Patent Application: US-14859903-A

Abstract:
more than 30 % of human malignancies harbor encogenic ras . both pro - apoptotic and anti - apoptotic pathways emanate from encogenic ras with survival being dominant . ras survival signaling is thought to be controlled by transcriptional and post - translational processes . the present invention shows that a repressor of cap - dependent translation initiation , 4e - bp1 , selectively activates apoptosis in ras - transformed fibroblasts and eliminates ras - induced chemoresistance . these effects of 4e - bp1 are strictly dependent on its ability to sequester translation initiation factor eif4e , thereby preventing its assembly into an active pre - initiation complex . these results suggest that translational control is critical for prevention of apoptosis and resistance to antitumor agents in ras - transformed cells .

Description:
herein , the inventors sought to identify the molecular target of frap kinase conferring ras - induced viability and chemoresistance . to that end , a cell system ( 12 ) was used , in which constitutively expressed oncogenic rasv12 enables cloned rat embryo fibroblasts ( cref ) to survive in otherwise lethal concentrations of cytostatic drugs ( non genotoxic , lovastatin ; genotoxic , camptothecin , fig1 ). rapamycin completely abrogated ras - dependent resistance to drug - induced cell death ( fig1 b ); and even when applied as a single agent , stimulated apoptosis in cells expressing activated ras . this pro - apoptotic effect of rapamycin was not observed in non - transformed fibroblasts . this observation confirms a dual pro - apoptotic and anti - apoptotic function for rasv12 , and implicates frap in ras - dependent rescue from both ras activated and drug - triggered apoptotic pathways . in accord with previous reports ( 9 ), rapamycin also caused a dose - dependent decline in protein synthesis which paralleled its ability to sensitize ras - transformed cells to lovastatin - induced apoptosis ( fig1 c ). of note , equipotent doses of the peptide elongation inhibitor cycloheximide actually blocked apoptosis . this is consistent with recent findings demonstrating that the execution of lovastatin - induced cell death requires global protein synthesis ( 13 ), and suggests that a generalized inhibition of mrna translation is not the means by which rapamycin exerts its pro - apoptotic effect . an alternative mechanism is that rapamycin predominantly inhibits translation of a specific set of mrna required for ras survival signaling and chemoresistance . in support of this idea , ras function has been closely linked to the initiation of cap - dependent protein synthesis . cell transformation by oncogenic ras requires increased activity of translation initiation factor eif4e ( 14 ), the mrna cap binding protein which functions during translation of cellular mrnas possessing the 5 ′ cap structure ( 15 ). the cap is bound by the initiation complex eif4f , which in mammalian cells consists of the bi - directional rna helicase eif4a , the docking protein eif4g , and the cap binding subunit eif4e . eif4e is considered to be rate - limiting for translation initiation under most circumstances , and a major target for regulation ( 16 ). to explore whether the 4e - bps modulate ras - dependent viability and chemoresistance , cref / rasv12 and cref were engineered to constitutively express wild type 4e - bp1 ( bp2wt ) linked to a puro selectable marker , and puromycin resistant clones were isolated ( see example 2 ). all mock - transfected , puromycin resistant clones ( n = 6 ) were stable . each displayed viability and biochemical characteristics similar to those of parental cref / rasv12 . four viable cref / ras / bpwt clonal lines were developed and assayed for steady state levels of 4e - bp1 . under conditions in which expression of endogenous 4e - bp1 in all mock - transfected cells was undetectable ( 70 μg cellular protein / lane ; 15 sec . development time ), cref / ras / bp1wt clones displayed a range of ectopic 4e - bp1 expression . western blot analysis performed on total cellular extracts ( fig2 a ) revealed human 4e - bp1 represented by hypo -, intermediate -, and hyperphosphorylated forms ( 19 ). many cells comprising the cref / rasv12 / bp1wt clonal lines displayed morphological hallmarks of apoptosis , such as cell shrinkage , chromatin condensation and fragmentation of nuclei ( fig2 b ). quantification of apoptosis by flow cytometry revealed a 2 to 8 - fold increase in basal apoptosis in complete medium , suggesting that ectopic 4e - bp1 shifted ras signaling from suppression to induction of apoptosis ( fig2 c ). cytostatic drugs approximately doubled the basal apoptotic frequency in all clones , indicating that ras - dependent chemoresistance was lost in cells ectopically