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Biomarker and Therapeutic Studies of Antibodies and Small Molecules that Target EGFRMutsaers, Anthony James 17 February 2011 (has links)
The field of targeted cancer therapy has progressed in recent years with the approval of new oncology drugs. Coupled with the benefits that these agents provide, has come an appreciation for challenges that occur when attempting to translate successful experiments from the laboratory into effective clinical trials. One such challenge has been predicting the optimal dose and schedule to take into clinical evaluation, given the possibility that certain targeted therapeutics may exhibit maximal anti-tumour efficacy well below maximum tolerated doses. Recent work with a targeted antibody to the mouse vascular endothelial growth factor receptor-2 demonstrated that detection of increased levels of its endogenous ligand in the plasma, namely VEGF, could address this issue, as maximal increases in VEGF paralleled optimal drug activity. The VEGF result has become recognized as a potential class effect for this family of inhibitors. This thesis summarizes experiments designed to build upon this discovery by investigating whether the utility of ligand measurement might also apply to drugs that inhibit the epidermal growth factor receptor (EGFR), which have also received recent regulatory approval, and inhibit angiogenesis as one of their mechanisms of action. In addition, we investigated the potential application of EGFR inhibitors to influence other markers of tumour angiogenesis, specifically their effects on levels of circulating endothelial progenitor cells (CEPs). Finally, we evaluated combination treatment of EGFR inhibition with anti-angiogenic scheduling of chemotherapy.
The EGFR ligand TGF-alpha increased in a dose dependent fashion following treatment with cetuximab, and levels in the circulation paralleled anti-tumour activity. This was a host-dependent effect that was not observed with the lower affinity antibody nimotuzumab. Inhibition of host EGFR also reduced plasma CEPs, but at higher doses these drugs increased off target growth factors VEGF and G-CSF, as well as CEPs. In a model of advanced triple negative breast cancer, the combination of nimotuzumab and metronomic cyclophosphamide was efficacious and well tolerated, leading to a potential new treatment strategy for this aggressive disease. Taken together, these studies identify new and useful applications for EGFR-targeted antibodies, and shed further light on their contributions within the field of tumour angiogenesis and antiangiogenic therapy.
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Biomarker and Therapeutic Studies of Antibodies and Small Molecules that Target EGFRMutsaers, Anthony James 17 February 2011 (has links)
The field of targeted cancer therapy has progressed in recent years with the approval of new oncology drugs. Coupled with the benefits that these agents provide, has come an appreciation for challenges that occur when attempting to translate successful experiments from the laboratory into effective clinical trials. One such challenge has been predicting the optimal dose and schedule to take into clinical evaluation, given the possibility that certain targeted therapeutics may exhibit maximal anti-tumour efficacy well below maximum tolerated doses. Recent work with a targeted antibody to the mouse vascular endothelial growth factor receptor-2 demonstrated that detection of increased levels of its endogenous ligand in the plasma, namely VEGF, could address this issue, as maximal increases in VEGF paralleled optimal drug activity. The VEGF result has become recognized as a potential class effect for this family of inhibitors. This thesis summarizes experiments designed to build upon this discovery by investigating whether the utility of ligand measurement might also apply to drugs that inhibit the epidermal growth factor receptor (EGFR), which have also received recent regulatory approval, and inhibit angiogenesis as one of their mechanisms of action. In addition, we investigated the potential application of EGFR inhibitors to influence other markers of tumour angiogenesis, specifically their effects on levels of circulating endothelial progenitor cells (CEPs). Finally, we evaluated combination treatment of EGFR inhibition with anti-angiogenic scheduling of chemotherapy.
The EGFR ligand TGF-alpha increased in a dose dependent fashion following treatment with cetuximab, and levels in the circulation paralleled anti-tumour activity. This was a host-dependent effect that was not observed with the lower affinity antibody nimotuzumab. Inhibition of host EGFR also reduced plasma CEPs, but at higher doses these drugs increased off target growth factors VEGF and G-CSF, as well as CEPs. In a model of advanced triple negative breast cancer, the combination of nimotuzumab and metronomic cyclophosphamide was efficacious and well tolerated, leading to a potential new treatment strategy for this aggressive disease. Taken together, these studies identify new and useful applications for EGFR-targeted antibodies, and shed further light on their contributions within the field of tumour angiogenesis and antiangiogenic therapy.
