Patent Application: US-200913131840-A

Abstract:
the invention describes a cytotoxic composition containing a drug combination targeting two or more functional elements in pancreatic cancer cells , the functional elements comprising egfr or src and stat3 or jaks . preferred drugs in the drug combination are selected from zd and s3i - 201 , das and s3i - 201 , zd and ag490 , das and ag490 , and combinations thereof . in a preferred embodiment of the invention , the drug combination further includes a nucleoside analog inhibitory for dna replication , for example , gemcitabine . disclosed is also a method of cytotoxically affecting pancreatic cancer cells using the described drug combination . a method of making the cytotoxic composition is additionally described .

Description:
the present invention will now be described more fully hereinafter with reference to the accompanying drawings , in which preferred embodiments of the invention are shown . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains . although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . any publications , patent applications , patents , and other references mentioned herein are incorporated by reference in their entirety . in case of conflict , the present specification , including any definitions , will control . in addition , the materials , methods and examples given are illustrative in nature only and not intended to be limiting . accordingly , this invention may be embodied in many different forms and should not be construed as limited to the illustrated embodiments set forth herein . rather , these illustrated embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . v - src - transformed mouse fibroblasts ( nih3t3 / v - src ), human pancreatic cancer ( panc - 1 ) and leukemic ( k562 ) lines have been described ( 14 - 16 ). the human pancreatic cancer lines , colo - 357 and mia - paca - 2 were kind gifts from drs . lancaster and mokenge ( moffitt cancer center ). the immortalized human pancreatic duct epithelial cell ( hpdec ) line was obtained from dr . tsao , oci , uhn - pmh , toronto ) ( 17 ). except for hpdec grown in keratinocyte - sfm media supplemented with 0 . 2 ng egf , 30 μg / ml bovine pituitary extract and containing antimycol , and k562 line in rpmi 1640 containing 10 % heat - inactivated fbs and 100 units / ml penicillin - streptomycin , all other cell lines were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) containing 5 % iron - supplemented bovine calf serum and 100 units / ml penicillin - streptomycin . recombinant human egf ( hegf ) is from creative biolabs , port jefferson station , n . y . ); gemcitabine is from ely lilly ( indianapolis , ind .). nuclear extract preparation and dna - binding with electrophoretic mobility shift assay ( emsa ) were carried out , as previously reported ( 14 , 15 ). the 32 p - labeled oligonucleotide probes used were hsie ( high affinity sis - inducible element from the c - fos gene , m67 variant ), 5 ′- agcttcatttcccgtaaatcccta ; ( seq id no : 1 ) that binds stat1 and stat3 ( wagner et al ., 1990 ) and the mgfe ( mammary gland factor element from the bovine β - casein gene promoter , 5 ′- agatttctaggaattcaa ; ( seq id no : 2 ) that binds stat1 and stat5 ( gouilleux et al ., 1995 ; seidel et al ., 1995 ). western blotting analysis was performed as previously described ( 15 , 18 ). primary antibodies used were anti - stat3 ( c20 ) ( santa cruz , santa cruz , calif . ), anti - py845egfr ( upstate biotech , millipore , billerica , mass . ), and antibodies against py705stat3 , stat3 , py1068egfr , py1086egfr , py1173egfr , egfr , py416src , src , c - myc , and β - actin from cell signaling ( danvers , mass .). sirna sequences for egfr and src were ordered from dharmacon rnai technologies , thermo scientific ( lafayette , colo .). sequences used are : egfr sense strand , 5 ′- gaaggaaacugaauucaaauu - 3 ′, seq id no : 3 ; egfr antisense strand , 5 ′- uuugaauucaguuuccuucuu - 3 , seq id no : 4 ′; control sirna sense strand , 5 ′- aguaauacaacgguaaagauu - 3 ′, seq id no : 5 ; and control sirna antisense strand , 5 ′- ucuuuaccguuguauuacuuu - 3 ′, seq id no : 6 . the c - src smartpool sirna reagent ( nm - 005417 , catalog # m - 003175 - 01 - 05 ) was used for src . transfection into cells was performed using 20 nm of egfr sirna or 25 nm of src sirna and 8 μl lipofectamine rnaimax ( invitrogen corporation , carlsbad , calif .) in opti - mem culture medium ( gibco , invitrogen ). proliferating cells in 6 - well or 96 - well plates were treated once with 0 . 