Pancreatic cancer: from molecular pathogenesis to targeted therapy

NON-THEMATIC REVIEW

Pancreatic cancer: from molecular pathogenesisto targeted therapy

Alexios Strimpakos & Muhammad W. Saif &Kostas N. Syrigos

Published online: 22 April 2008# Springer Science + Business Media, LLC 2008

Abstract Pancreatic cancer is a deadly malignancy withstill high mortality and poor survival despite the significantadvances in understanding, diagnosis, and access toconventional and novel treatments. Though cytotoxicchemotherapy based on the purine analogue gemcitabineremains the standard approach in adjuvant and palliativesetting the need for novel agents aiming at the mainpathophysiological abnormalities and molecular pathwaysinvolved remains soaring. So far, evidence of clinicalbenefit, though small, exists only from the addition of thetargeted agent erlotinib on the standard gemcitabinechemotherapy. Apart from the popular monoclonal anti-bodies and small molecules tyrosine kinase inhibitors, othernovel compounds being tested in preclinical and clinicalstudies target mTOR, NF-κB, proteasome and histonedeacetylase. These new drugs along with gene therapyand immunotherapy, which are also under clinical evalua-tion, may alter the unfavorable natural course of thisdisease. In this review we present the main pathophysio-logical alterations met in pancreatic cancer and the resultsof the florid preclinical and clinical research with regards tothe targeted therapy associated to these abnormalities.

Keywords Molecular pathophysiology .

Pancreatic adenocarcinoma .Monoclonal antibodies .

Tyrosine kinase inhibitors . Novel agents . Targeted therapies

1 Introduction

Pancreatic cancer was first described as a new entity in1836 by Mondiere [1]. According to cancer statistics datafrom the USA, the incidence of pancreatic cancer hasincreased from 2.9 per 100,000 in 1920 to 7.9 per 100,000in 1960 and up to 11.0 cases per 100,000 in 2003, with theblack race having the highest incidence (14.0/105) and theAmerican Indian and the Asian/Pacific islanders the lowest(6.7 and 7.9/105 respectively; [2] see also official statisticsat the website of United States Center for Disease Controland Prevention: http://apps.nccd.cdc.gov/uscs).

This incidence rise is to a certain extent associated withthe modern life style as among the established risk factorswe find age (especially >55 years), body mass index andcigarette smoking, all of which have showed an increasingtendency during the last century [3]. Pancreatic cancer isthe 4th most common cancer in the USA. Thoughpancreatic cancer represents only 2% of all cancers, it isthe most aggressive one accounting for the 6% of all cancerdeath, and with its mortality nearly equal to incidence(about 32.000 cases annually). It is the fourth mostcommon cause of death by cancer in men and the fifth inwomen. Pancreatic cancer has got the highest mortality rate(99%) and the lowest 5-year survival rate as it runsgenerally less than 5%.

Despite having been recognised for almost a century, nostandard treatment was available until 1930s when radicalsurgery in pancreatic cancer patients started to be per-formed by Whipple, a surgeon in New York, and actuallyoffered a curative option in this aggressive tumour. Theslow trend, though, in developing new treatments continuedfor decades. In 1960s chemotherapy and radiotherapystarted to be used in the palliative setting and showed asurvival benefit.

Cancer Metastasis Rev (2008) 27:495–522DOI 10.1007/s10555-008-9134-y

A. StrimpakosDepartment of Medicine, Royal Marsden Hospital,Surrey, UK

M. W. Saif :K. N. Syrigos (*)Department of Clinical Oncology,Yale Cancer Center, Yale School of Medicine,New Haven, CT, USAe-mail: [email protected]

http://apps.nccd.cdc.gov/uscs

Later, in 1984 a clinical study, with a phase II design,showed that continuous 5-FU (5-fluorouracil) infusion hada survival benefit in patients with metastatic cancer [4].Fluopyrimidines remained the standard palliative treatmentfor 10 years until 1997 when a randomised phase III studyof gemcitabine versus 5-FU was conducted and demon-strated a statistically significant clinical benefit responsewith gemcitabine over the 5-FU (23.8% versus 4.8%. p=0.0022). The 1-year survival rate with gemcitabine therapywas 18% versus 2% with 5-FU [5].

Since then, not much progress has been achieved despitea florid research in combinatorial treatment of chemother-apy agents, often along with the use of the popularbiological agents targeting specific receptors of cancercells, molecular pathways, gene epitopes or gene products,involved in pathogenesis of pancreatic cancer. Ideally, atreatment that would target the cause of cancer would leadto cure or at least to a major response. In reality, this is notfeasible as very few tumors types (or subtypes) are causedby a single event or by one cause. Unfortunately, even insituations where the single primary causal genetic alterationwas identified, additional epigenetic changes and shiftsoften occur, during the stages of tumor progression andmetastasis, obscuring our understanding further.

The multicausality of cancer is calling for combinations ofthe existing anticancerous weapons, but this approach is oftenlimited by unacceptable cumulative treatment side effects.

2 History of targeted treatments in cancer

After decades of laboratory research and drug development,targeted therapies have been now in use in clinical practiceover the last 10 years. Their initial successful applicationtook place in the field of haematology with the developmentof the monoclonal antibody Rituximab (MabtheraTN)targeting the CD20 protein on the surface of leukemic B-cells. Rituximab obtained its licence for use in refractory B-cell Non-Hodgkin’s lymphoma by the FDA in 1997. At thesame period alemtuzumab (MabCampathTN), an anti-CD52protein antibody, demonstrated good results in the treatmentof T cell lymphomas and T-cell leukemias. Few years later, asmall molecule was discovered targeting the protein productof the Philadelphia chromosome, from the fusion of BCR-ABL genes, in Chronic Myelogenous Leukemia (CML).This small molecule was the Tyrosine Kinase Inhibitor (TKI)Imatinib Mesylate (STI 571, GleevecTN, Novartis), whichhas led since to a revolution in the treatment of CML,changing its natural history. Imatinib was licenced for thetreatment of CML by the FDA in 2001.

In solid cancers, transtuzumab (HerceptinTN), an ErbB2(HER2/neu) kinase inhibitor was the first FDA approvedantibody against breast cancer in 1998. The last 5 years

many novel agents targeting specific molecules in thesurface of cancer cells, such as Epidermal Growth FactorReceptor (EGFR), Vascular Endothelial Growth FactorReceptor (VEGFR), metalloproteinases (MMPs) or intra-cellular components of these transcription factors (tyrosinekinase, farnesyl kinase, etc) have been developed andlicensed for use in patients with solid tumors.

In pancreatic cancer, the first biological agent studied in aclinical trial was marimastat, an MMP inhibitor, which failedhowever to demonstrate any clinical benefit. The firsteffective biological drug which was found to be effectiveand was granted approval for the treatment of advancedpancreatic cancer was erlotinib (TarcevaTN), a small mole-cule Tyrosine Kinase Inhibitor, licensed by FDA in 2006.

3 Molecular pathogenesis of pancreatic cancer

Despite pancreatic cancer (PC) constitutes a group ofmalignancies, including pancreatic ductal adenocarcinoma(PDAC), serous cystadenocarcinoma, neuroendocrinetumors, sarcoma, acinar cell carcinoma and lymphoma, inthe literature the term pancreatic cancer refers almostalways to the PDAC. In this review we are also dealingwith the PDAC (from now on will be referred as pancreaticcancer or pancreatic adenocarcinoma).

The development of pancreatic adenocarcinoma requiresthe transformation of normal pancreatic cells to precursorpancreatic intraepithelial neoplasia (PanIN). There are threestages of PanIN, PanIN1, PanIN2 and PanIN3 which areassociated with genes mutations, progressive changes of thenuclei (increasing atypia), loss of polarity (epithelial andnuclear) and changes in cellular architecture [6]. When allthese changes take place then PanIN3 or carcinoma in situdevelops [7].

It is well-known that chromosomic abnormalities areinvolved in the pathophysiology and development ofpancreatic cancer. These abnormalities usually present as aloss or gain of alleles in various chromosomes, in a ratherrandom appearance. Established allelic loss has beenshowed for chromosome arms 1p (50%), 6p (50%), 6q(50%), 8p (56%), 9p (76%), 10p (50%), 10q (50%), 12p(50%), 12q (67%), 17p (95%), 18q (88%), 21q (61%), and22q (61%). Chromosomal additions from the other handinvolve chromosomes 7 and 20. [8] The above chromo-somal changes are significant as they are related to specifictumor suppressor genes, for example chromosome 17p isthe location of p53 tumor suppressor gene, chromosome18q is the location of the DPC4 gene and chromosome 9pis the site of the p16INK4a(MTS1) gene.

Commonly mutated genes in pancreatic cancer includeK-ras (in 74–100% of cases), p16INK4a (up to 98%), p53(43 to 76%), DPC4 (about 50%), HER-2/neu (in about

496 Cancer Metastasis Rev (2008) 27:495–522

65%) and FHIT (found in 70% of cases) [9–15]. Othergenes involved are notch1, Akt-2, BRCA2 and COX-2.[16–25] K-ras, HER-2/neu, notch1, Akt-2 and likely COX-2 are proto-oncogenes whereas all the other genes are tumorsuppressors (Table 1).

3.1 K-ras

K-ras (Kirsten Rat sarcoma virus oncogene) is a proto-oncogene located in chromosome 12p12.1. The ras path-way is significant in growth-promoting signal transductionfrom the cell surface receptors to the nucleus, affecting theproduction and regulation of other key proteins. There arethree proto-oncogenes belonging to the RAS family (H-ras,K-ras and N-ras) and all of them are located in the innerplasma membrane, bind GDP and GTP and possess anintrinsic GTPase activity which cleaves the GTP to GDP(switch off position). K-ras protein is active and transmitssignals by binding to GTP (turn on), but it is inactive (turnoff) when GTP is converted to GDP.

Mutation of K-ras proto-oncogene leads to an inactivityof GTPase and therefore persistent activation (switch onposition). Such a mutation of K-ras is present in pancreaticadenocarcinoma, colorectal adenocarcinoma, lung cancerand other solid tumors. The frequency of the K-Rasmutations in PC ranges from 74% to 100% [26–30]. Inpancreatic cancer cell lines, point mutations of K-Ras incodons 12, 13 and 67 have been confirmed using single-stranded conformation polymorphism (SSCP) and slot-blotallele-specific oligonucleotide (ASO) hybridization analy-sis. The most common mutations occur in the codon 12[28]. Though, these mutation were not associated initiallywith any prognostic significance in patients harbouringthem,10 subsequent studies on specimens from patientswith pancreatic ductal adenocarcinoma showed reducedsurvival in patients with GaT, cGT and GcT K-Rasmutations compared to GtT, aGT and GaC mutations [31].

In colorectal carcinoma and non-small cell lung (NSCL)carcinoma, K-ras mutation is associated with poor survivaland low response to anti-EGFR antibodies [32, 33]. Noparallel association of K-ras with response to anti-EGFRtreatment has been yet reported for pancreatic cancer.

3.2 p16/INK4

Gene p16 (also known as INK4a, CDKN2 or MTS1-multiple tumor suppressor 1) is a tumor suppressor gene,located in chromosome 9p21. Gene p16 encodes forp16INK4a protein which inhibits the interaction of cyclinD with the CDK4 and CDK6 kinases, by competitivebinding, and represses the G1àS cell cycle progression. Innormal cells the cyclin D-CDK4 complex phosphorylatesthe retinoblastoma gene product (Rb1), preventing theformation of the E2F-Rb1 complex. E2F is a group ofgenes involved in cell cycle regulation, mainly the G1àSphase and DNA formation. As a result of Rb1 phosphor-ylation, E2F becomes free to act as a transcription factorfacilitating progression of the cell cycle to phase S.Mutation and loss of p16INK4a activity results in absenceof these inhibitory effects at the level of cyclin D-CDK4interaction, thereby promoting cell cycle progression.

In pancreatic cancer, point mutation, hypermethylationor homozygous deletion of this gene is a common finding(ranging from 27 to 96% of cases) [30, 34, 35]. Thesediscrepancies in gene alteration incidence may reflectmethodological variations; it was also felt by the research-ers that p16 suppression in in vitro cell lines was presentmore often than in primary tumors.