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Understanding EGFR Modification, Trafficking, and the Importance of its Juxtamembrane Domain in CancerHart, Matthew Robert January 2013 (has links)
Much of what is known about the role of the ERBB family in cellular biology and in cancer has to do with canonical downstream signaling cascades and modifications associated with their trafficking and degradation. The focus on canonical activity, while important, ignores a rapidly expanding number of separate, arguably equally important functions for which there is emerging knowledge. These include the translocation of ERBB family members to non-canonical sub-cellular locations including the nucleus and mitochondria. Of current interest is the elucidation of fate determination mechanisms for these proteins. How is one ERBB receptor designated to traffic to the nucleus or mitochondria, versus degradative lysosomes? A portion of the work presented here addresses the potential role of ubiquitin in EGFR nuclear translocation, a role which ubiquitin has been shown to play in the context of other proteins. This work demonstrates that while ubiquitinated EGFR can be translocated from the plasma membrane to the nucleus in response to ligand, efficient ubiquitination is not essential for this process. This work also broadens the potential roles for ubiquitin to include those involving EGFR nuclear biology. Additional work described aimed to exploit what was already known regarding the diverse roles played by the EGFR juxtamembrane domain in the non-canonical activities of EGFR and the ERBBs. This work involves the creation and evaluation of an EGFR juxtamembrane domain derived peptide designed to competitively interact with endogenous ERBB domains and inhibit their function in cancer cells. Termed EJ1, treatment induces cell death and promotes the formation of inactive ERBB dimers and reduces ERBB activation. While inactivating CaMKII signaling, myosin light chain dependent cell blebbing occurs, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, potentially contributing to a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of mouse models of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis, without observable side-effects/toxicities. These data demonstrate that a portion of the ERBB juxtamembrane domain, used as an intracellular decoy, can affect tumor growth and metastasis through ERBB-dependent and ERBB-independent mechanisms, representing a novel anti-cancer therapeutic.
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NUMB and Syncytiotrophoblast Development and Function: Investigation Using BeWo Choriocarcinoma CellsCarey, Julie 09 May 2012 (has links)
The role of NUMB, a protein important for cellular differentiation and endocytosis in non-placental cells, was investigated in syncytiotrophoblast development and function in the human placenta. The BeWo choriocarcinoma cell line was used as a model for villous cytotrophoblast cells and syncytiotrophoblast to investigate NUMB’s involvement in differentiation and epidermal growth factor receptor (EGFR) endocytosis. NUMB isoforms 1 and 3 were found to be the predominant isoforms and were upregulated following forskolin-induced differentiation. Overexpression of NUMB isoforms 1 and 3 did not mediate differentiation or EGFR signaling. Immunofluorescence analysis revealed that NUMB colocalized with EGFR at perinuclear late endosomes and lysosomes following EGF stimulation. We have demonstrated for the first time that NUMB isoforms 1 and 3 are expressed in BeWo cells, are upregulated in forskolin-differentiated BeWo cells and are involved in ligand-dependent EGFR endocytosis in BeWo cells.
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NUMB and Syncytiotrophoblast Development and Function: Investigation Using BeWo Choriocarcinoma CellsCarey, Julie January 2012 (has links)
The role of NUMB, a protein important for cellular differentiation and endocytosis in non-placental cells, was investigated in syncytiotrophoblast development and function in the human placenta. The BeWo choriocarcinoma cell line was used as a model for villous cytotrophoblast cells and syncytiotrophoblast to investigate NUMB’s involvement in differentiation and epidermal growth factor receptor (EGFR) endocytosis. NUMB isoforms 1 and 3 were found to be the predominant isoforms and were upregulated following forskolin-induced differentiation. Overexpression of NUMB isoforms 1 and 3 did not mediate differentiation or EGFR signaling. Immunofluorescence analysis revealed that NUMB colocalized with EGFR at perinuclear late endosomes and lysosomes following EGF stimulation. We have demonstrated for the first time that NUMB isoforms 1 and 3 are expressed in BeWo cells, are upregulated in forskolin-differentiated BeWo cells and are involved in ligand-dependent EGFR endocytosis in BeWo cells.