1 - 1 mm zd1839 ( iressa ), 100 nm dasatinib , 50 - 100 μm s3i - 201 , 1 μm gemcitabine , or combinations of inhibitors for up to 96 h . viable cells were counted by trypan blue exclusion / phase contrast microscopy or assessed by cyquant cell viability assay , according to manufacturer &# 39 ; s ( invitrogen ) instructions , or cells were processed for annexin v binding ( bd biosciences ) with flow cytometry for apoptosis . s3i - 201 is fully described in reference 30 ( see below ). single - cell suspension of panc - 1 and colo - 357 cells were seeded in 6 - cm dishes ( 500 cells per well ) and assayed as previously reported ( 19 ), treated the next day with inhibitors for 48 h , and allowed to grow until large colonies were visible . colonies were stained with crystal violet for 4 h and counted under phase - contrast microscope . cell migration and invasion experiments were carried out and quantified as previously described ( 20 ), using bio - coat migration chambers ( becton dickinson , franklin , n . j .) of 24 - well companion plates with cell culture inserts containing 8 μm pore size filters , according to the manufacturer &# 39 ; s protocol . statistical analysis was performed on mean values using prism graphpad software , inc . ( la jolla , calif .). the significance of differences between groups was determined by paired t - test at p & lt ; 0 . 05 *, & lt ; 0 . 01 **, and & lt ; 0 . 001 ***. consistent with published reports ( 6 , 7 ), stat3 activity , per dna - binding with emsa analysis in nuclear extract preparations is constitutive in panc - 1 and colo - 357 , low in mia - paca - 2 , and undetectable in the normal human pancreatic duct epithelial cells ( hpdec ), compared to aberrant levels in nih3t3 / v - src ( 15 ) ( fig1 a ( i )). per supershift analysis , the dna - protein complex contains stat3 ( fig1 a ( i ), lane 3 ). by contrasts , stat5 activity is undetectable in pancreatic cancer cells ( fig1 a ( ii )), compared to aberrant levels in the k562 leukemic cells ( 16 ). egfr and c - src are aberrant in many human cancers ( 2 , 4 ). immunoblotting analysis showed a moderate py416c - src level in mia - paca - 2 , but enhanced levels in panc - 1 and colo - 357 cells similar to levels in nih3t3 / v - src , compared to low levels in hpdec ( fig1 b ( i ), upper panel ). the elevated py416src levels parallel enhanced levels of the src - sensitive py845egfr motif ( 21 ) in panc - 1 and colo - 357 cells , compared to low levels of same in hpdec ( fig1 b ( i ), lower panel ). total src or egfr protein remained unchanged . immunoblotting analysis further showed elevated levels of the egfr autophosphorylation motifs ( 22 ), py1068egfr ( fig1 c ( i ), lanes 2 and 7 ), py1086egfr ( fig1 c ( ii ), lanes 2 and 7 ) and py1173egfr ( fig1 c ( iii ), lanes 2 and 7 ) in panc - 1 and colo - 357 , compared to basal levels of same in hpdec ( fig1 c ( i )-( iii ), lane 1 ). we next examined the functional relationship between the activated egfr and src . immunoblotting analysis showed treatment of cells with dasatinib ( das ) inhibited src activity ( py416src ) ( 23 ) and induced an early ( 1 h ) and a sustained ( 24 h ) decrease in py845egfr levels ( fig1 b ( ii )). by contrast , no detectable changes in py416src and py845egfr levels were induced by treatment with the pan - erbb inhibitor , pd169540 ( pd169 ) ( 24 ) ( data not shown ) or the selective egfr inhibitor , zd 1839 ( zd , iressa ) ( 25 ) ( fig1 b ( ii )). in confirmation , sirna knockdown of c - src abrogated py845egfr levels ( fig1 b ( iii ), src sirna ), while egfr knockdown by sirna had minimal effect on py416src level ( fig1 b ( iv ), egfr sirna ). scrambled sirna has no effect ( fig1 b ( iii ) and ( iv ), con sirna ). thus , elevated py845egfr levels in pancreatic cancer cells are sensitive to src activity . immunoblotting analysis further showed that treatment of panc - 1 and colo - 357 cells with zd diminished py1173egfr levels ( fig1 c ( iii ), lanes 3 , 4 , 8 and 9 ) by as early as 1 h and up to 24 h , with no effect on py1068egfr (/ fig1 c ( i ), lanes 3 , 4 , 8 and 9 ) or py1086egfr level ( fig1 c ( ii ), lanes 3 , 4 , 8 and 9 ), suggesting that egfr kinase is essential for the induction of py1173egfr levels , but not py1068egfr or py1086egfr . by contrast , das treatment decreased py1068egfr and py1086egfr levels ( fig1 c ( i ) and ( ii ), lanes 5 , 6 , 10 and 11 ), with minimal effect on pyegfr1173 ( fig1 c ( iii ), lanes 5 , 6 , 10 and 11 ). both the py1068egfr and py1086egfr levels are binding sites for stat3 ( 27 , 28 ). given the concurrent egfr and src activation in panc - 1 and colo - 357 cells , we sought to define the regulation of aberrant stat3 activation . by in vitro dna - binding assay with emsa analysis of nuclear extract preparations , we observe an early repression ( in the first 30 min to 1 h of treatment ) of constitutively - active stat3 by the pan - erbb inhibitor , pd169540 ( pd169 ), the erbb2 - selective inhibitor , ag879 ( 7 ), zd , or das ( fig2 a ( i ), lanes 4 , 5 , 7 , and 8 , and ( ii ), lanes 2 , 4 , 6 , and 11 , and fig2 b , 1 h ), or by a combined pd169 and das ( fig2 a ( i ), lanes 10 and 11 , and ( ii ), lane 8 ). however , the stat3 activity in panc - 1 cells consistently rebounded following 24 h treatments with das , zd , or pd169 ( fig2 a ( i ) and ( ii ), 24 h ), even though egfr or src activity remained inhibited ( fig1 b and 1c , 24 h ). twenty - four hour treatment with the ag879 moderately inhibited stat3 activity ( fig2 a ( ii ), lane 12 ), which we speculate may be due to its widespread activity as a pan - erbb inhibitor . by contrast , treatment with the jak inhibitor , ag490 for 1 h had no effect on constitutive stat3 activity , but surprisingly abolished stat3 activity at 24 h treatment ( fig2 a ( ii ), lanes 9 and 10 ). moreover , combined treatment with ag490 and zd , das or pd169 for 24 h similarly abolished constitutively - active stat3 ( fig2 a ( ii ), lanes 14 , 15 , and 16 ). in colo - 357 , stat3 activity was inhibited by both zd and das , with the effects more striking for dasatinib ( fig2 b ). these findings together reveal a pattern of constitutive stat3 activation in pancreatic cancer cells that is mediated by both egfr and src , and a compensatory , jak - dependent secondary stat3 activity . a similar pattern of stat3 activation has been observed in head and neck squamous carcinoma , mesothelioma , squamous cell skin carcinoma , and non - small cell lung cancer cell lines following the inhibition of src ( 29 ). in further support , the sirna knockdown of egfr ( egfr sirna ) or src ( src sirna ) led to pstat3 suppression , as assayed by immunoblotting analysis ( fig2 c ). scrambled sirna ( con ) has no effect . immunoblotting analysis also shows that egf stimulation induces py705stat3 , py1086egfr , py1173egfr , py845egfr and py416c - src ( supplemental fig . s 1 ( i )-( iii ), lane 4 ) over and above constitutive levels in panc - 1 cells , in a manner that is similar to the induction of same in response to the stimulation of normal hpdec ( supplemental fig . s 1 , lane 2 ), except for py1068egfr levels in panc - 1 ( fig . s 1 ( ii ), upper right panel ). in control studies , immunoblotting analysis showed elevated perk1 / perk2mapk and pakt in panc - 1 and colo - 357 cells compared to normal hpdec , neither of which was significantly affected by treatment with zd or das ( data not shown ). inhibition of stat3 sensitizes pancreatic cancer cells in vitro to egfr and src inhibitors . given the preceding data on the inter - relation between egfr , src and stat3 activation , we investigated the biological implications and the therapeutic potential of a combinatorial approach . dasatinib and zd were used at 100 nm and 0 . 1 - 1 μm , respectively , as in literature reports ( 23 , 24 ), while the stat3 inhibitor , s3i - 201 was used at the sub - optimum , 50 μm , or at the 100 μm required to inhibit stat3 activation ( 30 ). viable cell count by trypan blue exclusion / phase - contrast microscopy showed that treatment with 1 μm zd , 100 nm das , or 50 μm s3i - 201 alone minimally affected cell viability by 24 h ( fig3 a day 1 ). by contrast , treatment for 48 to 96 h with or das or s3i - 201 alone progressively decreased cell viability , while treatment for the same period with zd showed minimal effect ( fig3 a ), except at 96 h when the number of viable panc - 1 cells decreased ( fig3 a ( i ), zd , day 4 ). comparatively , the combined inhibition of stat3 ( by s3i - 201 ) and egfr ( by zd ) or src ( by das ) or the combined treatment with ag490 ( jaks inhibitor ) and zd or das