expressing 4e - bp1 . in contrast to results with transformed cref / rasv12 , overexpressed 4e - bp1 did not activate apoptosis in non - transformed parental cref lacking activated ras . four clonal lines representing a wide spectrum of 4e - bp1 expression were subjected to further analysis ( fig2 d ). ectopic expression of 4e - bp1 did not alter the morphology of cref ( fig2 e ), nor did it alter their viability ( fig2 f ). these findings indicate that physiologically regulated wild type ras does not cooperate with 4e - bp1 in sensitizing fibroblasts to apoptosis , but rather that overexpressed oncogenic ras is required . next , it was investigated whether the pro - apoptotic function of 4e - bp1 in cref / rasv12 was associated with its ability to sequester eif4e from the translationally active eif4e / eif4gi complex . cellular extracts were incubated with the cap - analog m 7 gtp - agarose to capture eif4e and its cellular binding partners . the levels of cap - bound eif4e , 4e - bp1 , and eif4gi were quantified by sequential immunoblotting and densitometry . each cref / rasv12 / bp1wt clone displayed eif4e associated with significantly increased amounts of fast migrating , hypophosphorylated 4e - bp1 ( fig3 a ). consistent with this , clones ectopically expressing 4e - bp1wt also manifested decreased amounts of eif4gi bound to eif4e , confirming the ability of ectopic 4e - bp1 to inhibit assembly of the eif4f pre - initiation complex . the apoptotic frequency in clones co - expressing activated ras and 4e - bp1 was proportional to the amount of 4e - bp1 complexed with eif4e ( fig3 a ), and inversely related to the eif4gi / eif4e ratio ( fig3 c ). thus , the ability of 4e - bp1 to stimulate apoptotic death was a function of its activity in competitively displacing eif4gi from eif4e . to determine whether the interaction of 4e - bp1 with eif4e was necessary for the pro - apoptotic function of 4e - bp1 in ras - transformed cells , a 4e - bp1 deletion mutant ( 4e - bp1δ ) which lacks the eif4e binding site ( 24 ) was used . transient transfection of cref / rasv12 with 4e - bp1wt enhanced spontaneous apoptosis and chemosensitivity to lovastatin in a manner similar to that observed in the stable cref / rasv12 / bp1wt clones . in marked contrast , transient transfection with 4e - bp1δ had minimal effects on viability , despite similar levels of gene expression . thus , the ability of 4e - bp1 to bind eif4e was essential for its blockade of ras - induced survival signaling . the finding that 4e - bp1 , eif4e and eif4g are implicated in the apoptotic pathway emanating from oncogenic ras through frap is of pharmacological value , as specific modulation of the 4e - bp1 - eif4e interaction , as well as the modulation of the formation of the eif4f preinitiation complex and of the level of eif4e complex to eif4g1 , could be used to modulate this apoptotic pathway . this possibility is particularly intriguing in light of the fact that overexpression of 4e - bp1 did not activate apoptosis in non - transformed cells lacking activated ras . furthermore , the present invention , having identified translation initiation through eif4e and its association with eif4g as a biochemical pathway involved in modulation of apoptosis , provides numerous assays and methods to screen and identify such apoptosis modulators and especially pro - apoptotic agents . as seen in fig5 and 6 , the eif4e binding sites ( or eif4e interaction domains ) of numerous protein from evolutionarily distant organisms show a significant homology / identity . in addition , the sequences of rat and mouse 4e - bp1 , 4e - bp2 and 4e - bp3 are 100 % identical to those of the human in the region presented here . indeed , consensus sequences which retain their eif4e binding activity are provided . these consensus sequences could be used as eif4e sequestering agents or as starting points to design other eif4e sequestering agents . of note , recombinant peptides derived from 4e - bp1 and eif4gii have been shown to inhibit translation of mrnas ( marcotrigiano et al ., 1999 , molecul . cell 3 : 707 - 716 ). the present invention is illustrated in further detail by the following non - limiting examples . parental cloned rat embryo fibroblasts ( cref ) and their derivatives expressing empty vector ( cref / neo ) or h - ras [ v12 ] ( cref / rasv12 ) as well as cref / rasv12 constitutively expressing eif4e antisense mrna ( as4e cells ) were kindly provided by dr . a . de benedetti ( louisiana state university , shreveport , la .). growth characteristics of cref and cref / rasv12 ( the latter are also known as cref t24 ) have been described ( boylan et al . 1990 ). inhibitory effects of eif4e antisense mrna on eif4e expression pattern , cell cycle transit , and tumorigenicity of cref / rasv12 have been previously documented ( rinker - schaeffer et al . 