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The small GTPase RhoG mediates glioblastoma cell invasionKwiatkowska, Aneta, Didier, Sebastien, Fortin, Shannon, Chuang, Yayu, White, Timothy, Berens, Michael, Rushing, Elisabeth, Eschbacher, Jennifer, Tran, Nhan, Chan, Amanda, Symons, Marc January 2012 (has links)
BACKGROUND:The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.RESULTS:We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.CONCLUSIONS:Our functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.
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Targeting the EBFR and PI3K pathways as a therapeutic strategy for prostate cancerMaleka, Sechaba 04 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Targeted therapy for prostate cancer may offer potential improvement over current
conventional therapies because of its specificity. Although conventional treatments
are effective, they are not curative and have several limitations. In prostate cancer,
activation of both the epidermal growth factor receptor (EGFR) and the
phosphatidylinositol 3 – kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)
pathway have been implicated in tumorigenesis and resistance to both conventional
and targeted anticancer therapies. Having a better understanding of the molecular
mechanisms involved in PCa development, progression and resistance to therapy,
could assist in the design of novel therapeutic strategies.
The objective of this study was to inhibit key molecular targets of the human
epidermal growth factor receptor signalling pathway and expose prostate cell lines to
doses of radiation, so as to establish potential therapeutic targets that may be
amenable to combined modality therapy, and formulate a cocktail of inhibitors to
evaluate its radiosensitising capability.
The EGFR/PI3K/mTOR pathway plays an important role in the radiosensitivity of the
human prostate carcinoma cell line (DU145) and the normal cell line (1542N). In our
study we have shown that AG-1478, an EGFR inhibitor, and BEZ-235, a dual
inhibitor of the PI3K/mTOR pathway, singly or in combination, at low and relatively
high radiation doses, resulted in radiosensitisation of DU145 cells. Radio-protection
was achieved in 1542N cells. AG-1478 had no effect on radiosensitivity. / AFRIKAANSE OPSOMMING: Geteikende terapies wens hul spesifisiteit teenoor konvensionele terapies vir
prostaat kanker, mag potensieel verbetering offer. Konvensionele behandeling is wel
effektief maar nie genesend nie wens ‘n aantal beperkings, sowel as die toksisiteit vir
normale selle. In prostaat kanker is die aktivering van beide die epidermiese groei
faktor reseptor (EGFR) en fosfatidielinositol 3-kinase/Akt/soogdier teiken vir rapamisien
(mTOR) seingewing baan sterk betrek by tumor groeisel en weerstand teen
konvensionele en geteikende anti-kanker terapies.
Beter begrip van die molekulêre meganismes betrokke by prostaat kanker
ontwikkeling, bevordering en weerstand teen terapie, kan die ontwerp van nuwe
terapeutiese strategies ondersteun.
Die doelwit van hierdie studie was om sleutel molekulêre teikens van die
epidermiese groei faktor reseptor seingewing baan te inhibeer en om prostaat selle
bloot te stel aan dosisse bestraling, om potensiële terapeutiese teikens te vestig wat
vatbaar is vir gekombineerde modaliteit terapie, om ‘n mengsel van stremmiddels te
formuleer, en om die straling gevoeligmaking bekwaamheid daarvan te evalueer.