induced greater losses of viability at 48 - 96 h ( fig3 a and b ). the effects on cell viability as captured by trypan blue exclusion were confirmed by the cyquant cell proliferation / viability assay . unlike 24 h treatment duration that showed minimal effect on viability ( fig3 a ), cyquant assay showed that 48 - h treatment with each inhibitor alone decreased viable cell numbers ( quantified as fluorescent unit , fu ) in a dose - dependent manner ( fig3 c , zd , das and s3i - 201 ). we infer from the graphs that treatment with 1 μm zd for 48 h has minimal effect on cell viability ( fig3 c ( i ) and ( iv )), as observed by the trypan blue exclusion assay ( fig3 a ). however , the observed effects of single agents were significantly weaker compared to the concurrent treatment with a stat3 inhibitor and an inhibitor of egfr or src . results show that the treatment with s3i - 201 increased the sensitivity of panc - 1 and colo - 357 cells to zd and das , shifting the dose - response curves to the left ( fig3 c , zd + s3i - 201 , and das + s3i - 201 ). concurrent treatment with s3i - 201 significantly decreased the ic50 values as follows : 17 to 0 . 4 μm , and 100 to 6 nm , respectively , for zd and das against panc - 1 viability ( fig3 c ( i ) and ( ii )); and 6 . 5 to 2 . 4 μm , and 90 to 8 nm , respectively for zd and das against colo - 357 viability ( fig3 c ( iv ) and ( v )). for the impact of zd and das on the sensitivity to s3i - 201 , cyquant cell viability assay showed that das , but not zd increased the sensitivity of both cell lines to s3i - 201 , decreasing its ic50 from 40 to 15 μm , and from 45 to 20 μm , respectively , for effects on panc - 1 and colo - 357 cells ( fig3 c ( iii ) and ( iv )). thus , treatment with s3i - 201 sensitized cells to zd and das , while treatment with das , but not zd similarly sensitized cells to s3i - 201 . given the clinical implications of these findings , we extended these studies to examine the effect of egfr src and stat3 pathway on the response to gemcitabine , the anti - metabolite agent used in the treatment of pancreatic cancer . cyquant cell proliferation / viability studies showed that inhibition of egfr , src or stat3 sensitized panc - 1 and colo - 357 cells to gemcitabine ( fig3 d ). more importantly , the combined inhibition of stat3 and egfr or src induced a higher sensitization of cells to gemcitabine than that induced by the inhibition of any one alone ( fig3 d ). as known to the skilled , gemcitabine is a nucleoside analog of cytidine which interferes with dna replication , arresting tumor growth and resulting in apoptosis of the cell . gemcitabine is also known to bind to the active site of the enzyme ribonucleotide reductase ( rnr ) to irreversibly inactive the enzyme , thus interfering with the cell &# 39 ; s ability to produce deoxyribonucleotides necessary for dna replication and repair . this also leads to apoptosis . as noted above , the combined inhibition of stat3 and egfr or src induces a higher sensitization of cells to gemcitabine , creating another possibility for combination therapy of tumors . to further explore the sensitization potential of inhibition of aberrant stat3 , we performed colony survival assay ( 19 ). results show that inhibition of src ( by das ) or stat3 ( by s3i - 201 ( s3i )), but not egfr inhibition ( by zd ) resulted in reduced colony numbers ( fig4 a ). more importantly , the concurrent inhibition of stat3 and egfr or src resulted in much lower colony numbers ( fig4 a ), consistent with the much greater loss of viable cells due to the combined inhibition of stat3 and egfr or src ( fig3 ). to extend these studies , we performed annexin v binding / flow cytometric analysis for apoptosis . higher percentages of panc - 1 and colo - 357 cells undergoing apoptosis were observed for concurrent inhibition of stat3 and egfr or src than for the inhibition any one signaling molecule alone ( fig4 b ( ii ) and ( iii )). similar results were obtained for the concurrent treatments with ag490 and zd or das ( fig4 b ( ii ) and ( iii )). by contrast , similar treatments of normal hpdecs showed no significant apoptosis ( fig4 b ( i )) with the combination treatments . thus , we establish that pancreatic cancer cells have higher sensitivity to concurrent inhibition of stat3 and egfr or src than to the inhibition of a single entity . egfr , src and stat3 