1993 ; graff et al . 1995 ). all cells were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % fcs , 100 units / ml penicillin , 100 g / ml streptomycin and 250 ng / ml amphotericin b ( gibco ). plasmids encoding eif4e antisense mrna , the transfection procedure , and isolation of as4e clones have been described ( de benedetti et al . 1991 ; rinker - schaeffer et al . 1993 ; graff etal . 1995 ). the coding sequence of human 4e - bp1 was amplified by pcr and directionally cloned into the ecor1 and bamh1 sites of the mammalian expression vector psrαpuro ( a kind gift from dr . p . jolicoeur , institut de recherches cliniques , montreal , pq , canada ). the insert was fully sequenced and no mutation were detected . transfections of cref and cref / rasv12 were performed by calcium phosphate precipitation technique . selective medium containing 4 g / ml puromycin was applied after 24 h and resistant clones were isolated after 12 to 16 days . apoptosis was induced by reducing serum concentration in the medium ( 0 . 1 to 0 . 5 %) or by addition of cytostatic agents . each cytostatic agent was used at a concentration that caused 75 to 80 % of cref to be reversibly arrested at the expected cycle locus ( assessed by facs , see below ) as follows : 5 μm lovastatin , late g1 ; hydroxyurea , 1 . 5 mm , late g1 / early s ; camptothecin , 75 nm , g2 ; colcemide , 50 ng / ml , m phase . frequency of apoptosis was quantified by flow cytometric analysis of the percentage of cells with hypodiploid dna content , as described ( polunovsky et al . 1994 ; polunovsky et al . 1996 ). adherent and nonadherent cells were pooled , washed in phosphate - buffered saline ( pbs ), and fixed with ice cold ethanol ( 70 %, 4 ° for 1 h ). fixed cells were washed with pbs and incubated in propidium iodide stain mixture ( 52 g / ml propidium iodide , 0 . 05 % triton x - 100 , 18 mg / ml edta , 100 u / ml ribonuclease in pbs ). after incubation ( 45 min , 37 °), dna content was determined by quantitative flow cytometry ( becton - dickinson facs star plus ). results were tabulated as the mean ± sd of 2 to 5 separate experiments . in each experiment , all conditions were examined in duplicate or triplicate . flow cytometric data were confirmed by analysis of apoptosis after acridine orange or tunel staining ( polunovsky et al . 1994 ; polunovsky et al . 1996 ). cell cycle distribution was monitored by flow cytometric analysis of dna content . entry into s phase was quantified by analysis of brdu incorporation into dna , as described ( polunovsky et al . 1996 ). exponentially proliferating cells ( 5 × 10 4 ) were pretreated with potent translational inhibitors for 4 h in standard growth medium and transferred for 3 h into methionine - free medium containing 10 % dialysed fcs and 15 ci / ml [ 35 s ] methionine ( sa = 6000 ci / mmole ). cell monolayers were rinsed twice with pbs ( 4 °), washed three times with 5 % tca ( 4 °), and lysed with 0 . 1 % naoh containing 0 . 1 % sds . tca precipitable radioactivity was quantified by liquid scintillation counting . for the western blot assay , cells were collected by scraping into buffer a ( 150 mm nacl , 50 mm tris ph 7 . 5 , 50 mm naf , 1 mm edta , 1 mm egta , 1 mm dtt , 50 mm β - glycerophosphate , 10 mm na pyrophosphate , 0 . 1 mm na orthovanadate , 50 nm okadaic acid , 1 mm pmsf , 1 g / ml pepstatin a , 1 g / ml leupeptin ), and cell membranes were disrupted by three successive freeze - thaw cycles . the cell extracts were microcentrifuged at 4 ° for 10 min and the supernatants were retained . for ras , eif4e and actin , supernatants were mixed with reducing sample buffer , boiled , and subjected to 14 % page , followed by electroblotting to nitrocellulose . for 4e - bp1 , it was necessary to first boil the samples ( 7 minutes ) to eliminate interfering proteins prior to electrophoresis . western blotting was performed by blocking the membrane in tris buffered saline , ph 7 . 5 containing 0 . 05 % tween 20 and 5 % nonfat dry milk for 1 hour . after incubation with primary and peroxidase conjugated secondary antibodies , blots were developed by ecl ( amersham ) and film images were scanned for densitometry using a biorad gs700 imaging densitometer with molecular analyst software . cells were lysed as described above . to quantitate 4e - bp1 bound to eif4e , lysates representing 2 × 10 6 cells were diluted with buffer a to a final volume of 100 μl and mixed with 25 μl of packed 7 - methyl - gtp agarose beads ( pharmacia biotechnology inc .) that were pre - equilibrated with buffer b ( 50 mm tris , ph 7 . 5 , 150 mm nacl ). each lysate was mixed with beads in a microcentrifuge tube for 1 . 5 h at 40 ° under constant gentle agitation , followed by a brief centrifugation to pellet the beads . the pelleted beads with bound material were washed twice with 0 . 