Die EGFR/PI3K/mTOR seingewingbaan speel ‘n belangrike rol in the radiosensitiwiteit van
die menslike prostaat kanker sellyn (DU145) en die normale prostaat sellyn (1542N). Die
studie bevind dat AG-1478, ‘n EGFR stremmer, en BEZ-235, ‘n tweevoudige beperker van
die fosforinositied 3-kinase (PI3K) en soogdier teiken vir rapamisien (mTOR)
seingewingbaan, enkel of in kombinasie die DU145 selle radiosensitiseer vir straling dosisse
van 2 en 6 Gy. Stralings beskerming was verkry met die 1542N sellyn. AG-1478 het geen
effek getoon op radiosensitiwiteit nie.
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Associations antimétabolites fluoropyrimidiques-thérapies ciblées : étude des déterminants moléculaires de réponse et non-réponse / Targeted therapy-fluoropyrimides combination : study of molecular determinantsChefrour, Mohamed 13 July 2010 (has links)
Associer les thérapies ciblées et la chimiothérapie cytotoxique constitue un axe thérapeutique extrêmement prometteur en oncologie clinique. La combinaison des fluoropyridines orales de dernière génération (capécitabine) aux inhibiteurs de tyrosine kinase (erlotinib, lapatinib)a ainsi été proposée dans divers contextes cliniques, avec une efficacité variable, allant de l'échec (pancréas) à la réponse thérapeutique (sein). L'objectif de ce travail de recherche est de dégager, sur des modèles expérimentaux non-cliniques, un rationnel pharmacologique d'utilisation de la capécitabine avec divers TKI. Ce travail se focalise notamment sur l'étude de la modulation des déterminants de réponse aux drogues dans les modèles pancréatiques(association capecitabine + erlotinib) et mammaires (capecitabine + lapatinib). Nous avons ainsi pu montrer les effets délétères de l'association erlotinib-capecitabine dans les modèles pancréatiques chimio-résistants, liés d'une part à un effet represseur de l'expression du récepteur à l'EGF par la capécitabine, combiné à une sécrétion exocrine de VEGF en réponse aux diverses séquences d'association testées. Ces effets se sont soldés par l'obtention de réponses antagonistes aux drogues combinées, comparativement aux monothérapies respectives. Nos données suggèrent que quelle que soit la séquence d'utilisation envisagée,capecitabine et erlotinib ne devraient jamais faire l'objet d'association. A contrario, l'étude de l'association lapatinib-capecitabine sur diverses lignées mammaires a permis de mettre en évidence un effet positif allant de la supra-additivité à la synergie. Nos données suggèrent que c'est d'une part par une dérégulation de la thymidylate syntahse par le lapatinib qu'une plus grande efficacité anti-proliférative est obtenue. Par ailleurs, une meilleure inhibition de la voie p-AKT après exposition à la capécitabine des cellules traitées par le lapatinib pourraitde surcroît expliquer la synergie observée. Nos données suggèrent qu'une utilisation concomitante de ces deux molécules devrait garantir un gain d'efficacité optimal. La synergie de l'association a été conservée sur un modèle de souris porteuses de tumeurs mammaires ectotopiques et traitées de façon concomitante, avec un net ralentissement de la croissance tumorale comparativement aux animaux traités par monothérapie, sans augmentation de la toxicité médicamenteuse.En conclusion, nos données illustrent le fait que les associations thérapiesciblées/antimétabolites doivent faire l'objet systématiquement d'un travail préalable de recherche des déterminants oncopharmacologiques d'efficacité afin d'anticiper le gain d'efficacité qu'on est en droit d'en tirer, et d'identifier au mieux les modalités d'association de ces drogues. / Optimizing cancer treatments remains an on going task. The rise over the last decade of a new class of anticancer agents (the so-called “targeted therapies”) has fuelled agreat hope in the search for achieving better response with lower toxicities. To date,the actual efficacy of targeted therapies used alone looks in some respect below the initial expectations, and associating them with classic cytotoxic drugs represents therefore a new promising combinational strategy in clinical oncology. However, littleis known on the pharmacologic rationale for combing these drugs, and identifying molecular determinants could help to rationalize the way the drugs should beassociated. Anti-EGFR erlotinib (Tarceva®) has been approved for the treatment of patients with metastatic pancreatic cancer, in combination with a standard