Effectivity of pazopanib treatment in orthotopic models of human testicular germ cell tumors

Background: Cisplatin (CDDP) resistance in testicular germ cell tumors (GCTs) is still a clinical challenge, and one associated with poor prognosis. The purpose of this work was to test pazopanib, an anti-tumoral and anti-angiogenic multikinase inhibitor, and its combination with lapatinib (an anti-ErbB inhibitor) in mouse orthotopic models of human testicular GCTs. Methods: We used two different models of human testicular GCTs orthotopically grown in nude mice; a CDDP-sensitive choriocarcinoma (TGT38) and a new orthotopic model generated from a metastatic GCT refractory to first-line CDDP chemotherapy (TGT44). Nude mice implanted with these orthotopic tumors were treated with the inhibitors and the effect on tumoral growth and angiogenesis was evaluated. Results: TGT44 refractory tumor had an immunohistochemical profile similar to the original metastasis, with characteristics of yolk sac tumor. TGT44 did not respond when treated with cisplatin. In contrast, pazopanib had an anti-angiogenic effect and anti-tumor efficacy in this model. Pazopanib in combination with lapatinib in TGT38, an orthotopic model of choriocarcinoma had an additive effect blocking tumor growth. Conclusions: We present pazopanib as a possible agent for the alternative treatment of CDDP-sensitive and CDDP-refractory GCT patients, alone or in combination with anti-ErbB therapies

ErbBs inhibition by lapatinib blocks tumor growth in an orthotopic model of human testicular germ cell tumor

In this work, we have analyzed the expression of different members of the ErbB family in human samples of testicular germ cell tumors (GCTs). We observed expression of ErbB1 or ErbB2 in different tumor subtypes, but we also found high expression of ErbB3 in all GCTs tested. This pattern of expression was maintained when primary tumors were orthotopically implanted in nude mice. We have chosen a choriocarcinoma model characterized by high levels of ErbB1, but also of ErbB2 and ErbB3, to assay the in vivo effect of ErbB inhibitors on tumoral growth. Our results showed a complete lack of effect (refractoriness) to the pure ErbB1 receptor inhibitors cetuximab and gefitinib. While these inhibitors blocked ErbB1 phosphorylation, ErbB2 phosphorylation was not affected, suggesting an ErbB1-independent activation of this receptor. To confirm the importance of ErbB2 activation, animals were treated with lapatinib, a dual ErbB1 and ErbB2 inhibitor. Lapatinib treatment caused a 50% inhibition in tumor growth, an effect correlated with a blockade of both ErbB1 and ErbB2 phosphorylation levels, and of downstream signaling pathways (Akt, ERKs and Stat3). ErbB2 activation could still occur due to the formation of ErbB2/ErbB3 heterodimers, and ErbB3 activation was completely inhibited by lapatinib. Finally, combined inhibition of ErbB1 (gefitinib) and ErbB3 activities (knockdown expression by shRNA) inhibited tumoral testicular cells proliferation in a similar way to lapatinib. Our results explain why lapatinib but not anti-ErbB1 agents might be effective for treatment of testicular GCT patients

The PDGFRβ-AKT pathway contributes to CDDP-acquired resistance in testicular germ cell tumors

Purpose: we examined whether PI3K-AKT or extracellular signal-regulated kinase (ERK) signaling pathways could play a role in the development of cisplatin (CDDP) resistance in testicular germ cell tumor (TGT) cells. Experimental design: we compared AKT and ERK activation levels in CDDP-sensitive testicular tumor cells and in their corresponding CDDP-resistant-derived cells. We also analyzed these pathways in orthotopic testicular tumors and human patient samples. Results: our results indicated that there was overactivation of AKT in CDDP-resistant cells compared with sensitive cells, but no effect on activated ERK levels. We observed an increase in mRNA and protein levels for platelet-derived growth factor (PDGF) receptor β and PDGF-B ligand. These were responsible for AKT overactivation in CDDP-resistant cells. When PDGFRβ levels were decreased by short hairpin RNA (shRNA) treatment or its activation was blocked by pazopanib, CDDP-resistant cells behaved like sensitive cells. Moreover, CDDP-resistant cells were more sensitive to incubation with PDGFRβ inhibitors such as pazopanib or sunitinib than sensitive cells, a finding consistent with these cells being dependent on this signaling pathway. We also found overexpression of PDGFRβ and pAKT in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterparts. Finally, we found high PDGFRβ levels in human testicular tumors, and overexpression in CDDP-resistant testicular choriocarcinomas compared with the CDDP-sensitive and nontreated tumors. Conclusions: the PDGFRβ-AKT pathway plays a critical role in the development of CDDP resistance in testicular tumoral cells