5 ml of buffer b and eluted with 50 μl of buffer c ( 25 mm tris , ph 7 . 5 ; 75 mm nacl ; 70 μm 7 - methyl - gtp ). the 50 μl of eluate were removed completely from the beads , subjected to sds - page and transferred to nitrocellulose for immunoblotting . expression of the eif - 4e sequestering agent 4e - bp1 stimulates apoptotic death in naturally occurring cancer cells major limiting factors in anti - neoplastic therapy are the failure of some tumor types to respond to anticancer treatments , and the appearance of resistant cell populations in originally responsive malignancies upon relapse . it is widely recognized that most cytotoxic antineoplastic therapies do not kill cells by causing catastrophic damage to critical structures , but rather by triggering intrinsic apoptotic pathways ( reviewed by kaufmann and gores , 2000 ). there is a large body of evidence showing that tumor cells harbor genetic changes leading to increased synthesis of a limited set of proteins which are encoded by messenger rnas containing specific elements in their 5 ′ and 3 ′ untranslated regions ( reviewed by de benedetti and harris , 1999 and zimmer et al . 2000 ). these alterations are often accompanied by cancer cell chemoresistance and are thus often associated with a poor prognosis . initiation of cap - dependent translation in mammals is positively regulated by the cap - binding protein eif4e and inhibited by the translational repressor 4e - bp1 , which prevents associations between eif4e and caped mrnas ( reviewed by sonenberg , 1996 ; sonenberg and gingras , 1998 ; raught and gingras , 1999 ). it has also been discovered that increased expression of eif4e rescues drug - induced apoptosis ( tan et al ., 2000 ). as shown above , transfer of the gene encoding 4e - bp1 into malignant cells activates the intrinsic apoptotic machinery , sensitizes these cells to anti - cancer therapy in vitro , and dramatically reduces their tumorigenicity in vivo . most importantly from a therapeutic point of view , 4e - bp1 specifically activates apoptosis in cancer cells , but not in normal cells , identifying the cap - dependent translational aparatus as a potential novel molecular target for anticancer drug discovery . although the studies show that gain and loss of translation initiation activity potently modulates viability in oncogene - transformed rodent fibroblasts , it was of interest to assess whether translational control is relevant to regulation of apoptosis in naturally occurring cancer cells . it was also of interest to assess whether translation control was relevant to the regulation of apoptosis in naturally occurring cancer cells having tumor - related gene alterations . as model systems to assess the relevance of translational control in the regulation of apoptotis , breast cancer cells which are of epithelial origin and posses diverse tumor - related gene alterations , and non - small cell lung cancer cells were chosen . breast carcinoma cell lines express activation states of both apoptotic machinery and cap - dependent translational apparatus to detect whether susceptibility to spontaneous and drug - induced apoptosis in different human breast cancer cell lines correlates with activity of the cap - dependent translational machinery , protein drug - induced apoptosis assays were performed in a set of human breast carcinoma cell lines ( table 1 ), the genetic profile of which have been documented previously ( sepp - lorensino et al ., 1995 ). the ras status , estrogen ( er ) dependence , and other characteristics of non - transformed breast epithelial cells and breast carcinoma lines are shown . one cancer cell line ( mda - md - 231 ) harbors mutated ki - ras , while others express activated upstream effectors of eif4e signaling pathways . extracts from non - transformed breast epithelial cells and breast cancer cells lines were tested to detect cellular levels of eif4e and 4e - bp1 , and to evaluate the association of eif4gi with eif4e to form an intact translation initiation complex ( fig7 ). steady state levels of eif4e were similar among the breast cancer cell lines and modestly increased compared to the non - transformed 184 a1 breast epithelial cells ( fig7 a ). steady state levels of eif4g1 were significantly increased in all breast cancer cell lines tested . while 4e - bp1 is predominantly represented in non - transformed cells by hypophosphorylated isoform a which actively represses translation , breast cancer cell extracts are enriched for slow migrating hyperphosphorylated 4e - bp1 ( isoforms β and γ ) which is much less active in repressing assembly of eif4f . consistent with this , breast cancer cell extracts manifested increased amounts of eif4gi associated with eif4e in the cap bound fraction ( fig7 b ). this indicates increased amounts of intact eif4f complex in all breast cancer cell lines tested , suggesting these cells function in a translationally activated state . apoptosis assays revealed elevated spontaneous apoptosis in mcf - 7 and , mda - mb453 cells as well as increased susceptibility to drug - induced apoptosis in all tested breast carcinomas ( fig7 c ). since upregulated cap - dependent translation antagonizes apoptotic death as described above and in polunovsky et al ., 1996 ; and in tan et al ., 2000 , it was hypothesized that breast cancer cells require a high level of cap - dependent translation to suppress the apoptotic apparatus that is activated in the course of cell malignant transformation . enforced expression of 4e - bp1 stimulates spontaneous and drug - induced apoptosis in mda - mb - 231 breast carcinoma . to test the hypothesis that breast cancer cells are dependent or an increase in cap - dependent translation to overcome the apoptosis pathway , the effects of inhibitors of cap - dependent translation on spontaneous and drug - induced apoptosis in normal and malignant cells were examined . since breast cancer cells significantly differ from normal epithelial cells in expression of 4e - bp1 ( fig7 a ), the focus was put first on developing cell lines in which cap - dependent translation is inhibited by enforced overexpression of 4e - bp1 . as shown above , the translational repressor , and eif4e -[ sequestering agent ] 4e - bp1 and 4e - bp1 phosphorylation - inhibitor rapamycin , sensitized transformed fibroblasts to apoptosis and suppresses ras - dependent tumorigenicity in a manner strictly dependent on their ability to sequester eif4e from a translationally active complex with eif4g . to determine whether the anti - apoptotic effect of 4e - bp1 observed in ras - transformed fibroblasts is also seen in naturally occurring cancer cells , breast cancer cells , mda - mb - 231 cells expressing oncogenic ras , were stably transfected with a neo selectable vector pactag - 2 engineered to encode haemagglutinin ( ha ) tagged human wt 4e - bp1 ( gingras et al ., 1999 ). twelve neomycin - resistant clones were isolated and assayed for steady state expression of ha ; subjected to cap - affinity chromatography to quantify the proportion of eif4e complexed with eif4gi and examined for chemosensitivity to lovastatin and camptothecin ( fig8 ). in the three clones shown ( fig8 a ), ha expression ( as a surrogate for ectopic 4e - bp1 ), eif4e captured by cap - analog , and eif4gi associated with cap - bound eif4e can be compared to these parameters in non - transfected mda - mb - 231 cells . ectopic 4e - bp1 displaced eif4gi from eif4e ( fig8 b ). this was accompanied by an increase in intrinsic apoptosis and substantially augmented apoptotic death in response to the lovastatin or camptothecin ( fig8 c ). to confirm activation of apoptosis - related biochemical events in 4e - bp1 expressing cells and independently verify the results of flow cytometry , immunomorphological techniques in which nuclear apoptotic rearrangements are identified along with expression of active capspase - 3 were used ( fig9 ). ectopic expression of 4e - bp1 activates apoptosis in non - small cell lung cancer ( nsclc ) cells . to further validate that the present invention is relevant to the clinical situation and more specifically to validate that translation control is involved in the modulation of apoptosis in naturally occurring cancer cells , the nsclc cell line 2009 harboring ki - rasv12 was stably transfected with the neo vector carrying ha - tagged 4e - bp1 or with empty vector . two ha - 4e - bp1 positive and two mock - transfected clonal cell lines were subjected to cap - affinity chromatography to detect expression levels of slow migrating ha - 4e - bp1 , eif4gi captured by the cap - complexed eif4e and apoptotic response . cells expressing high levels of exogenous ha - 4e - bp1 ( clone # 8 ), manifested significant suppression of the eif4g1 / eif4e / cap complex formation and were highly susceptible to the pro - apoptotic effect of both lovastatin and etoposide ( fig1 ). clone # 10 ( a mid - level expressor ) displayed intermediate values , while mock - transfected and control cells had a minimal response . taken together , the present data validates the hypothesis that levels of expression of the translational factor eif4e determine chemoresistance in human breast and lung cancer cell lines . indeed , it was found that both the apoptotic and translational machinery are activated in all tested breast carcinomas when compared to non - transformed breast epithelial cells . together with the data presented above that activated cap - dependent translation can rescue cells from apoptotic death , these findings demonstrate the proof of principle that in