chemotherapy, gemcitabine, both in the USA and in Europe. Its combination with capecitabine has recently been tested in patients with gemcitabine-refractorypancreatic tumours, with limited success. Lapatinib (Tykerb®) is an innovative small molecule with unique dual-TK inhibitory action against both HER1 and HER2.Lapatinib showed clinical efficacy in various models and has heightened the expectation for new breast cancer therapies. Associating lapatinib with capecitabine(Xeloda®) has been recently approved for the treatment of patients with HER2+metastatic breast cancer, after failure of trastuzumab treatment, both in the USA andin Europe as well. The aim of our work was to study, either in vitro or in tumor bearing animals, the effects of these drugs used either alone or in combination. In this preclinical study, we studied the molecular determinants of this new targeted therapies + antimetabolite association on various, representative human pancreatic and breast cancer cell lines. Efficacy and apoptosis experiments were performed after concomitant and various sequential combinations, along with study ofresponse/resistance determinants such as dihydropyrimidine dehydrogenase (DPD),thymidylate synthase (TS), thymidine phosphorylase (TP), Bax, Bcl-2, P21 andHER1/2 signaling pathway. Determination of Chou & Talalay combination indices (CI)allowed us to determine whether the tested associations led to synergism, additivity or antagonism. The best sequences found were transposed to tumor-bearing animal model for in vivo confirmation of the efficacy observed in vitro. Lapatinib andcapecitabine showed additive to moderate synergistic effects in our breast cancer celllines. Further experiments were performed to identify the mechanisms underlyingthis efficacy. We first found that lapatinib down-regulates thymidylate synthase (-30%) but did not have any effect on dihydropyrimidine dehydrogenase levels, thussuggesting theoretically a better cytotoxic effect of capecitabine within tumor cellspreliminary exposed to Lapatinib. Besides, capecitabine, when added to lapatinib,strongly down-regulates P-AKT and p-P42/44, thus allowing a deeper inhibition of the downstream signaling pathway after inhibition of HER1 and HER2. Muchinterestingly, we showed as well an increase of the Bax/Bcl2 ratio in cells exposed toboth drugs, leading to the enhancement of apoptosis induction and cell death. Thisfinding is fully consistent with the fact that the association of lapatinib andcapecitabine led to an increase of the P21 protein expression and consequently tothe arrest of cell cycle indeed. Besides, in pancreatic cancer cell lines, wedemonstrated that erlotinib up-regulated thymidine phosphorylase (+50%) anddown-regulated dihydropyrimidine dehydrogenase (-55%), thus suggestingtheoretically a better activation of capecitabine within tumor cells preliminaryexposed to erlotinib. We checked as well that Bax, P27, p-AKT were fully functionalin our models. Nevertheless, when not antagonist, only moderate additive effectswere achieved. To understand these rather surprising results, further experimentswere performed to identify the putative causes of this lack of efficacy. We first foundthat capecitabine down-regulates EGFR (-35%) in capan-1 cells with no effect on itsdownstream signalisation pathway nor antiproliferative related effects, thussuggesting a possible loss of efficacy when cells were subsequently treated witherlotinib due to the diminution of its target. Much interestingly, we showed as well adramatic overexpression of both cytosolic (+100 up to +1900%) and extra-cellular(+100 up to 3500%) VEGF secretion in response to our treatments, leading possibly to a boost in cell proliferation. Taken together, this in vitro study suggests that themore-than-additive efficacy observed when associating lapatinib and capecitabine inbreast cancer is based upon TS modulation and down-regulation of HER1/2 signalingpathways, resulting in apoptosis and cell death eventually. In vivo experiments are ongoing to fully confirm these data. Concerning the erlotinib/capecitabineassociation in pancreatic cancers, we suggest that VEGF secretion could be a new,major mechanism of resistance to the erlotinib + capecitabine association inpancreatic cancers, and, possibly, to other targeted therapy + antimetabolitescombinations. To test this hypothesis, further experiments including the use of anti-VEGF bevacizumab will be undertaken.