Teràpia dirigida a receptors ErbBs i teràpia antiangiogènica en tumors germinals testiculars

[cat] En aquesta tesi s’ha realitzat l’avaluació de l’eficàcia de la teràpia antiangiogènica i la teràpia dirigida a receptors ErbBs en tumors germinals testiculars (TGTs). Tot i que els TGTs són dels tumors amb taxes de curació més elevades, la incidència de la malaltia va augmentant en les poblacions caucàsiques i encara hi ha pacients que presenten resistència al cisplatí, amb l’agreujant de que són pacients joves entre 15 a 35 anys. En primer lloc hem observat que els receptors ErbB1, ErbB2 i ErbB3 s’expressen de manera generalitzada en els TGTs ortotòpics de diferent component histològic, mantenint a més a més el patró d’expressió del tumor primari a xenògraft. En un d’aquest models, el coriocarcinoma TGT38, es va avaluar l’eficàcia de les teràpies dirigides contra els receptors ErbBs. En estudis previs s’havia observat que aquest tumor no responia a les teràpies dirigides a ErbB1, malgrat presentava uns elevats nivells d’expressió del receptor. En aquest estudi, per contra, hem observat que el tractament dirigit de manera dual al bloqueig d’ErbB1 i d’ErbB2, tractant amb lapatinib, comprometia en un 50% el creixement tumoral, així com provocava la inhibició de les vies de senyalització efectores ERK1/2, AKT i STAT3. Demostrem doncs que l’activitat d’ErbB2 dimeritza amb ErbB3, i aquest heterodímer té un paper clau en el creixement tumoral del TGT38, així com en la proliferació de cel•lular de tumor testicular in vitro, i que confereix la resistència de novo a les teràpies dirigides a ErbB1. L’altra teràpia estudiada en aquesta tesi és l’antiangiogènica, que hem avaluat utilitzant pazopanib, inhibidor dels receptors de VEGF, dels receptors de PDGFalfai -beta,i c-KIT. Hem observat com aquest fàrmac bloqueja significativament el creixement tumoral del tumor sensible a cisplatí TGT38, així com una activitat antitumoral sinèrgica amb lapatinib. Per altra banda també hem observat un bloqueig significatiu en el TGT44, un model tumoral que presenta resistència a cisplatí, característica mantinguda del tumor primari on ja va presentar refractorietat al quimioterapèutic. En ambdós models tumorals hem observat que pazopanib tenia una activitat antiangiogènica i antitumoral, així com també hem observat com bloquejava la proliferació de cèl•lules tumorals de TGTs in vitro presentant més efecte en les cèl•lules amb resistència a cisplatí que en les sensibles. En aquesta línia d’investigació hem observat com PDGFRbeta, un dels receptors diana de pazopanib, es sobre expressa en els fenotips resistents de parelles sensibles-resistents a cisplatí, tant de línies cel•lulars de TGTs com de tumors ortotòpics testiculars. Hem demostrat també que el principal efector d’aquest receptor és AKT, observant també una sobre activació d’aquest en les cèl•lules resistents a cisplatí comparat amb les sensibles; així com una activació en presència de PDGF-BB i una disminució en presència de shRNAs pel receptor o del tractament amb pazopanib. En aquest estudi hem descrit doncs l’activació de la via de PDGF-BB/ PDGFRbeta/AKT com a nou mecanisme de resistència adquirida a cisplatí. A més a més hem observat una elevada expressió de PDGFRbetaen la majoria de TGTs humans analitzats. El conjunt dels resultats presenten a pazopanib com a possible agent per una teràpia alternativa al tractament dels pacients de TGT sensibles o resistents a cisplatí, sol o en combinació amb teràpies dirigides a receptors ErbBs.[eng] Cisplatin resistance in testicular germ cell tumor (TGT) patients is still a clinical challenge, and the aim of this study has been the study of different therapies for TGT. On one hand we’ve detected high expression of ErbB1, ErbB2 and ErbB3 receptors in different human TGTs and that the expression pattern was maintained to the orthotopic animal models generated. We’ve described that the heterodimer ErbB2/ErbB3 has an important role in tumor growth of a TGT model in vivo and in in vitro tumor testicular cells and its activity might explain the de novo resistance to pure ErbB1 receptor inhibitors. We’ve shown that treatment with lapatinib caused a 50% inhibition in TGT38 tumor growth, a coriocarcinoma ortotopic tumor sensitive to cisplatin. Moreover, in this model we observed that lapatinib treatment in combination to pazopanib had an additive effect blocking tumor growth. Furthermore, pazopanib had an important antitumoral effect in a new orthotopic model generated from a metastatic TGT refractory to first-line cisplatin chemotherapy (TGT44). Pazopanib main targets are the VEGFRs, PDGFRs and c-KIT receptor, and in this work we’ve observed that not only had antiangiogenic effect in vivo, besides it had antitumoral activity also in in vitro TGT cells. Moreover the antiproliferative effect was stronger in cisplatin-resistant cells than in the sensitive counterparts. Our results indicated that there was an overexpression of PDGFRbetaand an AKT overactivation in cisplatin-resistant cells, compared with sensitive cells. When PDGFRbetalevels were decreased CDDP-resistant cells behaved like sensitive cells, and PDGF-BB stimuli was the only that activated AKT phosphorilation. These results suggested that PDGFRbetaoverexpression was responsible for AKT overactivation, presenting the PDGFRbeta-AKT pathway as a pathway involved in the development of cisplatin resistance in TGT cells. Furthermore we also found overexpression of PDGFRβ in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterpart, and we found high PDGFRbetalevels in human testicular tumors. All together, these results present pazopanib as possible agent for an alternative treatment of cisplatin-sensitive and cisplatin-refractory TGT patients, alone or in combination with anti-ErbB therapies