Interestingly, p16INK4a deletion is associated withdecreased survival in non small cell lung cancer (NCSLC)and transformation of follicular centre cell lymphoma tohigh grade lymphoma. In pancreatic cancer cell linesthough, the results are inconsistent regarding the prognosticvalue of p16 [36, 37].

Table 1 Main gene alterations,their frequency and signifi-cance in pancreatic cancer

Mutated Genes Frequency References Clinical significance

Proto-oncogenesK-Ras 74–100% [26–33] Its protein consists therapeutic

target (FTIs)HER2/neu 16–65% [60–62] Its protein consists therapeutic

target (mAbs, TKIs)Akt2 10–72% [18, 72] UnknownTumor suppressor genesp16INK4a 27–96% [30, 34–35] Controversialp53 43–76% [10, 30, 38–39] Resistance to chemotherapy [40–44]

Probably poor prognosis [9, 11, 39, 45]DPC4 50% [14, 51] ControversialBRCA2 10–17% [17, 19, 57–59] Mainly in Familial casesFHIT 70% [16] Unknown

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3.3 p-53

Tumor suppressor gene p53 is among the most studied andfrequently mutated genes in cancer. While it was firstdiscovered as a gene in 1979, its role as a tumor suppressorgene was first published in 1989 by researchers at The JohnHopkins’s Hospital, USA. This 393 amino acid long gene islocated on chromosome 17p13. In normal cells it is usuallyinactive, bound to the protein mdm (HDM2 in humans),which prevents its action and promotes its degradation byubiquitination (attachment of ubiquitin and degradation byproteasome). In cells with abnormal or damaged DNA (asseeing in ageing or ionising radiation), p53 inhibitsproliferation and growth by blockage of the G1 to S phasetransition of the cell cycle, leads to cell-cycle arrest andpromotes a programmed cell death. These actions areachieved by inhibition of CDK4 regulatory protein or viaactivation of the inhibitory protein p21/WAF1. p53 muta-tions cause loss of the above functions and are central incarcinogenesis leading to uncontrolled cell growth andproliferation, increased cellular survival and chromosomalinstability.

In pancreatic cancer, p53 mutations are found in upto76% of pancreatic cancer cell lines [10, 30, 38, 39].Usually, mutations of p53 are not specific but rathersporadic and are resulting to the production of a mutantp53 protein which lives longer than the wild type (normal)p53 protein. Furthermore, p53 mutations may lead toresistance to chemotherapy treatments due to impairedp53-induced apoptosis [40–42]. The latest was supportedby studies in cancer cell lines where apoptosis was restoredafter introduction of the wild-type p53 [43, 44]. Whetherthese mutations have also a prognostic significance remainsunclear. Few researchers have suggested a shorter overallsurvival in pancreatic cancer patients carrying p53 mutationcompared to p53(-) patients [9, 11, 39, 45]. In other studiesthough no correlation of p53 with prognosis was demon-strated [46, 47].

3.4 DPC4

Another tumor suppressor gene which is involved inpancreatic cancer pathogenesis is the DCP4 (Deleted inPancreatic Cancer, locus 4) gene also known as SMAD4,found in chromosome 18, location 18q21.1. Smad 4 is amember of the smad family which contains nine members.[48] In normal cells the product of this gene, a 64-kDaprotein, plays a role in TGF-β (Tissue Growth Factor-beta)mediated signal transduction, gene transcription and growtharrest. The exact mechanism of the aforementioned pro-cesses involves binding of the TGF-β to its receptor TGF-βRII and subsequent activation of the TGF-βRI byphosphorylation. The activated TGF-βRI causes further

phosphorylation intracellularly of its targets smad2 and 3,which upon activation form a heterodimer complex withsmad4 [49, 50]. This complex interacts with DNA directlyor indirectly via other DNA-binding proteins, regulatingtranscription of the target genes, such as c-myc, p21 andp15 and thus leading to the regulation of cellular prolifer-ation. The ultimate cellular events in the normal cells willbe growth arrest, apoptosis and cell differentiation byblockage of the cell cycle in phase G1. Inactivation ofDPC4, hence, facilitates uncontrolled cellular growth andproliferation. DPC4/smad4 gene is inactivated most com-monly by homologous deletion with point mutations butalso by loss of heterozygosity (LOH).

Up to 50% of pancreatic adenocarcinoma cases harbourmutated/abnormal DPC4 [14, 51]. Germline mutation ofDPC4 is the causative factor in Familial Juveline polyposis[52]. Mutated DPC4 is also found in other adenocarcino-mas (colon, breast) though not as often as in pancreaticcancer. It is thought that loss of the DPC4 expression is arather late event in the pathogenesis of pancreatic cancer, asthis gene was expressed normally in PanIN1 and 2 and onlyin 30% of cases with PanIN3 [15, 51]. Whether the DPC4status has a prognostic value remains controversial, as in afew studies positive DPC4 status was associated with betteroutcome and survival post resection, [15, 53] but in otherstudies DCP4 expression was associated with worseoutcome after surgery or adjuvant chemotherapy [54, 55].

Additional antitumor effect of DPC4/smad4 is exertedthrough reduction of angiogenesis by decreasing VEGFexpression and induction of thrombospondin (an anti-angiogenetic factor) expression [56].

3.5 BRCA2

BRCA2 (BReast CAncer type 2) is a tumor suppressor genediscovered in 1995 and its mutant allele is related withfamilial but also sporadic cases of breast cancer. It islocated in chromosome 13q12.3 and, in normal cellsparticipates in DNA damage repair.

In pancreatic cancer, germline mutation of BRCA2 gene(locations 6174delT and 6158insT) have been reported bytwo published studies with an up to 17% familial link, [18,57, 58] though in other studies same germline mutationwere seen in up to 10% of sporadic cases [16, 59].

So far, no correlation of BRCA2 with prognosis orsurvival has been reported. Therefore, in clinical practiseregular screening for these gene mutations doesn’t seem tobe of help.

3.6 Erb family genes (HER-2/neu—EGF)

The proto-oncogene HER-2/neu, known also as ErbB2,encodes for the HER-2/neu protein, a member of the


epidermal growth factor receptor family (ErbB protein family)which consists of four receptor tyrosine kinases (Erb1/EGFR,erb2/HER-2/neu, erb3/HER3 and erb4/HER4). Her-2/neu asthe rest of the ErbB family members consists of three differentparts, an extracellular region, a transmembrane region and theintracellular tyrosine kinase domain.

Growth factors bound to these receptors, facilitate signaltransduction via activation of the PI3/Akt-MAPK (phos-phatidylinositol-3 kinase and mitogen-activated proteinkinase) pathway and consequently promote cell growthand differentiation. Amplification or overexpression ofthese proto-oncogenes and persistent activation of any oftheir protein receptors leads to uncontrolled proliferation,which is the case actually in many cancers such as breast,colon, lung, ovary as well as pancreas.

Despite the fact that HER-2/neu is overexpressed inbreast cancer in about 35% and is related with moreaggressive disease and poor prognosis, patient with thisgenetic abnormality do respond well to the monoclonalantibody transtuzumab (HerceptinTN), targeting the over-expressed HER-2/neu receptor.

In pancreatic adenocarcinoma, amplification of the HER-2/neu gene has been demonstrated by many researchers but theincidence rate ranges from 16% in one study, [60] to 27% [61]and up to 65% in a third one [62]. A further research workon pancreatic carcinomas demonstrated overexpression ofthe HER-2/neu in 45% of tumors, with no gene amplificationbut elevated levels of HER-2/neu mRNA [63].

So far, no association of HER2/neu gene amplificationand overexpression with prognosis or survival has beenestablished [13, 64].

There is evidence from preclinical and clinical studies inpancreatic cancer, that EGF and EGFR are both overex-pressed in the majority of the tumors; [65–67] therefore,there is growing scientific interest and research on the useof EGFR and HER2/neu inhibitors in clinical practise (e.g.trastuzumab, erlotinib, cetuximab).

3.7 Notch1 and Hedgehog

Notch1 is a proto-oncogene (location 9q34.3) normallyinvolved in cell differentiation, proliferation and apoptosis.In mammalian systems notch signaling pathway playssignificant role in embryogenesis as it controls epithelialstem/progenitor cells differentiation, survival, self-renewaland cell fate. In adult tissues Notch seems to contribute inmaintenance of homeostasis. It is important to mention thatNotch collaborates with other genes and related signallingpathways such as Wnt and Hedgehog (Hh), controllingtogether the proliferation of stem cells and their cellulardifferentiation and organogenesis. It has been demonstratedin recent studies that deregulation or constitutive activationof one or more of these pathways and their related genes

can cause cancer stem cells initiation and tumorigenesis.Upon sustained activation notch1 and Hedgehog have beenfound to play a role in the pathogenesis of pancreaticcancer. One of the main modes of action of Notch is byinduction of the activity of NF-κB and by up-regulation ofits related signalling pathway. Persistent activation of NF-κB is present in the majority of pancreatic cancer cases.NF-κB is also interacting with Hedgehog as we explainlater in this review. The notch1/NF-κB interaction wasestablished in studies in which down-regulation of notch-1by small interfering RNA or natural phytochemicals(genistein, curcumin) resulted in inhibition of cancerinvasion and metastases via inactivation of NF-kB and itsdown stream genes MMP-9 and VEGF [20, 21, 68, 69]. Inanother study, transfection of pancreatic cancer cells with aconstitutive active Notch-1 mutant (Notch-IC) resulted inincreased levels of VEGF, bFGF (i.e. beta fibroblast growthfactor) and angiogenin. Increased expression of notch-1was also noted in the intratumoral nerves The researchersconcluded that notch pathway most likely regulates theneurovascular development of pancreatic cancer [70]. Asexpected, cross-talk and interactions of the above molecularpathways is far more complicated and therefore synchro-nous targeting of various abnormalities may improvetherapeutic efficacy. Novel treatment approaches, such ashumanised monoclonal antibodies and small moleculeinhibitors against Hedgehog, gamma-secretase inhibitorsagainst Notch in combination with the current biologicalagents and cytotoxics may overcome the treatment resis-tance and hit the cancer problem closer to its roots.

3.8 COX-2 and other genes

Cyclooxygenase (COX), a key enzyme involved in thecyclooxygenase pathway, converts arachidonic acid tothromboxanes and prostaglandins. Two isozymes of COXexist, called COX-1 and COX-2. COX-1 is a constitutiveisoform expressed in most tissues; its inhibition results inadverse effects such as gastrointestinal ulcers or impairmentof renal blood flow. COX-2 on the other hand, is inducibleby cytokines and intracellular signals produced at sites ofinflammation; it can also be induced in various normaltissues by the hormones of ovulation and pregnancy,growth factors, oncogenes and tumor promoters. Constitu-tively, COX-2 is expressed only in brain and spinal cordtissue. COX-2 overexpression has been implicated in thecarcinogenesis of many tumors such as colon, rectum,breast, head and neck, lung, pancreas, stomach and prostate[71].

Preclinical studies testing the COX-2 expression onpancreatic cancer cells in comparison to PanINs, chronicpancreatitis specimens and normal pancreatic cells, demon-strated overexpression of COX-2 in adenocarcinomas in


contrast to non-malignant tissues; additionally, in adeno-carcinomas overexpressing COX versus non-expressingones there was a positive association with severity andpoor outcome [22–25]. The above findings have led to theinvestigation of COX-2 inhibitors (celecoxib) in clinicalstudies along with conventional cytotoxic treatment butalso in cancer chemoprevention.

Another gene implicated in pancreatic carcinogenesis isthe Akt-2 gene, which is often amplified and overexpressedin pancreatic tumors, varying from a 10% in one study [72]up to 70% in a second study on resected pancreaticadenocarcinomas [17]. Inhibition of Akt-2 in pancreaticcancer cells leads to decreased activity of NF-κB, increasedlevels of the pro-apoptotic gene Bax and down-regulationof the antiapoptotic gene bcl-2. Furthermore, inhibition ofAkt-2 resulted in increased sensitization of cancer cells tochemotherapy-induced apoptosis [73].