naturally occurring cells which have acquired metabolic alterations leading to increased cap - dependent translation to oppose transformation - related activation of their intrinsic apoptotic program . in addition , ectopic expression of wild type 4e - bp1 stimulates apoptosis and abrogates chemoresistance in breast carcinoma cells expressing oncogenic ras and in clonal lung cancer cell lines . activation of apoptosis by translational inhibitors parallels their ability to disrupt eif4e / eif4g assembly and repress function of the cap - dependent translation apparatus . these results suggest that the integrity of the cap - dependent translational apparatus is critically important for viability and chemoresistance in naturally occurring cancer cell . they also demonstrate that targeted disruption of the cap - binding complex by transferring the 4e - bp1 gene can be used as a novel approach to block malignant progression in carcinomas and / or tumors whose growth depends on an activation of cap - dependent translation and more particularly in breast or lung carcinoma . the present invention thus opens the way to the use of preclinical models for cancer in which there is an activation of cap - dependent translation ( e . g . breast and lung cancer ) to test the ability of upregulated 4e - bp1 to collaborate with well - tolerated doses of available cancer therapeutics to inhibit xenograft growth in athymic mice . these data identify a novel survival pathway from ras through frap to 4e - bp1 - inhibitable translation initiation , providing new insights into the biology of cancer . many cancers and tumor cell lines have increased levels of eif4e ( 25 ). ectopic eif4e transforms immortal fibroblasts and cooperates with myc in the transformation of primary fibroblasts , whereas reduction of eif4e level ( 26 ) or suppression of eif4e function by overexpressed 4e - bp1 ( 21 ) reverts ras - transformed cells to a non - malignant phenotype . available data , therefore , suggest that eif4e is a powerful oncogene and that 4e - bp1 has the potential to function as a tumor suppressor gene . although further studies are required to discover the downstream effectors of the eif4e / 4e - bp1 regulated survival pathway and to delineate how they interact with the apoptotic apparatus , the present findings add translational control to the established transcriptional and post - translational mechanisms that regulate cell viability downstream of ras . more broadly , the present findings add translational control to the established transcriptional and post - translational mechanisms that regulate apoptosis in cells . the inatant invention therefore has broad implications to all diseases or conditions in which a perturbation of apoptosis is encountered . the findings that ectopic expression of 4e - bp1 selectively kills and chemosensitizes ras - transformed cells have clear implications for cancer therapeutics . non - malignant cells can apparently function normally over a wide range of 4e - bp1 expression . its absence in knockout mice results in no apparent phenotype ( 27 ). moreover , it is herein shown that even a dramatic overexpression in non - transformed fibroblasts is compatible with normal physiological function . however , against the background of oncogenic ras , 4e - bp1 exerts a powerful control over growth and viability . taken together , these findings suggest that translational repressors may constitute a significant component of the mammalian tumor surveillance system , and that they might be safely augmented for cancer treatment . of equal therapeutic importance , since overexpressed eif4e has been detected in many tumors and malignant cell lines ( 25 ), the present invention suggests a novel mechanism whereby tumor cells can acquire resistance to genotoxic and non - genotoxic anticancer agents . many properties of eif4e and 4e - bp1 , including their ability to regulate proliferation , apoptosis and drug resistance make them potential therapeutic targets in human malignancy . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified , without departing from the spirit and nature of the subject invention as defined in the appended claims . 1 . c . y . chenn et al ., j . biol . chem . 273 , 16700 ( 1998 ); j . downward , curr . opin . gen . dev . 8 , 49 ( 1998 ); a . b . vojtek and c . j . ders , j . biol . chem 273 , 19925 ( 1998 ). 3 . p . rodriquez - viciana et at ., nature 370 , 527 ( 1994 ); p . rodriquez - viciana et al ., embo j . 15 , 527 ( 1994 ). 4 . m . w . mayo et al ., science 278 , 1812 ( 1997 ); c . beraud et al ., biochemistry 96 , 429 ( 1999 ). 5 . j . downward , curr . opin . cell biol . 10 , 262 ( 1998 ). 6 . r . s . datta et al ., cell 91 , 231 ( 1997 ); l . peso et al ., science 278 , 687 . 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