The PDGFRβ-AKT pathway contributes to CDDP-acquired resistance in testicular germ cell tumors

Purpose: we examined whether PI3K-AKT or extracellular signal-regulated kinase (ERK) signaling pathways could play a role in the development of cisplatin (CDDP) resistance in testicular germ cell tumor (TGT) cells. Experimental design: we compared AKT and ERK activation levels in CDDP-sensitive testicular tumor cells and in their corresponding CDDP-resistant-derived cells. We also analyzed these pathways in orthotopic testicular tumors and human patient samples. Results: our results indicated that there was overactivation of AKT in CDDP-resistant cells compared with sensitive cells, but no effect on activated ERK levels. We observed an increase in mRNA and protein levels for platelet-derived growth factor (PDGF) receptor β and PDGF-B ligand. These were responsible for AKT overactivation in CDDP-resistant cells. When PDGFRβ levels were decreased by short hairpin RNA (shRNA) treatment or its activation was blocked by pazopanib, CDDP-resistant cells behaved like sensitive cells. Moreover, CDDP-resistant cells were more sensitive to incubation with PDGFRβ inhibitors such as pazopanib or sunitinib than sensitive cells, a finding consistent with these cells being dependent on this signaling pathway. We also found overexpression of PDGFRβ and pAKT in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterparts. Finally, we found high PDGFRβ levels in human testicular tumors, and overexpression in CDDP-resistant testicular choriocarcinomas compared with the CDDP-sensitive and nontreated tumors. Conclusions: the PDGFRβ-AKT pathway plays a critical role in the development of CDDP resistance in testicular tumoral cells

The PDGFRβ-AKT pathway contributes to CDDP-acquired resistance in testicular germ cell tumors

Purpose: we examined whether PI3K-AKT or extracellular signal-regulated kinase (ERK) signaling pathways could play a role in the development of cisplatin (CDDP) resistance in testicular germ cell tumor (TGT) cells. Experimental design: we compared AKT and ERK activation levels in CDDP-sensitive testicular tumor cells and in their corresponding CDDP-resistant-derived cells. We also analyzed these pathways in orthotopic testicular tumors and human patient samples. Results: our results indicated that there was overactivation of AKT in CDDP-resistant cells compared with sensitive cells, but no effect on activated ERK levels. We observed an increase in mRNA and protein levels for platelet-derived growth factor (PDGF) receptor β and PDGF-B ligand. These were responsible for AKT overactivation in CDDP-resistant cells. When PDGFRβ levels were decreased by short hairpin RNA (shRNA) treatment or its activation was blocked by pazopanib, CDDP-resistant cells behaved like sensitive cells. Moreover, CDDP-resistant cells were more sensitive to incubation with PDGFRβ inhibitors such as pazopanib or sunitinib than sensitive cells, a finding consistent with these cells being dependent on this signaling pathway. We also found overexpression of PDGFRβ and pAKT in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterparts. Finally, we found high PDGFRβ levels in human testicular tumors, and overexpression in CDDP-resistant testicular choriocarcinomas compared with the CDDP-sensitive and nontreated tumors. Conclusions: the PDGFRβ-AKT pathway plays a critical role in the development of CDDP resistance in testicular tumoral cells

The PDGFRβ-AKT pathway contributes to CDDP-acquired resistance in testicular germ cell tumors