Genes cyclin D1 and cyclin D3, which are modulators ofcell cycle at phases G1àS, were also found overexpressed inpancreatic cancer and reported to be associated with poorprognosis [74–76].

Lastly, other genes involved in pancreatic carcinogenesisand possibly in tumor metastasis and invasion are MUC4,Scr, Bcl-6, mdm2 and S100P [39, 77, 78]. Though we havelimited yet knowledge about their clinical importance, someof these genes products have been spotted as therapeutictargets in some preclinical studies as we will demonstratelater in this review.

3.9 Molecular pathways involved

Nuclear factor-kappa B (NF-κB) is a family of cytoplasmicmolecules which, in normal conditions, is in inactive statusby binding to proteins IkBa and p100. There are five proteinsbelonging to the NF-κB family (p50, p52, p65, c-Rel andRelB) which appear as heterodimers. NF-κB is activatedthrough phosphorylation of IκBα by IKKβ and/or phos-phorylation of p100 by IKKα. The phosporylation will leadIκBα to degradation and p100 to process into the small formp52. As a result, two heterodimers of NF-κB (p50/p65 andp52/RelB) become free to translocate as active now NF-κBheterodimers to the nucleus. In nucleus, the NF-κB activeheterodimers bind to NF-κB-specific DNA-binding sites andgene promoters regulating gene transcription and alteringtheir expression. Affected genes include survivin, VEGF,EGF and MMP-9 which are involved in cellular survival,apoptosis, progression, invasion and tumor metastasis.

As in many other cancers, NF-κB is activated inpancreatic cancer cells, playing central role in its patho-genesis, progression, invasion and metastasis. [20, 21]

A second common pathway involved in pancreaticcarcinogenesis is the Ras-Raf-MEK-MAPK (mitogen-activated protein kinase) pathway, in which the mutated Ras

gene translates the constitutively activated Ras proteins and,via up-regulation of the MAPK cascade, causes uncontrolledcellular proliferation, differentiation and survival.

During the last few years a new molecular pathway, thehedgehog (hh) signalling, has been found to play a role inpancreatic carcinogenesis. Hedgehog pathway is normallyinvolved in pancreas embryogenesis/development, but itsderegulation and particularly overexpression of its sonichedgehog (Shh) ligand has been found to about 70% ofpancreatic adenocarcinomas [79]. Inhibition of Shh bycyclopamine (a naturally occurring steroidal alkaloid)resulted in apoptosis induction and blockage of pancreaticcancer cell lines proliferation, both in vivo and in vitro. [80]It is postulated that Shh overexpression is regulated byactivation of NF-κB and consequently inhibition of NF-κBresults to down-regulation of Shh [81].

The family of matrix metalloproteinases (MMPs) playsan essential role in cancer progression, invasion andmetastases. These are enzymes-proteases able to breakdown the extracellular matrix, its proteins and the basementmembrane and therefore to facilitate the cancer cellsimplantation and migration to distal sites. There are manyMMPs, but MMP-2 and MMP-9 are likely the mostimplicated in cancer invasion and metastases. [82] Asshowed in a study by Juuti et al., MMP-2 expressioncorrelates with advanced stages of disease and poorprognosis [83]. Along with overexpression of specificMMPs in advanced cancer, there is production of the tissueinhibitor of the metalloproteinases (TIMP) which aims tocounteract the untoward effects of MMPs in pathologicalsituations [82].

The described pathways outline the perplexity of themolecular pathways and their cross talk especially in highlyuncontrolled conditions as in malignancies.

4 Molecular targets

Based on the above basic knowledge new treatmentstargeting the implicated abnormalities have been developed.The main biological agents against pancreatic canceravailable at present are the epidermal growth factor receptor(EGFR) inhibitors, HER2/neu receptor inhibitors, metal-loproteinases (MMP) inhibitors, vascular endothelialgrowth factor (VEGF) inhibitors, farsenyl transferaseinhibitors (FTI) aiming to inhibit the activated K-rasprotein, and many other novel agents which will bediscussed later in our review (Fig. 1.).

4.1 EGFR inhibitors

Particular attention at present is paid on the EGFRinhibitors. There are two types of inhibitors against the


EGFR; monoclonal antibodies (MAbs) and small moleculestyrosine kinase inhibitors (TKIs).

4.1.1 Tyrosine kinase inhibitors

Erlotinib Erlotinib (CP-358774, OSI-774, Tarceva™) is anoral tyrosine kinase inhibitor (TKI) which is blockingselectively the EGFR, often overexpressed in NCCLC andpancreatic cancer in up to 70% of cases [12, 84]. Itseffectiveness in inhibiting the EGFR and also the MAPK(previously known as ERK1/2) molecular pathway wasfirstly demonstrated in vivo in mice whose pancreas wasinoculated with pancreatic cancer specimens, taken frompatients having undergone radical pancreatectomy forpancreatic cancer. In this early study OSI-774 significantlyenhanced tumor apoptosis when it was added on thecombination of wortmannin (a PI3K inhibitor) withgemcitabine [85].

Passing to the next step of drug development, the maximumtolerated dose (MTD) of erlotinib was determined in aphase I study, on patients with locally advanced pancreatic

cancer, in combination with concurrent chemoradiotherapy(gemcitabine, paclitaxel and RT). Three dose levels oferlotinib were tested (from 50 to 100 mg) during the RTperiod followed by a maintenance dose of 150 mg postradiotherapy, until disease progression. During chemo-radiotherapy, at doses of ≥75 mg of erlotinib, dose-limitingtoxicities (DLTs) reported were diarrhoea, dehydration, rashand small bowel stricture. The maintenance dose of 150mgwas well tolerated [86].

In another phase I/II study on patients with metastaticpancreatic cancer and other solid malignancies, the tolera-bility, pharmacokinetics and preliminary efficacy of erloti-nib in combination with gemcitabine was tested [87].Similar toxicities with the previous study were reported,as well as neutropenia and transaminitis. Again, themaximum tolerated dose (MTD) of erlotinib was set at150 mg, once a day, and no significant interaction withgemcitabine, in terms of side effects, was reported. In thisstudy, the pancreatic cancer subgroup (15 patients) had anoverall survival (OS) of 12.5 months and a progression freesurvival (PFS) of 9.6 months. The 1-year survival rate wasas high as 51% [87].

Fig. 1 The main biological molecules that currently consist targets ofpancreatic cancer therapies are the epidermal growth factor receptor(EGFR) inhibitors, the HER2/neu receptor inhibitors, the metal-

loproteinases (MMP) inhibitors, the vascular endothelial growth factor(VEGF) inhibitors and the farsenyl transferase inhibitors (FTI)


More recently, the final data from the first phase III,randomised, placebo controlled, study testing the efficacyof erlotinib along with gemcitabine versus gemcitabinealone were published by Moore et al. [88]. This study,sponsored by the Canadian National Cancer Institute(CNCI), recruited 569 patients and showed that patientson the combinatorial treatment arm demonstrated a statis-tically significant survival benefit (6.24 months versus5.91 months, p=0.038). In addition, the 1-year survival wasin favour of the erlotinib arm (24% versus 17%, p=0.023).Possibly, the most important information from this studywas a survival benefit seen on the subgroup of patients onerlotinib arm who developed high grade (≥2 according tothe NCI Common Terminology Criteria for Adverse Eventsversion 3.0) acneiform rash, similarly to cetuximab effect incolorectal cancer [88, 89]. The recommended dose forerlotinib in this study was down to 100 mg/day, mainly dueto slightly higher grade 1 and 2 toxicities in the combina-torial treatment. (Table 2)

Whether the survival benefit demonstrated in the laterstudy is significant in clinical practise and, the use oferlotinib justified by pharmaco-economic terms for broaderuse is difficult to say. Nevertheless, this was the first largepositive study of a combinative therapy in pancreatic cancerin the last 5 years.

Though targeting of the epidermal growth factor receptor(EGFR/HER1) is the main action of erlotinib, it was shown ina recent in vitro study that cancer cell lines from varioustissues sensitive to erlotinib express also the HER3 receptor(known to regulate the PI(3)K/Akt molecular pathway) [90,

91]. Insensitive to erlotinib cell lines were found to lackHER3 receptors. It was postulated thus that erlotinib inhibitssimultaneously the EGFR and HER3 receptors, and thereforeHER3 status could serve as a biomarker of response toerlotinib [91]. As multiple molecular pathways are involvedin carcinogenesis, rather than a single molecular abnormality,the concomitant use of erlotinib with genistein (a naturalpolyphenol with chemopreventive properties) and gemcita-bine was tested in another preclinical study on pancreaticcancer cell lines. Significant down-regulation of EGFR,activated Akt, NF-kB and survivin in pancreatic cancer cellwas demonstrated with the combination of all three agentscompared to erlotinib alone, resulting to growth inhibitionand induction of apoptosis; these findings suggested thatbetter results may be achieved with simultaneous inhibitionof the numerous involved targets [92].

Combination of erlotinib with rapamycin may be alsoclinically beneficial. Rapamycin is an inhibitor of mTOR, amolecule which controls cell growth; mTOR is regulated byAkt and participates in the phosphatidylinositol 3′-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway(PI3K-PIDK1-Akt-mTOR) which in turn is controlled bythe EGFR. In an in vitro study, combination of erlotinib withrapamycin resulted in a synergistic inhibitory effect on thecell lines growth from various tissues including pancreatic.This synergisms was seen in xenograft models as well buthasn’t been confirmed yet in clinical studies [90].

Lately, the role of erlotinib in combination withcapecitabine as a second line regimen after failure ofgemcitabine therapy was investigated in a clinical study on

Table 2 Published phaseIII studies of targeted agents inadvanced pancreatic cancer*

This refers toP value for all linesexcept 4.9–6.5 and 5.0–6.9

Agent (reference) Study arms n Responserate (%)

MedianOS(months)

P*95%CI

1-yearsurvival(%)

ERLOTINIB [88] EGFRTyrosine Kinase Inhibitor

Gem +Erlotinib vsGem

569 8.6 6.24 0.038 238.0 5.91 (HR

0.82)17

CETUXIMAB [118]EGFR Inhibitor

Gem +Cetuximab vsGem

735 7 6.5 0.014 n/a7 6.0 (HR

1.09)BEVACIZUMAB(132)

VEGFR InhibitorGem +Bevacizumabvs Gem

602 13.1 5.7 *4.9–6.5

n/a

11.3 6.0 *5.0–6.9

MARIMASTAT [141]MMP inhibitor

Gem +Marimastatvs Gem

239 11 5.51 1816 5.46 17

TANOMASTAT [143](BAY 12–9566) MMP inhibitor

Tanomastat vsGem

277 6 3.74 <0.001 n/a6 0.9 6.59

TIPIFARNIB [146] (R115777)Farnesyl Transferase Inhibitor(FTI)

Gem +Tipifarnib vsGem

688 6 6.43 <0.75 278 6.06 24


patients with advanced/metastatic pancreatic cancer. Thirtygemcitabine-refractory patients were treated with thiscombination and showed an overall radiological responseof 10% with a median survival of 6.5 months. Biochemicalresponse (decrease of the tumor marker CA19-9 level by >50%) was demonstrated in 17% of patients though. Apartfrom the modest efficacy, of concern were also the highincidence of diarrhoea (77% of patients) and the need fordose reduction in 2/3 of participants [93].

Many more phase I/II/III studies on pancreatic cancerpatients are in progress at present trying to answer bothcorrect dosing and efficacy of erlotinib in combination withvarious anticancer and biological agents.

4.2 Gefitinib

Gefitinib (ZD1839, IressaTN), is also a small moleculetyrosine kinase inhibitor which similarly to erlotinib blocksthe phosphorylation of the EGFR and downregulates thecascade molecular phenomena described previously. Gefi-tinib, similarly to erlotinib, is most effective in EGFRoverexpressing tumors, such as lung cancer, but its activitywas also tested in preclinical models in pancreatic cancercell lines. It was found that gefitinib at concentrations of2.5–10 μM could inhibit completely EGF-induced cellproliferation but no insulin growth factor (IGF)-inducedmitogenesis. Additionally, gefitinib caused inhibition ofEGF-induced phosphorylation of EGFR and mitogen-activated protein kinase (MAPK), rendering both inactive,and also inhibition of EGF-induced anchorage-independentcell proliferation and invasion [94].