together promote pancreatic cancer cell migration and invasion . aberrantly - active src and stat3 have both been implicated in tumor cell motility , migration , invasion and metastasis ( 4 , 23 ). in vitro matrigel assay confirmed that inhibition of src or stat3 alone suppresses migration and invasion ( fig5 a ). however , concurrent inhibition of stat3 and egfr or src for 24 - h has a stronger effect on colo - 357 migration and panc - 1 invasion , except for src inhibition , which showed a similar effect on panc - 1 migration ( fig5 a ). at the 24 - h treatment when these studies were done , there is no significant effect on cell viability ( fig3 ). these findings are further evidence that pancreatic cancer lines are more sensitive to concurrent inhibition of stat3 and src or egfr . egfr , src and stat3 module regulates c - myc over - expression in pancreatic cancer cells . for insight into the underlying molecular mechanisms by which the egfr , src and stat3 pathway may support the cancer phenotype , we studied the regulation of key cancer - relevant genes . we show that c - myc is over - expressed in pancreatic cancer lines compared to normal hpdec ( fig5 b ). furthermore , the concurrent inhibition of stat3 and egfr or src consistently repressed c - myc expression . these findings suggest a functional synergy between egfr , src and stat3 in inducing c - myc expression in the context of pancreatic cancer phenotype and that the stronger repression of c - myc expression contributes to the antitumor cell effects of and the increased sensitivity of pancreatic cancer lines to concurrent stat3 and egfr or src inhibition . concurrent inhibition of stat3 and egfr or src induces human pancreatic tumor growth inhibition in xenografts . subcutaneous xenografts of colo - 357 , a metastatic pancreatic adenocarcinoma line were used to study the therapeutic implication of the stat3 , egfr and src inter - relationships and to evaluate the in vivo antitumor effects of concurrent inhibition of stat3 and egfr or src . data showed that in general , xenografts of colo - 357 cells showed low responsiveness to treatment with inhibitor of egfr , src or stat3 alone , although , as the therapy progressed , those tumors treated with only one inhibitor alone appeared to show reduced growth , which was statistically not significant from the control , non - treated tumors ( fig6 ). by contrast , tumors from mice treated with combined s3i - 201 and das or s3i - 201 and zd consistently showed reduced growth and smaller tumor sizes throughout the entire study ( fig6 ). thus , the residual tumor volumes ( sizes ) for tumors in mice treated with combination inhibitors were significantly different ( p & lt ; 0 . 05 ) from tumor volumes for tumors in control mice at days 20 and upwards post treatment . these in vivo antitumor effects of combination treatment with inhibitors of s3i - 201 and das or s3i - 201 and zd are consistent with the in vitro antitumor cell data and together indicate that aberrant stat3 cooperates with hyperactive egfr or src to sustain human pancreatic cancer . within the context of aberrations in the egfr , src and stat3 pathway in pancreatic cancer , present study reveals a strong role for src in supporting aberrant egfr activation by not only inducing the phosphorylation of y845egfr motif ( 31 ), but also promoting the induction of py1068egfr and py1086egfr motifs . these src - promoted events will greatly influence the status of egfr in pancreatic cancer cells . a role for egfr in aberrant stat3 activation in cancer cells has previously been reported in other tumor cells , including head and neck squamous cell carcinoma and breast cancer ( 26 , 32 ). present study extends those findings to pancreatic cancer and show that egfr is key in facilitating aberrant stat3 activation . moreover , the py1068egfr and py1086egfr induction by src is likely to have significant impact on the activation of stat3 , given that these two motifs are essential sites for the binding of stat3 to egfr in order to promote its phosphorylation and activation ( 27 , 28 ). furthermore , src may not only facilitate stat3 activation via the induction of those two tyr motifs of egfr , but it can also directly phosphorylate stat3 , as has been previously reported in other systems ( 18 ). it is therefore consistent that both hyperactive egfr and src promote baseline constitutive