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Short hairpin RNA-directed knockdown of epidermal growth factor receptor in human oesophageal squamous carcinoma cell linesKillick, Mark Andrew 27 May 2008 (has links)
Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which
activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein
kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of
EGFR is a common feature in variety of human cancers including lung, colorectal, breast,
pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic
potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has
been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi)
pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in
human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated
through the transfection of three specifically designed short hairpin RNAs (shRNAs) against
human EGFR. The shRNAs were specifically designed using bioinformatics tools and their
individual knockdown efficacy determined through the introduction of an exogeneous based
target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined
using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all
three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite
low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR
sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in
WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all
three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show
substantial potential for the specific and efficient targeting of EGFR in human cancer cells.
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Fonctionnalité de la signalisation en aval des récepteurs HER : implication dans la réponse cellulaire et tumorale aux thérapies ciblées / Human Epidermal Growth Factor Receptor (HER) downstream signalling functionality : implications in cell and tumour response to targeted therapiesChrétien, Anne-Sophie 20 June 2011 (has links)
Les thérapies ciblées dirigées contre les récepteurs de la famille des HER représentent un progrès majeur en cancérologie. Leur efficacité repose sur leur capacité à inhiber la signalisation oncogénique notamment recrutant les voies RAS/RAF/MAPK et PI3K/AKT. Indépendamment de l'expression de la cible moléculaire, l'activité des thérapies ciblées est tributaire de la présence dans le tissu tumoral d'anomalies génétiques capables de perturber la signalisation cellulaire tumorale en aval des récepteurs. Notre travail a consisté à évaluer l'intérêt de l'évaluation de la fonctionnalité des voies de signalisation par analyse multiplexe des niveaux d'expression des formes phosphorylées des principales kinases intracellulaires en aval des récepteurs HER. La preuve de concept a été tout d'abord apportée sur des modèles cellulaires génétiquement modifiés, en montrant l'impact d'anomalies génétiques tumorales sur la fonctionnalité des voies de signalisation RAS/RAF/MAPK et PI3K/AKT et sur la réponse au cétuximab. Dans la seconde partie de ce travail, après validation des techniques utilisées en situation clinique, nous avons montré la pertinence d'une approche de biologie intégrative combinant analyse de génétique moléculaire et analyses fonctionnelles avec analyse en composante principale. Nos résultats, obtenus au cours d'une étude clinique rétrospective dans les cancers colorectaux métastatiques, suggèrent que l'utilisation des données issues de l'évaluation de la fonctionnalité des voies de signalisation en aval des récepteurs HER, pourraient à terme trouver des applications en qualité de marqueurs prédictifs de réponse clinique aux thérapies ciblées anti-HER / Targeted therapies, monoclonal antibodies and tyrosine kinase inhibitors, directed against the Human Epidermal Growth Factor Receptors (HER) family, represent a major advance in oncology. Their efficacy depends on their ability to inhibit oncogenic signalling, including the RAS/RAF/MAPK and PI3K/AKT signalling pathways. Regardless of the expression of the molecular target, the efficacy of targeted therapies is independent on the genetic abnormalities of the tissue which are able to disrupt tumour cell signalling, and the research of these abnormalities is now included in the prescription criteria. The aim of this work was to evaluate the usefulness of assessing HER downstream signalling functionality using multiplex analysis of the phosphorylated forms of key intracellular kinase signalling. Proof of concept was first introduced with genetically modified cell lines models, showing the consequences of tumour genetic abnormalities on RAS/RAF/MAPK and PI3K/AKT signalling pathways and on response to cetuximab. In the second part of this work, after validation of the techniques used in clinical situation, we demonstrated the relevance of system biology approach, combining molecular biology and functional analysis with principal component analysis. Our results obtained in a retrospective clinical study in metastatic colorectal cancer, suggests that the use of data from the evaluation of HER downstream signalling pathways functionality could eventually find applications as predictive markers of clinical response to anti-HER targeted therapies
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