The synergism of gefitinib with cytotoxics has beentested in vitro on pancreatic cell lines and has showed adifferential cytotoxic effect [95, 96]. The authors of thestudy where no response was demonstrated suggested thatonly EGF receptors with specific mutations are responsiveto gefitinib which was not the case in the tested two celllines used in this study [96].

The tolerability of gefitinib (250 mg/day) along withcapecitabine and radiotherapy (RT) (50.4 Gy in 28fractions) was tested in a phase I clinical study on patientswith pancreatic cancer. Dose-limiting toxicities (DLTs)were mainly diarrhoea, which occurred in 60% of patients(6/10), and arterial thrombosis in 20% of patients; thereforefurther research of this combination was recommendedbefore phase II studies are planned [97]. A second, phase I,study of combination of gefitinib (250 mg/day) withgemcitabine and RT (45Gy in 25 fr) was published in2006 and showed no significant DLTs [98]. In the laterstudy, the decreased incidence of diarrhoea was explainedpossibly by the fact that lower RT dose (45Gy vs. 50.4) wasadministered with no concomitant fluopyrimidines [98]. Ina recently published phase I trial, data regarding the

maximum tolerated dose (MTD) and DLTs of gemcitabinein combination with gefitinib were provided. With regardsto the secondary end points of this study, overall survival(OS) was found to be 7.13 months and the time toprogression (TTP) 4.57 months [99].

On the contrary, in a clinical phase II study thecombination of docetaxel with gefitinib, as a salvageregimen in gemcitabine pretreated patients with advancedor metastatic pancreatic cancer, failed to demonstrate anyclinical benefit (median survival 2.9 months, TTP2.1 months) [100]. Many more studies of gefitinib arecurrently in progress.

4.3 Other TKIs

Novel tyrosine kinase inhibitors have been developed andtested in preclinical and currently in clinical studies as well.

PKI-166 is a small molecule TKI targeting both ErbB1and ErbR2 (EGFR and HER2/neu) receptors. In an earlypreclinical study, nude mice, whose pancreas was implantedwith human pancreatic carcinoma cells, were tested forgrowth and metastasis after treatment with intraperitoneal(i.p.) gemcitabine alone, oral PKI-166 alone or theircombination [101]. The authors reported that pancreatictumor size was reduced by 59% with gemcitabine and 45%with PKI-166 treatment alone but by 85% with theircombination. Furthermore, the lymph nodal disease andliver metastases were reduced only in the combinatorialarm, reflecting to a survival improvement. Therefore, thisbiological agent showed efficacy both as a single agent aswell as in conjunction with cytotoxic treatment [101]. Thesame team of researchers reproduced the above results withfurther preclinical studies on mice transfected with humanpancreatic carcinoma cell lines, and also concluded that oraladministration of PKI-166 thrice weekly was the optimalschedule of administration with minimal toxicities [102,103]. Yet, no clinical studies have been conducted in orderto confirm the above promising effects of PKI-166.

Lapatinib (GW572116) is another TKI blocking revers-ibly both EGF (ErbB1) and HER2/neu (ErbB2) kinases.Though most of the research regarding this agent has beenconducted in breast cancer patients, a recent two stage,phase I study on biliary and pancreatic cancer waspublished in the American Society of Clinical Oncology(ASCO) gastrointestinal symposium in 2006. The aims ofthe study were to determine tolerance, dosing and efficacyof lapatinib in combination with either gemcitabine or withgemcitabine and oxaliplatin in patients with advancedpancreatic and biliary cancer. In this study the TKI waswell tolerated (at the full dose of 1,500 mg/day) andshowed significant efficacy in pancreatic cancer patientswith liver or peritoneal metastases calling for furtherevaluation in phase II–III studies [104].


Currently, a phase II single arm study of lapatinib andgemcitabine, in metastatic pancreatic patients, is in progressaiming to complete recruitment in March 2009 (details ofthis study can be found in US National Institute of Healthwebsite: http://clinicaltrials.gov/).

5 Monoclonal antibodies (MAbs)

5.1 Trastuzumab

As mentioned previously in this article, HER2/neu is oftenoverexpressed in pancreatic cancer [60, 61, 63]. Blockageof this receptor by the recombinant humanised antibodyTrastuzumab (HerceptinTN) can switch off the persistentactivation of signal transduction and tumor growth, accord-ing to results of a preclinical study on pancreatic cancer celllines in vitro and on orthotopic mouse model in vivo [105].It was found in this study that only cell lines overexpressingthe HER2/neu oncogene did respond to treatment withtrastuzumab [105].

Similar conclusions were drawn from subsequent pre-clinical study on sixteen pancreatic cell lines, which investi-gated the level of HER2/neu overexpression along with theantiproliferative effect of trastuzumab, either alone or incombination with gemcitabine [106]. Trastuzumab failed toshow any inhibitory effects of cancer cells proliferation invitro, regardless their HER2/neu status. In vivo however, inorthotopic implanted nude mouse xenograft models, thecombination of trastuzumab with gemcitabine was highlyeffective in the inhibition of growth and prolongation ofsurvival, in those models (Capan-1) which demonstratedhigh HER2/neu expression (+3) [106]. The synergistic effectof trastuzumab with the humanized anti-EGFR monoclonalantibody matuzumab was tested in mice bearing humanpancreatic carcinoma xenografts, which exhibit high EGFRand low HER2/neu expression. The combination of the twoantibodies demonstrated a significant antitumor effect com-pared to each antibody alone or no treatment [107].

Significant synergism was found as well with transuzu-mab and the fluopyrimidines, on pancreatic cancer celllines, both in vivo and in vitro (5-fluorouracil was used invitro and S-1, an oral compound, in vivo) [108]. Theexamined pancreatic cancer cell lines TRG were over-expressing the HER2/neu receptor and treatment with theantibody and the cytotoxic drug significantly inhibited thetumor volume and growth [108].

In patients with pancreatic cancer though, the results werenot that satisfactory. In a phase I clinical study by Safran et al.,pancreatic cancer patients with avid HER2/neu oncogeneoverexpression by immunochemistry criteria, were treatedwith gemcitabine along with trastuzumab and were tested fortoxicities and efficacy. Toxicities of the combinatorial

therapy were acceptable and similar to gemcitabine alone.In this trial the overall response rate (clinical and biochem-ical) was 41%, though only two patients (6%) demonstratedconfirmed partial response. Interestingly, only 12% ofpatients enrolled showed a (+)3 overexpression of Her2/neu. The median survival reported was 7 months, no muchdifferent than gemcitabine monotherapy [109].

5.2 Cetuximab

Cetuximab (IMC-C225, ErbituxTN) is a chimeric antibody(fusion of mouse and human protein) against the EGFreceptor (or ErbB1). Cetuximab has already been approvedby the FDA for treatment of colorectal adenocarcinoma andof Head and Neck squamous cell carcinoma, which alsooverexpress the EGF receptor.

The effect of cetuximab in pancreatic cancer has beeninvestigated in both preclinical models and clinical studies.In an early study on orthotopic mice models transfectedwith the human pancreatic cancer cells L3.6pl, treatmentwith cetuximab inhibited EGFR activation and resulted inup to 20% cytostasis [110]. Furthermore, cetuximab causedreduced levels of interleukin-8 (IL-8) and of vascularendothelial growth factor (VEGF). Concurrent treatmentwith gemcitabine, in the same study, resulted in enhancedantitumor effect (cytotoxic and cytostatic) and reduced livermetastases [110]. Similarly, treatment of athymic mice,bearing subcutaneous BxPC-3 xenografts, with IMC-C225alone (up to 33 mg/Kg every 3 days) or along with 5-fluorouracil (17 mg/kg twice per week) resulted insignificant in vitro inhibition of DNA synthesis by 23.8%,and suppression of pancreatic tumor growth in vivo. Again,the combinatorial treatment showed superior results com-pared to either treatment alone [111].

A further preclinical study investigated the effect ofIMC-C225, Gemcitabine and radiation, alone or in combi-nations, in vitro on the BxPC-3 and MiaPaCa-2 humanpancreatic carcinoma cell lines, and in vivo on athymicnude mice, bearing subcutaneous tumor xenografts of theabove cells. The authors reported inhibition of the EGFRphosphorylation by IMC-C225 in both cell lines, althoughlow levels of EGF receptor expression were noted. Thecombinatorial therapy of IMC-C225, gemcitabine andradiation caused complete tumor regression in the Mia-PaCa-2 models and the greatest response in the BxPC-3models, better than any single or doublet treatment [112].The same team of researchers investigated, on the samecell lines (MiaPaCa-2 and BxPC-3), the effect of pro-longed IMC-C225 treatment to the sensitivity of cells togemcitabine with or without radiotherapy, and alsomechanisms of resistance. They reported increasingsensitivity of MiaPaCa-2 cells to chemotherapy andradiation therapy after long-lasting exposure to the EGFR


http://clinicaltrials.gov/

antibody, but no effect whatsoever to the BxPC-3 cell line.Downregulation of EGFR levels and upregulation of thepro-apoptotic Bax gene were associated with a betterresponse of the MiaPaCa-2 cells. The postulated mecha-nisms of differential response included possible alternativeRas-MAPK pathway activation, persistent activation ofthis pathway by constitutive ErbB3 signaling, transactiva-tion of the Ras-MAPK pathway by the fibroblast growthfactor (FGF), and also persistent MAPK activation andimpaired internalization of the EGFR receptor in theresistant BxPC-3 cells [113, 114].

In another study, Sklabas et al. reported that treatment ofthe pancreatic cell line MDA Panc-28 with IMC-C225inhibited the activation of EGFR, resulting in downregulation of NF-κB and subsequently decrease of theanti-apoptotic genes bcl-2 and bfl-1 expression, reinstatingthus the apoptosis [115].

The synergistic effect of IMC-C225 with the VEGFR-2inhibitor (CD101) was tested on BxPC-3 pancreatic cancercells and colon (GEO) cancer cell xenograft models. Incontrast to the previous studies, the authors here reportedtumor response of the BxPC-3 models to the combinationof EGFR and VEGFR-2 inhibitors. This effect was likelydue to integration of the EGFR and VEGFR-2 inhibitors atthe level of the Hypoxia Inducible Factor-1 (HIF-1)pathway [116].

The promising results from the preclinical field justifiedthe study of cetuximab in clinical trials. In 2004 con-clusions of a phase II clinical study were published, testingefficacy and toxicity of cetuximab in combination withgemcitabine in pancreatic cancer patients with advanced ormetastatic disease, who were chemotherapy-naive and alsotested positive for EGFR expression [117]. Cetuximab wasadministered intravenously once weekly for 7 weeks (atinitial dose of 400 mg/m2 and 250 mg/m2 subsequently)followed by a week rest on the first cycle; thereafter for3 weeks on and 1 week off. Gemcitabine (1,000 mg/m2)was also given as an i.v. infusion, following the sameadministration schedule as cetuximab. Out of the 41patients enrolled in the study, five (12.2%) demonstrated apartial response to treatment, whereas another 26 (63.4%)showed stable disease by Response Evaluation Criteria inSolid Tumors (RECIST) criteria. The median time toprogression (TTP) was only 3.8 months and the medianoverall survival (OS) 7.1 months. The 1-year progressionfree survival (PFS) and overall survival rate were 12% and31.7%, respectively. The regimen was well tolerated withmost common grade 3 or 4 toxicities been neutropenia(39%), asthenia (22%), abdominal pain (22%) and lowplatelets (17%), while acne-like rash (87.8%) was the mostfrequent among all grades side effect [117].

