Clin Cancer Res. 3, 2269 –2274 the more advanced, aggressively growing androgen-independ- 7. Yang, R. M., Naitoh, J., Murphy, M., Wang, H. J., Phillipson, J., deKernion, ent hPCa cells (e.g. T1.16). J. B., Loda, M., and Reiter, R. E. (1998) J. Urol. 159, 941–945 8. Tsihlias, J., Kapusta, L. R., DeBoer, G., Morava-Protzner, I., Zbieranowski, I., Increased AKT activity may play a profound role in the Bhattacharya, N., Catzavelos, G. C., Klotz, L. H., and Slingerland, J. M. progression of human prostate cancers. AKT regulates many of (1998) Cancer Res. 58, 542–548 the processes associated with metastatic progression and the 9. De Marzo, A. M., Meeker, A. K., Epstein, J. I., and Coffey, D. S. (1998) Am. J. AKT Activity Increases with Prostate Tumor Progression 24505 Pathol. 153, 911–919 (1998) Genes Dev. 12, 502–513 10. Ittmann, M. M. (1998) Oncol. Rep. 5, 1329 –1335 27. Delcommenne, M., Tan, C., Gray, V., Rue, L., Woodgett, J., and Dedhar, S. 11. Whang, Y. E., Wu, X., Suzuki, H., Reiter, R. E., Tran, C., Vessella, R. L., Said, (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11211–11216 J. W., Isaacs, W. B., and Sawyers, C. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 28. Lynch, D. K., Ellis, C. A., Edwards, P. A., and Hiles, I. D. (1999) Oncogene 18, 95, 5246 –5250 8024 – 8032 12. Vlietstra, R. J., van Alewijk, D. C., Hermans, K. G., van Steenbrugge, G. J., 29. Attia, M. A., and Weiss, D. W. (1966) Cancer Res. 26, 1787–1800 and Trapman, J. (1998) Cancer Res. 58, 2720 –2723 30. Graff, J. R., Boghaert, E. R., De Benedetti, A., Tudor, D. L., Zimmer, C. C., 13. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Chan, S. K., and Zimmer, S. G. (1995) Int. J. Cancer 60, 255–263 Miliaresis C, Rodgers, L., McCombie, R., Bigner, S. H., Giovanella, B. C., 31. Graff, J. R., De Benedetti, A., Olson, J. W., Tamez, P., Casero, R. A., Jr., and Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M. H., and Parsons, R. Zimmer, S. G. (1997) Biochem. Biophys. Res. Commun. 240, 15–20 (1997) Science 275, 1943–1947 32. Nakatani, K., Thompson, D. A., Barthel, A., Sakaue, H., Liu, W., Weigel, R. J., 14. Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C., and Roth, R. A. (1999) J. Biol. Chem. 274, 21528 –21532 Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., and Mak, T. W. 33. Craft, N., and Sawyers, C. L. (1998 –99) Cancer Metastasis Rev. 17: 421– 427 (1998) Cell 95, 29 –39 34. Webber, M. M., Bello, D., and Quader, S. (1997) Prostate 30, 58–64 15. Cantley, L. C., and Neel, B. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 35. Nakatani, K., Sakaue, H., Thompson, D. A., Weigel, R. J., and Roth, R. A. 4240 – 4245 (1999) Biochem. Biophys. Res. Commun. 257, 906 –910 16. Wu, X., Senechal, K., Neshat, M. S., Whang, Y. E., and Sawyers, C. L. (1998) 36. Craddock, B. L., Orchiston, E. A., Hinton, H. J., and Welham, M. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 95, 15587–15591 J. Biol. Chem. 274, 10633–10640 17. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, 37. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. Science M. E. (1997) Cell 91, 231–241 (1997) 278, 687– 689 18. Cardone, M. H., Roy, N., Stennicke, H. R., Salvesen, G. S., Franke, T. F., 38. Ramaswamy, S., Nakamura, N., Vazquez, F., Batt, D. B., Perera, S., Roberts, Stanbridge, E., Frisch, S., and Reed. J. C. (1998) Science 282, 1318 –1321 T. M., and Sellers, W. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 19. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, 2110 –2115 M. J., Arden, K. C., Blenis, J., and Greenberg, M. (1999) Cell 96, 857– 868 39. McConkey, D. J., Greene, G., and Pettaway, C. A. (1996) Cancer Res. 56, 20. Kops, G. J., de Ruiter, N. D., De Vries-Smits, A. M., Powell, D. R., Bos, J. L., 5594 –5599 and Burgering, B. M. (1999) Nature 398, 630 – 634 40. Russell, P. J., Bennett, S., and Stricker, P. (1998) Clin. Chem. 44, 705–723 21. Tang, E. D., Nunez, G., Barr, F. G., and Guan, K. L. (1999) J. Biol. Chem. 274, 41. Culig, Z., Hobisch, A., Cronauer, M. V., Radmayr, C., Hittmair, A., Zhang, J., 16741–16746 Thurnher, M., Bartsch, G., and Klocker, H. (1996) Prostate 28, 392– 405 22. Takaishi, H., Konishi, H., Matsuzaki, H., Ono, Y., Shirai, Y., Saito, N., 42. Raught, B., and Gingras, A. C. (1999) Int. J. Biochem. Cell Biol. 31, 43–57 Kitamura, T., Ogawa, W., Kasuga, M., Kikkawa, U., and Nishizuka, Y. 43. De Benedetti, A., and Harris, A. L. (1999) Int. J. Biochem. Cell Biol. 31, 59 –72 (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11836 –11841 44. Gioeli, D., Mandell, J. W., Petroni, G. R., Frierson, H. F., Jr., and Weber, M. J. 23. Medema, R. H., Kops, G. J., Bos, J. L., and Burgering, B. M. (2000) Nature 404, (1999) Cancer Res. 59, 279 –284 782–787 45. Craft, N., Shostak, Y., Carey, M., and Sawyers, C. L. (1999) Nat. Med. 5, 24. Muise-Helmericks, R. C., Grimes, H. L., Bellacosa, A., Malstrom, S. E., Tsichlis, 280 –285 P. N., and Rosen, N. (1998) J. Biol. Chem. 273, 29864 –29872 25. Coffer, P. J., Jin, J., and Woodgett, J. R. (1998) Biochem J. 335, 1–13 46. Yeh, S., Lin, H. K., Kang, H. Y., Thin, T. H., Lin, M. F., and Chang, C. (1999) 26. Gingras. A. C., Kennedy, S. G., O’Leary, M. A., Sonenberg, N., and Hay, N. Proc. Natl. Acad. Sci. U. S. A. 96, 5458 –5463

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Published: Aug 1, 2000

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