stat3 activation in pancreatic cancer , as revealed by our study . the present study is also in agreement with an earlier report of erbb - 2 - dependent constitutive stat3 activation in mia - paca - 2 and uk pan - 1 cells ( 7 ) and another study that showed that the full induction of stat3 activation by erbb2 required both src and jaks ( 33 ). our findings indicate that jaks contribute to the maintenance of constitutive activation in revealing a jak - dependent compensatory mechanism of stat3 activation upon inhibition of egfr and src . given that jaks inhibition did not abolish aberrant stat3 at the earliest time point , we deduce that this family of cytoplasmic tyrosine kinases may not be the predominant mediators of the baseline aberrant stat3 . thus , in pancreatic cancer cells , a two - phase model of activation of stat3 signaling emerges composed of an egfr - and src - dependent baseline , constitutive stat3 induction , and an induced stat3 activation that is dependent on jaks . the observed secondary induction of stat3 activation via jaks has similarly been reported in head and neck squamous cell carcinoma line ( 29 ) and could be due to growth - stimulatory factors released from tumor cells ( 34 ), which in turn would induce the activation of jaks and thereby promote stat3 activation . egfr , src and stat3 has each independently been established to have critical roles in malignant transformation ( 6 , 14 , 23 , 26 , 35 ), while their collective roles in promoting tumorigenesis have not been explored . while the inhibition of the activity of each of the three proteins induced antitumor cell response to some degree , data presented here strongly indicate that the multiple targeting of stat3 and egfr or src together has a higher potential to inhibit growth , viability , survival , malignant transformation , and migration and invasion in vitro . significantly , hyperactivation of the egfr signaling has been deemed a prognostic indicator of low survival among pancreatic cancer patients ( 36 - 38 ). also , there is evidence to indicate that the concurrence with aberrant src signaling potentiates the effects of aberrant egfr and induces biological synergy ( 3 , 21 , 39 ). given the potential collective roles of stat3 , egfr and src in promoting and supporting pancreatic cancer , the inhibition of any single entity alone is unlikely to be insufficient to impact the disease . present data that simultaneous inhibition of stat3 and egfr or src induced greater antitumor cell effects and a higher sensitization to gemcitabine provides a strong support for the opinion that stat3 may cooperate with egfr and src to support the malignant phenotype . indeed , the inhibition of stat3 seemed to sensitize pancreatic cancer cells to the antitumor cell effects of zd and das . multiple targeting of stat3 and egfr or src therefore has the potential to induce a greater antitumor efficacy . this is supported by our present data that concurrent treatment with thestat3 inhibitor , s3i - 201 and zd or das induced greater regression of xenografts of colo - 357 than treatment with either inhibitor alone . such a multiple - targeted therapy has received a strong interest in recent times , particularly given the dismal results in certain cases of molecular targeted monotherapy , such as anti - egfr monotherapy ( 40 , 41 ). thus , a combined gemcitabine and erlotinib ( egfr tk inhibitor ) therapy has recently been approved for patients with locally advanced / metastatic pancreatic cancer ( 42 , 43 ), although we note by our data that the inhibition of stat3 and egfr or src together induces a higher gemcitabine sensitivity than inhibition of egfr alone . the enhanced antitumor effects due combined stat3 and egfr or src inhibitors may in part be due stronger repression of the expression of c - myc oncogene . altogether , present study provides support for a multiple - modality therapeutic approach and lays the foundation for concurrent targeting of aberrant stat3 and egfr or src as a more effective approach for achieving an enhanced antitumor therapeutic efficacy in pancreatic cancer . accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims . 1 . tzeng c w , frolov a , frolova n , et al . egfr genomic gain and aberrant pathway signaling in pancreatic cancer patients . j surg res 2007 ; 143 : 20 - 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