Recently, the results of a large phase III study ofgemcitabine plus cetuximab versus gemcitabine were

reported by the Southern Western Oncology Group (SWOGS0205 Study) in the ASCO’s Annual meeting of 2007[118]. Seven hundred and sixty-six patients with locallyadvanced or metastatic pancreatic cancer were enrolled(735 eligible) and randomised to either gemcitabine aloneor in combination with cetuximab. The median survivalwas 6 months for gemcitabine monotherapy and 6.5 monthsfor the combinatorial therapy (HR 1.09, 95% CI 0.93–1.27,p=0.14), whereas the progression free survival (PFS) was 3and 3.5 months, respectively. The confirmed responseprobability was 7% in both arms [118]. Therefore, noclinical and statistically significant benefit from the additionof EGFR inhibition was demonstrated in this large study.

In the same meeting, the Eastern Cooperative OncologyGroup (ECOG) presented the results of its phase IIrandomised study of Irinotecan and Docetaxel with orwithout cetuximab [119]. Based on previous results of aphase II study of irinotecan and docetaxel in advancedpancreatic cancer, which showed a median survival of9 months, the researchers investigated whether efficacyimproved by adding an EGFR inhibitor. Ninety-twopatients were assigned to receive either chemotherapy(35 mg/m2 of docetaxel and 35 mg/m2 of irinotecan weeklyon a 6 weeks cycle) alone (arm A) or in combination withweekly cetuximab (arm B; 400 mg/m2 on week 1 and250 mg/m2 afterwards). Grade 3/4 neutropenia was ob-served in 26% of patients in arm A and in 33% in arm B.Other common toxicity was grade 3/4 diarrhoea in 33% ofarm A patients and 44% of arm B. At the time ofpublication, the median overall survival was 6.5 monthsfor arm A (95% CI, 4.8–8.6) and 7.4 months for arm B(95% CI, 4.4–10.7), while there were still patients alive inboth arms. From the above results it seems that irinotecanand docetaxel, plus/minus cetuximab could be anotheractive choice in advanced pancreatic cancer, but withcommon grade 3/4 toxicities [119].

At present numerous studies, mainly phase I and phaseII, are in progress, investigating the combination ofcetuximab with other biological agents (erlotinib, bevaci-zumab) cytotoxics (oxaliplatin, gemcitabine, capecitabine,cyclophosphamide), radiotherapy or vaccines.

5.3 Matuzumab

Matuzumab (EMD 72000) is a fully humanised monoclonalantibody with affinity to the EGF receptor. Matuzumab iscurrently investigated in gastrointestinal cancer but also innon-small cell lung cancer (NSCLC) where it has showedgood tolerance and some response.

In pancreatic cancer, a phase I study assessing thesafety and efficacy of matuzumab in combination withgemcitabine was recently published [120]. Seventeen


patients with no previous exposure to cytotoxics weredivided in three cohorts of escalating dose of matuzumabin combination with standard dose of gemcitabine.Agranulocytosis (grade 3 neutropenia in 3 patients,leucopenia in 1 and decreased white cell count in 1patient) was the only Dose-Limiting Toxicity (DLT), butfew patients experienced low grade rash and fever. Eightout of 12 evaluated patients showed response (66% OR,partial response and stable disease), and three of sixpatients with the partial response (50%) were treated with800 mg matuzumab weekly [120].

The combination of matuzumab with the HER2/neuinhibitor, trastuzumab, was assessed in preclinical models(nude mice) bearing pancreatic cancer cells xenografts(MiaPaCa-2 and BxPC-3). A significantly higher inhibitionof tumor progression was found in both models by thecombination of the two antibodies as compared to a singleantibody or no treatment [107].

Therefore, further studies need to be conducted forevaluation of this agent in combination with other biolog-ical agents or chemotherapy drugs.

5.4 Panitumumab

Panitumumab (ABX-EGF, E7-6-3, VectibixTN) is anothernovel fully humanised monoclonal antibody against theEGF receptor. It binds to the EGFR with high affinity invarious human cancer cell lines overexpressing the EGFR,preventing thus the EGF and the transforming growthfactor-alpha (TGF-α) from binding to this receptor.

Tumor xenografts from various solid cancers, includingpancreatic, showed significant inhibition of growth andproliferation, even eradication, after treatment with panitu-mumab [121, 122]. A phase II clinical study comparinggemcitabine plus erlotinib with or without panitumumabwill soon start recruitment in the USA.

6 VEGFR inhibitors

The role of angiogenesis in tumor growth and progressionin pancreatic cancer cells, through overexpression ofvascular endothelial growth factor (VEGF), is known forover a decade [123, 124]. As stated earlier in this review,VEGF is overexpressed in pancreatic cancer inasmuch as80% of cases. Overexpression of VEGF and VEGFreceptors is often accompanied by co-expression ofEGF, HER2/neu, transforming growth factor-alpha(TGF-α) and platelet-derived endothelial cell growthfactor (PD-ECGF) and is associated with advancedstaging and with poorer prognosis [125, 126]. The latestwas not the case in all published studies, as no correlationto prognosis or even better outcome has been also

reported in studies with VEGF positive pancreatic tumors[55, 127]. Therefore, researchers have been attracted totarget the VEGFR (via antibodies, TKIs or other agents)often simultaneously with other overexpressed receptorsin pancreatic cancer, aiming to tackle this devastatingdisease [102, 128, 129].

6.1 Bevacizumab

Bevacizumab (AvastinTN) is the first anti-VEGF receptorantibody which has shown merit in clinical practise againstvarious types of cancer. Bevacizumab is humanised andbinds to both VEGF receptors 1 and 2. AvastinTN has beenlicensed by the Food and Drug Administration (FDA) ofUSA for use in metastatic colorectal cancer (mCRC) andnon-small cell lung cancer (NSCLC).

In pancreatic cancer, a multicenter phase II studyinvestigated the efficacy (response rate and overall survival)of the combination of bevacizumab with gemcitabine [130].Fifty-two patients with advanced pancreatic cancer, previ-ously untreated, received gemcitabine (1,000 mg/m2 i.v.over 30 min on days 1, 8 and 15 on a 4 weeks cycles) andbevacizumab 10 mg/kg on days 1 and 15. The response ratewas 67% (partial response in 21% and stable disease in46%), the median survival 8.8 months and the median PFS5.4 months. Toxicities of notice were grade 3/4 hyperten-sion in 19% of patients, thromboembolic events in 13%, GIperforation in 8% and bleeding which occurred in 2% ofpatients [130].

Interim analysis from a further phase II study assessingthe efficacy of the combination of bevacizumab, oxaliplatinand gemcitabine in advanced pancreatic cancer waspresented in 2007 ASCO’s meeting [131]. The early results(60 out of the 82 patients enrolled) showed a response rateof 13.3% (one complete response and seven partialresponses) and a median survival of 9.3 months, thoughthree grade 5 events (resulting in death) occurred andshould draw awareness for future studies [131].

Subsequent randomised phase III study, double-blindplacebo controlled, was conducted by Kindler et al. andcompared overall survival of Gemcitabine-bevacizumab(GB) versus gemcitabine (G) alone. A preliminary reportfrom interim analysis was announced in the ASCO meetingof 2007 and showed no survival benefit in the combinato-rial arm (5.7 months in GB versus 6.0 months in G) [132].

In a slightly different context, a phase I study tested thesafety of bevacizumab in combination with capecitabine-based chemoradiotherapy in locally advanced and inoper-able pancreatic cancers [133]. Forty-eight patients wereenrolled and scheduled to be treated with escalation dosesof bevacizumab until dose-limiting toxicities (DLTs) andthe maximum-tolerated dose (MTD) were established.Bevacizumab was administered as an infusion (2.5–


10 mg/kg) every 2 weeks, starting 2 weeks prior tochemoradiotherapy initiation. Addition of bevacizumabdid not add on any significant toxicities and was welltolerated. Twenty patients (43%) though experienced agrade 2 (Common Terminology Criteria for AdverseEvents, v3.0) gastrointestinal toxicities such as nausea,vomiting and diarrhoea with two (4%) experiencing a grade3 toxicity requiring inpatient admission for supportive care.Eleven patients (23%) developed a grade 2 palmar plantarerythema (PPE) and four (8%) a grade 3 GI ulcerationassociated with perforation or bleeding in the radiotherapyfield, due partly to bevacizumab treatment. The dose of5 mg/kg was finally selected for patients who wereresponders at the completion of chemoradiotherapy. Theauthors reported a 20% partial response rate with a mediansurvival of 11.6 months (CI 95%, 9.6–133.6). Of interestfour patients (8%) were satisfactorily downstaged toundergo radical surgery to clear tumor margins [133].

Many more clinical trials including bevacizumab arecurrently in progress and expected with interest.

6.2 Vatalanib

Vatalanib (PTK787, ZK222584) is a small molecule TKItargeting selectively the VEGF Receptors 1, 2 and 3.Results from a preclinical study showed that this smallmolecule alone or in combination with gemcitabine wasable to inhibit the tumor volume of nude mice transfectedwith pancreatic cancer cells (L3.6pl) by 60 and 81%,respectively. Furthermore, the compinational therapyresulted in reduction of liver metastasis and lymph nodesinvolvement [134]. The same team of researchers con-firmed the above results and additionally demonstrated atherapeutic advantage by the synergism of concomitantinhibition of EGF and VEGF by PTK787 and PKI166 asmentioned in previous section as well [102].

In a phase I study, presented as an abstract in ASCO2006, eleven patients with advanced pancreatic cancer weretreated with vatalanib along with gemcitabine and assessedfor dosing, tolerability and toxicities [135]. The combina-tion was tolerated without major adverse events, and two ofthe patients (18%) showed a good response to treatment byRECIST and five stable disease. At the time of presentation,accrual to the final study cohort continued [135].

6.3 Other agents

Many more small molecules, TKIs and multikinase inhib-itors are in the drug development pipeline from thelaboratory to the clinical field.

Of those, ZactimaTN (ZD6474), a small moleculeinhibiting strongly the VEGFR-2 receptor, but also at alesser extent VEGFR-3, ErbB1(EGF) and RET kinase, has

shown some promising activity against pancreatic cancer inanimal models alone or in combination with chemotherapyand radiotherapy [136, 137]. Nevertheless no clinicalstudies have yet been conducted in humans.

Sorafenib (BAY43-9006) from the other hand, a Rafkinase inhibitor with activity also against PDGFR-beta,VEGFR-2,-3 and c-kit has demonstrated antitumor activityagainst several cancer cell lines in vitro, including pancre-atic cancer. Sorafenib has showed promising results in solidtumors, with classic example renal cell carcinoma andlately hepatocellular carcinoma. Sorafenib exhibits itsaction through downregulation of the MAPK pathway.

Recently, a phase I study tested safety and compatibilityof sorafenib with gemcitabine, and after initial recruitmentto dose escalating cohorts of patients with various advancedsolid malignancies, an expanded cohort of patients withadvanced pancreatic cancer was commenced at the recom-mended doses i.e. sorafenib 400 mg twice daily andgemcitabine 1000 mg/m2 weekly for 7 weeks initiallyfollowed by a week rest and thereafter for 3 weeks on a4 week cycle [138]. Significant grade 3/4 toxicitiesincluded thrombocytopenia (28.6%), lymphopenia(21.4%), neutropenia (16.7%), lipase increase (19%) andfatigue (14.3%). Thirteen patients in the pancreatic cohort(56.5%) achieved disease stabilization, and interestingly 2patients with ovarian cancer (10.5%) showed partialresponse. Therefore, further evaluation of this combinationis warranted in pancreatic cancer and of course in ovariantumors [138].

7 Metalloproteinases (MMPs)

7.1 Marimastat

Marimastat is the first and most studied MMP inhibitor insolid cancers, with broad activity against MMP-1, -2, -3, -7and MMP-9. In a phase 1 study on patients with advancedpancreatic tumors after previous standard treatment failure,dosing, safety and toxicities of marimastat was assessed.[139] Patients were recruited in different cohorts ofescalating dose of Marimastat (from 5 mg BD to 75 mgBD orally). The drug was well tolerated with a fewtoxicities such as arthralgia, musculoskeletal pain andstiffness. The overall median survival of the 64 enrolledpatients was 5.3 months and the 1-year survival rate 21%.The optimal dose recommended for subsequent trialsranged from 5 mg to 25 mg twice a day orally [139].