Purpose: we examined whether PI3K-AKT or extracellular signal-regulated kinase (ERK) signaling pathways could play a role in the development of cisplatin (CDDP) resistance in testicular germ cell tumor (TGT) cells. Experimental design: we compared AKT and ERK activation levels in CDDP-sensitive testicular tumor cells and in their corresponding CDDP-resistant-derived cells. We also analyzed these pathways in orthotopic testicular tumors and human patient samples. Results: our results indicated that there was overactivation of AKT in CDDP-resistant cells compared with sensitive cells, but no effect on activated ERK levels. We observed an increase in mRNA and protein levels for platelet-derived growth factor (PDGF) receptor β and PDGF-B ligand. These were responsible for AKT overactivation in CDDP-resistant cells. When PDGFRβ levels were decreased by short hairpin RNA (shRNA) treatment or its activation was blocked by pazopanib, CDDP-resistant cells behaved like sensitive cells. Moreover, CDDP-resistant cells were more sensitive to incubation with PDGFRβ inhibitors such as pazopanib or sunitinib than sensitive cells, a finding consistent with these cells being dependent on this signaling pathway. We also found overexpression of PDGFRβ and pAKT in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterparts. Finally, we found high PDGFRβ levels in human testicular tumors, and overexpression in CDDP-resistant testicular choriocarcinomas compared with the CDDP-sensitive and nontreated tumors. Conclusions: the PDGFRβ-AKT pathway plays a critical role in the development of CDDP resistance in testicular tumoral cells

Effectivity of pazopanib treatment in orthotopic models of human testicular germ cell tumors

Background: Cisplatin (CDDP) resistance in testicular germ cell tumors (GCTs) is still a clinical challenge, and one associated with poor prognosis. The purpose of this work was to test pazopanib, an anti-tumoral and anti-angiogenic multikinase inhibitor, and its combination with lapatinib (an anti-ErbB inhibitor) in mouse orthotopic models of human testicular GCTs. Methods: We used two different models of human testicular GCTs orthotopically grown in nude mice; a CDDP-sensitive choriocarcinoma (TGT38) and a new orthotopic model generated from a metastatic GCT refractory to first-line CDDP chemotherapy (TGT44). Nude mice implanted with these orthotopic tumors were treated with the inhibitors and the effect on tumoral growth and angiogenesis was evaluated. Results: TGT44 refractory tumor had an immunohistochemical profile similar to the original metastasis, with characteristics of yolk sac tumor. TGT44 did not respond when treated with cisplatin. In contrast, pazopanib had an anti-angiogenic effect and anti-tumor efficacy in this model. Pazopanib in combination with lapatinib in TGT38, an orthotopic model of choriocarcinoma had an additive effect blocking tumor growth. Conclusions: We present pazopanib as a possible agent for the alternative treatment of CDDP-sensitive and CDDP-refractory GCT patients, alone or in combination with anti-ErbB therapies

ErbBs inhibition by lapatinib blocks tumor growth in an orthotopic model of human testicular germ cell tumor

In this work, we have analyzed the expression of different members of the ErbB family in human samples of testicular germ cell tumors (GCTs). We observed expression of ErbB1 or ErbB2 in different tumor subtypes, but we also found high expression of ErbB3 in all GCTs tested. This pattern of expression was maintained when primary tumors were orthotopically implanted in nude mice. We have chosen a choriocarcinoma model characterized by high levels of ErbB1, but also of ErbB2 and ErbB3, to assay the in vivo effect of ErbB inhibitors on tumoral growth. Our results showed a complete lack of effect (refractoriness) to the pure ErbB1 receptor inhibitors cetuximab and gefitinib. While these inhibitors blocked ErbB1 phosphorylation, ErbB2 phosphorylation was not affected, suggesting an ErbB1-independent activation of this receptor. To confirm the importance of ErbB2 activation, animals were treated with lapatinib, a dual ErbB1 and ErbB2 inhibitor. Lapatinib treatment caused a 50% inhibition in tumor growth, an effect correlated with a blockade of both ErbB1 and ErbB2 phosphorylation levels, and of downstream signaling pathways (Akt, ERKs and Stat3). ErbB2 activation could still occur due to the formation of ErbB2/ErbB3 heterodimers, and ErbB3 activation was completely inhibited by lapatinib. Finally, combined inhibition of ErbB1 (gefitinib) and ErbB3 activities (knockdown expression by shRNA) inhibited tumoral testicular cells proliferation in a similar way to lapatinib. Our results explain why lapatinib but not anti-ErbB1 agents might be effective for treatment of testicular GCT patients