In a phase III study performed in the United Kingdom,414 patients with advanced unresectable pancreatic cancerwere randomised to receive marimastat (5, 10 or 25 mgtwice a day) or gemcitabine (1,000 mg/m2 weekly) andwere subsequently assessed for survival, clinical benefit


and safety [140]. The authors reported statistically signif-icant difference (p<0.003) of median survival in favour ofgemcitabine (167 days) compared to low dose marimastat5 mg and10 mg (111 and 125 days) but no significantdifference (p<0.78) compared to marimastat 25 mg (medi-an survival of 125 days). The 1-year survival rate wassimilar in the gemcitabine and marimastat 25 mg subgroup(about 20%) but both superior to lower doses of marima-stat. Both drugs were generally well tolerated but in themarimastat 25 mg group up to 55% of patients reportedmuscoloskeletal toxicity (grade 3 or 4 in 12%). In general,the novel drug had an acceptable profile as more treatment-related withdraws occurred in the gemcitabine arm (8%)than the marimastat group (3%) [140].

A second phase III study performed by the same team ofresearchers compared the combination of marimastat andgemcitabine versus gemcitabine alone. Two hundred andthirty-nine (239) patients were randomised to receivegemcitabine in combination with either marimastat orplacebo. There was no statistical difference in terms ofmedian survival (165.5 vs 164 days) overall response rate(11% vs. 16%), 1-year survival (18% vs. 17%), PFS (p=0.68 log-rank test) or TTF (p=0.70 log-rank test). Again thenovel agent was well tolerated with commonest toxicitybeen musculoskeletal symptoms [141].

Though marimastat does not seem to add on anysignificant advantage in the treatment of pancreatic cancer,given it possesses some activity may be useful in othermultimodality combinations in future.

7.2 Other MMP inhibitors

Ro 28-2653 is an oral MMP inhibitor with selective activityagainst MMP-2 and MMP-9. Studies on male Syrianhamsters induced with pancreatic cancer by subcutaneousinjections of N-nitrosobis-2-oxopropylamin (BOP; a carci-nogenesis inducer) tested the effect of Ro 28-2653 on livermetastasis and on the levels of MMP-2 and 9. The studyshowed that both the levels of MMP-2 and 9 and also theincidence of liver metastases were reduced by the Ro 28-2653 inhibitor. No clinical studies have yet been publishedregarding this agent [142].

BAY 12-9566 (tanomastat) is another matrix metal-loproteinase inhibitor which has been studied in clinicaltrials in patients with solid tumors. After many phase Istudies had demonstrated its safety and the appropriate dosewas established, a phase III trial comparing Bay 12-9566 togemcitabine in patients with pancreatic cancer was con-ducted few years ago by the National Cancer Institute ofCanada (NCIC). Patients with untreated advanced pancre-atic cancer were enrolled to receive either oral BAY 12-9566 800 mg BD daily or gemcitabine 1000 mg/m2 i.v ondays 1, 8 and 15 on a 28-day cycle. While 350 patients

were initially planned to be recruited, the study was closedprematurely after an interim analysis was performed, with277 patients enrolled at that point. The reason of this earlytermination was a statistical significant advantage ofgemcitabine in terms of median survival (6.59 vs. 3.74, p<0.001) and progression free survival (3.5 vs. 1.68, p<0.001). Both agents demonstrated a low toxicity profile, butin quality of life (QoL) assessment gemcitabine was foundagain superior [143].

Following the above results, BAY 12-9566 has not beentested since in other clinical trial in pancreatic cancer patients.

8 Farnesyl transferase inhibitors (FTIs)

8.1 Tipifarnib

Tipifarnib (R115777, ZanestraTN) is a selective non-peptidomimetic competitive inhibitor of farnesyl transfer-ase. Farnesyltransferase (FTase) is one of several enzymesinvolved in cell survival and regulation of apoptosissignalling. This enzyme is required for the function ofp21 (Ras), RhoB and other proteins (e.g. PI3K/Akt).Without farnesylation Ras cannot attach to cell membrane,which is significant for signal transmission from themembrane receptors to intracellular proteins.

Many preclinical studies showed activity of this agentagainst pancreatic cell lines and xenograft models.

A phase II study tested the efficacy of tipifarnib inpatients with previously untreated advanced pancreaticcancer. [144] Twenty patients were treated with oraltipifarnib 300 mg twice daily for 3 weeks on a 4 weekcycle. Though the treatment caused a partial inhibition ofFTase in mononuclear cells, the authors reported noobjective antitumor activity of this agent. Main toxicitiesobserved included gastrointestinal (elevation of liverenzymes, nausea, vomiting), fatigue and myelosuppression[144].

In addition, a phase II study was designed by theSouthwest Oncology Group (SWOG) to assess the efficacyof tipifarnib as a single agent in advanced pancreatic cancer.Again, tipifarnib as a monotherapy was found to beineffective (median survival was only 2.6 months) in thisaggressive disease [145].

A well designed phase III study was subsequentlyconducted in Belgium, Europe, and tested whether thecombination of gemcitabine with tipifarnib improvessurvival and outcome as compared to gemcitabine alone[146]. Six hundred and eighty-eight patients were rando-mised in this study to receive weekly gemcitabine infusion(initially for 7 weeks with a week rest and subsequently for3 weeks followed by a week rest) with either oral tipifarnib(200 mg BD continuously) or placebo. The median overall


survival (OS) with the combination was 193 days comparedto 182 days in the gemcitabine arm (p<0.75) and the 1-yearsurvival rate 27% versus 24%. Regarding toxicities, moreevents of myelosuppression and drug related deaths (10 v7), were reported in the experimental treatment, though theauthors reported an overall acceptable toxicity profile inboth treatment arms [146]. Once more, no added benefitwas demonstrated by the concomitant use of tipifarnib andgemcitabine.

8.2 Lonafarnib

Lonafarnib (SCH66336) is an oral FTI known to causetumor growth suppression in mouse cancer models andhuman xenografts in vivo. [147] Treatment of many cancercell lines in vitro, including MiaPaCa-2 and PC-1 pancre-atic cancer cells, with SCH66336 and SCH58500 (areplication-deficient, recombinant adenovirus, whichexpresses the human p53 tumor suppressor) showedsynergism and significant antiproliferative activity [148].Subsequently, many phase I studies in patients withadvanced solid tumors have been conducted since, anddemonstrated a good safety profile. Main toxicities reportedwith SCH66336 at the high dose of 400 mg twice a daywere a grade 4 myelosuppression and vomiting (accordingto Common Terminology Criteria for Adverse Events v3.0)and also grade 3 anorexia, fatigue and diarrhoea. Fewpatients experienced a grade 2/3 elevation of creatinine.Therefore, the recommended dose for further evaluationwas 200–300 mg twice daily [149, 150].

In a phase II study, patients with advanced pancreaticcancer were randomised to receive either oral SCH66336200 mg BD or weekly gemcitabine infusion. It was foundthat the lonafarnib (SCH66336) was inferior in terms ofoverall survival (3.3 vs. 4.4) and progression free survival(PFS) at 3 months (23 vs. 31%). The experimental treatmentwas well tolerated (Proc Am Soc Clin Oncol 20: 2001, abstr608). Based on these results more studies are warranted forfurther evaluation in combinatorial treatments.

9 COX-2 inhibitors

9.1 Celecoxib

Cyclooxygenase-2 (COX-2) is overexpressed in manycancers such as colorectal, breast, lung and pancreas, inboth early and late stages. Inhibition of COX-2 is beingextensively investigated as a chemopreventive and antican-cer treatment.

Celecoxib is the most studied COX-2 inhibitor, tested inmany preclinical and clinical trials. Studies on pancreaticcell lines in vitro and on xenografts models in vivo have

showed that treatment with celecoxib induces apoptosis,inhibits angiogenesis and reduces tumor growth andmetastasis [151, 152]. There are other studies though,celecoxib failed to demonstrate antitumor activity [153].Interestingly, synergistic anticancer effect was shown withthe combination of celecoxib with gemcitabine or withfluopyrimidines and radiotherapy in preclinical pancreaticcell lines or in xenografts overexpressing COX-2 [154,155]. Synergism was also noted by combination ofcelecoxib with either erlotinib or curcumin. In the firstexample celecoxib potentiated the erlotinib induced apo-ptosis and growth inhibition by down regulation of Her2/neu, EGFR and COX-2 expression with parallel NF-kBinactivation [156]. The combination of celecoxib withcurcumin resulted in growth inhibition and induction ofapoptosis in pancreatic cancer cell lines, by down-regulationof COX-2 expression [157].

Many clinical studies have been conducted already andmany more are in progress at present. In a pilot study,celecoxib in combination with protracted 5-FU infusionwas used as a second line chemotherapy in patients withadvanced pancreatic cancer after progression to gemcita-bine treatment [158]. Of the 17 patients enrolled, twoexperienced a partial response and two stable disease(overall response rate 12%, 95% CI 0–27%). The mediansurvival was 3.75 months and the median time toprogression 2 months. Four patients stopped celecoxibtreatment due to upper gastrointestinal toxicities. Further-more, the most common laboratory toxicity was anasymptomatic elevation of transaminases [158].

In a subsequent phase II study, patients with advancedinoperable pancreatic cancer were treated with a combina-tion of gemcitabine, irinotecan and celecoxib [159]. Of the20 patients enrolled, 18 were evaluable for toxicity and 17for tumor response. Patients were administered 1,000 mg/m2

of gemcitabine and 100 mg/m2 of irinotecan intravenous-ly, on days 1 and 8 on a 3 week cycle, along withcontinuous oral celecoxib 400 mg twice a day. At the timeof publication, three patients had a partial response (18%),twelve stable disease (70%) and two disease progression.The majority of patients reported significant pain im-provement (69%) and better quality of life (76%). Themost common grade 3–4 toxicities were neutropenia(50%), anaemia (39%), diarrhoea (17%), fatigue (17%),nausea (11%), one event of upper GI bleeding (6%) andalso one of neutropenic sepsis which led to death. Themedian time to progression (TTP) was 8 months and theoverall survival (OS) 13 months. At 1 year, 64% ofpatients were alive [159].

In another phase II study on advanced pancreatic cancerpatients, the efficacy of the combination gemcitabine atfixed-dose-rate infusion (FDR), cisplatin and celecoxib wastested. The authors reported no benefit from the addition of


this COX-2 inhibitor to the cytotoxics as the mediansurvival was only 5.8 months and the probability of6 months survival 46%. Furthermore 65% of patientsexperienced a grade 3 or 4 neutropenia [160].

A third phase II trial, in a similar group of patients withthe previous studies, examined the efficacy of gemcitabine(1,000 mg/m2 i.v. on days 1,8 every 3 weeks) withcelecoxib (400 mg BD daily). Four patients from the 42recruited achieved partial response (9%) and 26 (62%)stable disease by RECIST criteria. By clinical terms, twentythree patients reported benefit from the treatment (54.7%).There was no grade 4 toxicity, while grade 3 neutropeniaoccurred in 19% and grade 3 of hepatotoxicity in 7% ofpatients. Therefore this combination merits further evalua-tion in phase III studies [161].

In the most recent clinical study, patients with resectablepancreatic cancer were assigned to receive either celecoxibor placebo 5 to 15 days prior to their operation. In the samestudy, tumors from other pancreatic cancer patients weretransplanted in nude mice and subsequently treated withcelecoxib or vehicle. Unfortunately no growth inhibition orantitumor effect was seen in the resected specimens or inthe xenograft tumors after treatment with single agentcelecoxib, though synthesis of prostaglandin E2 wassufficiently inhibited [162].

A concern that has arisen lately regarding the use ofCOX-2 inhibitors is the increasing risk of cardiovasculardiseases, as shown in a few meta-analysis assessing the riskof MI with selective COX-2 inhibitors including celecoxib[163–166]. For that reason, cautious use may have to beconsidered in high risk patients for cardiovascular disease.Nevertheless, celecoxib is still studied in clinical trials invarious cancers including cancer of pancreas.

10 NF-kB Inhibitors

Nuclear factor-κB (NF-κB) is an important pathway in signaltransduction persistent activation of which is implicated inthe pathophysiology of many chronic and malignant dis-eases. Therefore, ongoing research, mainly preclinical, aimsto develop ways or agents that can block the overactivationof NF-κB and reverse its deleterious effects.

Recently, a dose-dependent inhibition of NF-κB inpancreatic cancer cell lines PANC1 and SW1990was reportedafter treatment with lidamycin, an antibiotic and cytotoxicdrug of the enediyne family. NF-κB inhibition resulted ininduction of apoptosis, growth arrest and cell cycle arrest ofthe lidamycin treated cancer cells. Lidamycin managed toproduce these cell effects at a much lower concentration(IC50) than the required dose of cytotoxics gemcitabine, taxol,mitomycin and adriamycin [167]. Moreover, lidamycincaused down-regulation of K-ras mRNA and reduction of

metalloproteinase MMP-9 which has been related with theinvasive and metastatic potential of pancreatic cancer.

Curcumin, a phytochemical antioxidant found in curry,has demonstrated activity against pancreatic cancer inpreclinical and clinical studies [168]. In pancreatic cancercell lines curcumin inhibited the overexpressed NF-κB andNF-κB-related molecules and proteins. In addition, on thesame cancer cell lines curcumin potentiated the cytotoxiceffect of gemcitabine [169]. Similarly, genistein, a soyisoflavone phytochemical, was found to downregulate NF-κB and thus to increase the antitumor activity of cytotoxics(cisplatin, gemcitabine) and of biological agents (erlotinib)[92, 170, 171].

The COX inhibitors aspirin and sulindac, in combinationwith parthenolide (a natural phytochemical), were able toinhibit growth of the pancreatic cancer cell lines in vitroand of animals injected with orthotopic tumor cells in vivo,via down-regulation of the NF-κB pathway [172, 173].

DCB-3503 (a tylophorine analog) is another agent withactivity against NF-κB, which when administered toPANC-1 and HPAC pancreatic cancer cells caused inhibi-tion of NF-κB and its related protein cyclin D, and thussuppressed cell cycle progression and tumor growth. [174]

Apart from the above agents, many more compoundsand natural products are able to inhibit NF-κB and thereforeto influence the myriad of genes regulated by this importantpathway which are also involved in various stages ofpancreatic cancer development.

11 Proteasome inhibitors

Proteasome is a cellular protein complex involved in thedegradation of many molecules such as proteins, transcrip-tion factors and cyclins. Since, mutated or up/down-regulated tumor suppressor genes (e.g. p53), proteins andtranscription factors are involved in cancer pathogenesisand progression, proteasome inhibitors aim to restore someimportant cellular functions, mostly apoptosis. Vortezomib(PS-341, VelcadeTN) is a proteasome inhibitor licensed bythe United States Food and Drug Administration (FDA) fortreatment of multiple myeloma in 2003. In pancreaticcancer, preclinical studies on cell lines and animal modelsdemonstrated induction of apoptosis and improvement ofthe cytotoxic effect of chemotherapy [175–177]. Enhancedapoptosis was also found with combination of bortezomiband trichostatin A (TSA), a histone deacetylase inhibitor[178]. A number of phase I studies in various solid tumorshave tested and established the safety and dosing ofbortezomib in combination with cytotoxic agents.

In pancreatic cancer patients, a phase II study assessedthe efficacy of bortezomib alone or in combination withgemcitabine. The bortezomib monotherapy was proven


ineffective (response rate 0%) but even the combinationtreatment didn’t add any survival benefit (median survival4.8 months, 95% CI 2.4–7.4) to that expected fromgemcitabine alone [179].

Currently, two phase II studies on pancreatic cancerpatients are open for recruitment in the United States. Thefirst trial is testing the efficacy of combination ofcarboplatin with bortezomib. The second trial is assessingan individualized drug treatment selection process testingbortezomib and another nine anticancer drugs, initially inmice implanted with the resected human pancreatic cancerspecimens, and at a second stage in patients who progressafter surgery, using the most effective drug based on thepreclinical data. End points in this study include time totreatment failure, 6 months survival and response rate afterthe most effective agent (as identified in animal models) foreach individual has been used.

12 Src inhibitors

The Src family kinases (SFKs) are non receptors kinasesoverexpressed in the majority of pancreatic cancers andinvolved in cancer progression and metastasis [180].Inhibition of these kinases has been studied the last fewyears and has showed promising results in preclinicalcancer models. Ito et al. investigated the effect of the Srcinhibitor pyrazolopyrimidine, on three different pancreaticcell lines (BXPC-3, PANC-1, and MIAPaCa-2) and found avariable but substantial inhibition of cell growth and ofinvasiveness of these cell lines (50%, 22% and 7%,respectively) [181]. Src overexpression may be associatedwith pancreatic cancer cell chemoresistance to gemcitabinethrough activation of Akt; therefore, inhibition of Src mayimprove cytotoxic effect of treatment, as demonstrated ina preclinical study on pancreatic cancer cell lines [182].The importance of Src kinase was also demonstrated in astudy on L3.6pl pancreatic tumor cells in which Srcexpression was reduced by use of small interfering RNA(siRNA), resulting in a significant reduction of tumorgrowth and metastasis [183]. In the same study, similarwith the above results were observed in vivo on male nudemice bearing pancreatic tumors after treatment withdasatinib (BMS-354825) which selectively inhibited Src/Abl kinases [183].

Treatment of orthotopic pancreatic cancer animal modelswith the Src inhibitor AZD0530 led to a significant growthinhibition and increase of survival [184]. Another kinaseinhibitor, GN963, with activity against PDGFR, Src andAkt kinases demonstrated antitumor activity in L3.6plpancreatic cancer cell orthotopic mice models both as amonotherapy and in combination with gemcitabine [185].Treatment with GN963 single agent (100 mg/kg three times

a week) resulted in 52% tumor volume reduction, whereastogether with i.p. gemcitabine twice weekly caused an 81%tumor volume reduction and also complete inhibition ofliver metastases incidence [185]. Analogous synergisticeffect in terms of tumor volume and metastases reductionwas demonstrated after treatment of pancreatic cancer cellswith the Src inhibitor AZM475271 alone or along withgemcitabine [186].

Currently, there are clinical studies in progress withphase II design testing the efficacy of the Scr inhibitorsdasatinib and AZD0530 in metastatic pancreatic cancer.

13 MUC4

MUC4 is a member of the MUC genes (other membersinclude MUC 1,-7) which are often found in normalpancreatic tissue but are apparently overexpressed inpancreatic cancer [187]. MUC4 in particular seems to beexpressed only in pancreatic cancer but not in benignsituations, such as chronic pancreatitis, and therefore maywell serve as a diagnostic biomarker [77]. The level ofMUC4 expression correlates with the advanced stage of themalignant disease [188], its invasiveness and increasedproliferation, [189] but may also correlate with a poorprognosis [190]. Though MUC4 mucin gene and proteinwere known for over a decade, it was not until recently thattreatment of pancreatic cancer cells in vitro and in vivo (inmice models) with an antisense MUC4 RNA demonstrateda significant suppression of tumor growth and metastasis[191]. Therefore, MUC4 is a potential target for treatmentrequiring further clinical exploration.

14 FAK

Focal adhesion kinase (FAK) is a non-receptor tyrosinekinase located in the cellular cytoplasm. FAK participatesin signal transduction, cell cycle regulation, apoptosis andmigration. It was found that up to 50% of tumors frompatients operated for pancreatic cancer expressed thiskinase [192]. In a preclinical study on three pancreaticcancer cell lines, all lines expressed FAK and demonstratedenhanced cell adhesion and invasion [193]. In vitroinhibition of FAK in MiaPaCa-2 cancer cells treated withthe flavonoids quecertin and luteonil or small-interferingRNA (siRNA) resulted in significant inhibition of cellmigration and invasion [194]. Another study on nudemouse orthotopic pancreatic cancer xenograft modelsreported that inhibition of FAK with small interferingRNA (siRNA) results in reversal of chemoresistance togemcitabine and therefore enhanced cytotoxicity [195]. Thesame team of researchers, in another preclinical study,


suggested that silencing of FAK gene facilitated apoptosisand could suppress metastasis in pancreatic cancer cell linesand in mice xenograph models [196].

Therefore, this kinase may serve as a future therapeutictarget of pancreatic cancer and it also merits furtherinvestigation in the clinical setting.

15 mTOR inhibitors

The mammalian target of rapamycin (mTOR), previouslyknown as FRAP-p70s6K, is a serine/threonine kinase proteininvolved in a signalling pathway which regulates cell cycle,cell growth and cell proliferation. This pathway is deregu-lated and constitutively active (persistently phosphorylated)in pancreatic cancer cells [197]. Inhibition of mTORphosphorylation by rapamycin (an immunosuppressant alsoknown as sirolimus) resulted in cell cycle arrest at the G1àSphase and inhibition of pancreatic cancer cell proliferationand cancer growth without any effect on apoptosis [198].

Similar results, but in addition induction of apoptosis,were observed after treatment of the pancreatic cell linesBxPC-3 (p53-defective) and MiaPaCa-2 with the rapamy-cin analog CCI-779 (temsirolimus) [199]. Researchers fromKyoto, Japan, investigated the activity of CCI-779 alone orin combination with gemcitabine on pancreatic cancer cellsin vitro and on xenograft models in vivo. They suggestedthat the combinatorial treatment resulted in significantsynergistic antigrowth activity in vivo but in contrast CCI-779 showed no activity (alone or in combination) in vitro[200]. In this study the authors found that up to 65% of thepancreatic cancer cells expressed an activated mTORpathway [200]. In another paradigm of therapeutic syner-gism, combination treatment of rapamycin with erlotinib(an EGFR tyrosine kinase inhibitor) resulted in improvedantitumor effect on various cancer cell types includingpancreatic cancer cells [90]. In a recent published study onpancreatic cancer cells B13LM, which are prone tolymphatic metastasis, in vitro and on BALB/c mice in vivo,treatment with rapamycin caused reduction of both lym-phatic vessels production and lymph nodes metastasis[201].

With regards to human studies, there are currently twoclinical trials of mTOR inhibitors in the United Statesrecruiting patients with advanced pancreatic cancer. Thefirst study, with a phase II design, is testing efficacy ofsirolimus as a second line treatment after failure ofgemcitabine based chemotherapy. The second active phaseI/II study is investigating safety, toxicity and activity of themTOR inhibitor RAD001 (everolimus) in combination withgemcitabine. The results of these trials will be expectedwith great interest.

16 TRAIL

TNF-related apoptosis-inducing ligand (TRAIL) is a mol-ecule of the TNF family which induces apoptosis inmalignant cells with no effect on normal cells. Pancreaticcancel cells are often resistant to apoptosis induced byTRAIL for quite a few reasons explained below [202]. In apreclinical study on various pancreatic cancer cell lines(HPAF, Panc1, Miapaca2, Bxpc3, Panc89 and SW979)treatment with TRAIL only, resulted to a significantincrease of apoptosis, whereas in TRAIL resistant Aspc1cells significant cytotoxic effect was achieved only afterconcomitant treatment of TRAIL with actinomycin D (anantibiotic with anticancer activity). [203] According toother in vitro study on pancreatic cancer cell lines,synchronous treatment with TRAIL and gemcitabineresulted in increased apoptosis and cell death via inductionof the proapoptotic actions of caspase-3 and caspase-8 [204]. The chemoresistance of pancreatic cancer cells isoften associated with overexpression of the anti-apoptoticprotein Bcl-XL. This chemoresistance was overcome invitro with knockdown of the Bcl-XL overexpression by thecombinatorial treatment of TRAIL, geldanamycin (anantibiotic which binds to Hsp90 and induces degradationof proteins mutated in cancer cells) and PS-341 (protea-some inhibitor, widely known as bortezomib or VelcadeTN).[178] Pancreatic cancer cells may be also resistant toTRAIL apoptosis due to overexpression of the FLICE-inhibitory protein (FLIP)-S and -L, variants of FLIP, andoverexpression of the receptor-interacting protein (RIP)which may cause inhibition of caspase-8 cleavage andinhibition of the subsequent activation of mitochondrialpathway. This resistance to TRAIL was overcome aftertreatment with the protein synthesis inhibitor cyclohexi-mide, the anticancer drugs camptothecin, cisplatin and alsowith celecoxib which led to a reduction of the highexpression of FLIP [205, 206]. In a recently publishedstudy, a third mechanism of pancreatic cancer cellsresistance to TRAIL was reported. In this study, the authorsclaimed that the X-linked inhibitor of apoptosis (XIAP) wasinvolved in pancreatic cancer cell survival by regulatingsensitivity to TRAIL, thus down-regulation of XIAP byinterfering RNA resulted in increasing TRAIL inducedapoptosis [207]. Other potential mechanism of resistance toTRAIL apoptosis may be through activation of NF-κBpathway. This was suggested after inhibition of the IKK(NF-κB activator) by bortezomib, PS-1145 or curcumin.The inhibition of NF-κB resulted then in downregulation ofBcl-XL and XIAP, and subsequently in increasing sensitiv-ity and antitumorigenesis of TRAIL in previously resistantpancreatic cancer cells [208]. In another study, though,which also showed a similar link between NF-κB activation


and XIAP, the researchers reported that only constitutivelyactivated NF-κB is associated with XIAP upregulation andresistance to TRAIL, in contrast to induced NF-κB [209].

With regards to safety profile of TRAIL treatment, it wasreported recently that concomitant use of TRAIL with highbut clinically relevant doses of bortezomib may causehepatotoxicity. Nevertheless, lower doses of bortezomibmay be just enough for sensitization of the TRAIL resistantcancer cells, without carrying the risk of liver toxicity [210].

The previous results in pancreatic cancer cell linesregarding the cytotoxic effect of TRAIL therapy wererepeated and rather validated in mice transplanted withpatients-derived pancreatic tumors [211]. In this researchwork, treatment of the patient cancer xenograft/SCIDmouse models with TRAIL alone or TRAIL in combinationwith gemcitabine caused cancer growth inhibition in someof the tumors but not all of them, with much better resultsseen after the combinatorial therapy. This differentialapoptotic response was due to the fact that the resistant toapoptosis tumors expressed high levels of the Bcl-XL anti-apoptotic molecule and therefore were resistant to TRAILtreatment [211]. TRAIL has also been tried in gene therapyshowing evidence of cytotoxic activity against pancreaticcancer cells and tumor models targeted by Ad/g-TRAIL, anadenovirus vector in which TRAIL gene expression isdriven by the human telomerase reverse transcriptase(hTERT) promoter [212, 213].

Phytochemicals have long demonstrated activity aschemopreventive agents and their efficacy in cancer treat-ment is currently under evaluation in many human clinicaltrials. The simultaneous treatment of TRAIL with phyto-chemicals such as genistein (an isoflavoin found in soyproducts) or resveratrol (a phytoalexin found in red grapesand red wine) resulted in enhanced apoptosis and cytotox-icity [214, 215]. Though the mechanism of synergism withgenistein was not clear, resveratrol was thought to causesensitization of the cancer cells to TRAIL-induced apopto-sis by depleting survivin, a known antiapoptotic protein[215]. Similarly, sensitization of pancreatic adenocarcinomacells to TRAIL-induced apoptosis, due to survivin declineby transcriptional down-regulation, was seen after concom-itant treatment of TRAIL with a cyclin-dependent kinase 4(CDK4) inhibitor {2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione} [216].

In constrast to all previous preclinical studies whichsuggested a beneficial role of TRAIL therapy, there hasbeen a single published study on pancreatic cancer cells invitro and on immunosuppressed mice models transplantedwith human pancreatic tumors in vivo, which showed thattreatment with TRAIL increased the incidence of distalmetastases. For example, the authors reported a four-foldincrease of the number and six-fold increase of the volume

of liver metastases. The postulated mechanism of thisuntoward effect of TRAIL was the strong induction of thepro-inflammatory cytokines IL-8 and monocyte chemo-attractant protein 1 expression, and also the upregulation ofthe urokinase-type plasminogen activator which facilitatedtumor invasion in vitro [217]. Fortunately, these resultshave not been reproduced in subsequent studies, but ofcourse they cannot be ignored and further evaluation ofTRAIL is required prior to its use in the clinical setting.

In conclusion, TRAIL therapy seems promising inpancreatic cancer, mainly in combination with cytotoxicsand other agents used to beat cancer cell resistance, but ofcourse further studies may be required in order to curtailany risk of facilitating invasion and metastasis. Equally, ofspecial interest would be to investigate the role of TRAILin gene therapy aiming to reduce the resistance tochemotherapy, as suggested by a very recent study inwhich up-regulation of the pro-apoptotic Bax and TRAILgene expression, by two adenoviruses using the humantelomerase reverse transcriptase (hTERT) promoter incombination with gemcitabine, resulted in significant tumorregression and survival benefit of the treated animal models[218].

17 MEK inhibitors

Mutations of K-Ras gene are seen in the majority ofpancreatic cancers. Ras exerts its effects mainly through theRasàRafàMEKàMAPK (mitogen activated protein kinase)àERK (extracellular receptor kinase) àFos pathway.

Blockage of this pathway by the MEK inhibitor UO126,caused cell cycle arrest on human pancreatic cancer celllines and inhibition of cancer cells proliferation. Theseeffects were achieved by up-regulation of protein p27Kip1,an inhibitor of cdc2 which is involved in the regulation ofcell cycle [219].

In a phase I clinical trial on patients with advanced solidcancers, the MEK inhibitor CI-1040 was tested for safety,pharmacokinetics, pharmacodynamics, dosing and efficacy[220]. Of the 77 patients recruited in various cohorts, from100 mg CI-1040 QID to 800 mg TID, the majority (98%)developed only grade 1 or 2 toxicities, such as diarrhoea,nausea, vomiting, fatigue and rash. Dose-limiting toxicity(DLT) of the cohort of 800 mg TID was a grade 3 fatigue.The recommended dose for future phase II studies was setat 800 mg BID. Out of the 66 patients assessed forresponse, a pancreatic cancer patient achieved partialresponse and 19 patients (28%) with various solid tumorsstable disease [220].

Subsequent phase II study of CI-1040 on various solidmalignancies again including pancreatic cancer, sixty seven


(67) patients (counting fifteen with pancreatic cancer) weretreated with CI-1040 800 mg twice daily [221]. None of thepatient reached a partial response and only eight patientsdemonstrated stable disease (two patients with pancreaticcancer out of the 15 or 13.3%). Though, no major toxicitieswere observed, the clinical benefit was not significant withthis particular MEK inhibitor [221].

18 HDAC inhibitors

Histone deacetylases (HDAC) is a group of enzymescausing deacetylation of histone. Deacetylated histonebinds around the DNA and interferes with gene transcrip-tion and expression, resulting mainly to a suppressed geneexpression. Normal cellular function requires coordinationbetween the histone deacetylases (HDAC) and histoneacetyltransferases (HAT). Inhibition of the HDAC enzymesby HDAC inhibitors is aiming to modulate gene transcrip-tion and expression and to alter cellular functions, partic-ularly cell cycle progression, cell differentiation, apoptosisand angiogenesis [222].

Inhibition of HDAC, in nine pancreatic cancer cell lines invitro, by Trichostatin A (TSA) resulted in cell growth arrestand induction of apoptosis. These effects were mediated byinduction of p21(WAF1/CIP1) expression which in turninhibited the cyclin-dependent kinases (cdk), through a p53-independent pathway [223]. Similar results were alsoobserved in other preclinical studies on pancreatic cancercell lines after treatment with the HDAC inhibitors SK-7041(a hybrid synthetic compound) [224] and FR901228 [225].The latter agent facilitated in addition apoptosis by activationof caspace-3 and reduction of survivin levels [225].

Interestingly, the HDAC inhibitors Trichostatin A (TSA)and suberoylanilide hydroxamic acid (SAHA, Vorinostat,ZolinzaTN), were found to induce apoptosis of pancreaticadenocarcinoma cells either as single agents or in syner-gism with cytotoxic chemotherapeutic drugs. For example,combination of TSAwith irinotecan resulted in 80% growthinhibition of the treated cancer cell lines [226]. Nude micemodels with pancreatic cancer xenografts treated with TSAand gemcitabine demonstrated an up to 50% reduction ofthe tumor mass [227]. Similarly, inhibition of growth wasreported with co-administration of SAHA and gemcitabinein vitro [228]. These studies suggested that HDACinhibitors may be able to sensitise cancer cells that werepreviously resistant to chemotherapy.

Combination of the HDAC inhibitors with other biolog-ical agents seemed to enhance apoptosis and cancer growthinhibition; e.g. synergism between SAHA and 5-Aza-2′-deoxycytidine (an inhibitor of DNA methylation), [229]and between TSA and the proteasome inhibitor PS-341(Bortezomib, VelcadeTN) [178].

According to the preclinical in vivo data and based on theexperience from the use of HDAC inhibitors in other humandiseases so far, there have been no particular safety or toxicityconcerns, leading to the assumption that these agents will belikely well tolerated in pancreatic cancer patients as well.

Up to now, there are no published clinical studies aboutthe use of HDAC inhibitors in pancreatic cancer patients, butsafety and lack of toxicities have been confirmed in phase Istudies conducted on other solid malignancies. Currently,more than forty (40) clinical trials, testing the combination ofSAHA (Vorinostat) with other agents in solid tumors andhaematological malignancies are in progress.

19 Conclusion and Perspectives

There has long been a need for effective therapy for patientswith pancreatic cancer. With the comprehensive understandingof its biology, a number of molecularly targeted agents haveevolved through logical design, which specifically interferewith major pathways involved in pancreatic cancer pathogen-esis. Although these agents are in their late phase ofdevelopment, the first results with anti-EGFR, anti-VEGFmonoclonal antibodies as well as with tyrosine kinase inhibitorsare promising, especially in terms of disease stabilization.Future clinical trials evaluating the integration of these agentsinto combined modality treatment schedules for advanced andearly-stage tumours, along with the identification of patientswho will most likely benefit are expected to provide newopportunities in the treatment of pancreatic cancer.

It is becoming increasingly apparent that the complexityof the EGFR signaling cascade provides a wealth ofmechanisms for resistance to EGFR targeted agents inpatients with pancreatic cancer. Mechanisms that mediateresistance to anti-EGFR therapies include the presence ofredundant tyrosine kinase receptors, increased angiogene-sis, and the constitutive activation of downstream media-tors. Most recently, investigators have identified thatspecific mutations in the kinase domain of EGFR in somelung carcinomas are associated with markedly improvedresponse rates to an EGFR tyrosine kinase inhibitor.Mutations in the EGFR receptor seem to play a significantrole in determining the sensitivity of tumor cells to EGFRinhibitor therapy by altering the conformation and activityof the receptor. Conversely, as development proceeds withadditional targeted agents for moieties located downstreamof EGFR or in pathways which “crosstalk” with EGFR, theopportunities arise for combination targeted therapies whichmay overcome resistance or provide synergy with thoseagents which are currently available. A number of studieshave already been done based on prior observations ofdifferent pancreatic cancer cell lines manifesting markedlydisparate sensitivities to EGFR antagonism.


In conclusion, the therapeutic landscape for the treatmentof pancreatic cancer is rapidly changing with new, logicallydesigned agents with high response rates and in some, asurvival benefit. So far there is little to suggest high degreesof cross resistance unlike chemotherapy, but optimaladministration regimes remain to be defined. New andspecific toxicities have emerged with each type of newtargeted therapy, which themselves need to be understoodand managed to maintain optimal quality of life for thepatient. The ultimate goal would be a tailored-madetreatment for maximal therapeutic results.

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Pancreatic cancer: from molecular pathogenesis to targeted therapy

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