Regorafenib Alteration of the BCL-xL/MCL-1 Ratio Provides a Therapeutic Opportunity for BH3-Mimetics in Hepatocellular Carcinoma Models

Background: The multikinase inhibitor regorafenib, approved as second-line treatment for hepatocellular carcinoma (HCC) after sorafenib failure, may induce mitochondrial damage. BH3-mimetics, inhibitors of specific BCL-2 proteins, are valuable drugs in cancer therapy to amplify mitochondrial-dependent cell death. Methods: In in vitro and in vivo HCC models, we tested regorafenib's effect on the BCL-2 network and the efficacy of BH3-mimetics on HCC treatment. Results: In hepatoma cell lines and Hep3B liver spheroids, regorafenib cytotoxicity was potentiated by BCL-xL siRNA transfection or pharmacological inhibition (A-1331852), while BCL-2 antagonism had no effect. Mitochondrial outer membrane permeabilization, cytochrome c release, and caspase-3 activation mediated A-1331852/regorafenib-induced cell death. In a patient-derived xenograft (PDX) HCC model, BCL-xL inhibition stimulated regorafenib activity, drastically decreasing tumor growth. Moreover, regorafenib-resistant HepG2 cells displayed increased BCL-xL and reduced MCL-1 expression, while A-1331852 reinstated regorafenib efficacy in vitro and in a xenograft mouse model. Interestingly, BCL-xL levels, associated with poor prognosis in liver and colorectal cancer, and the BCL-xL/MCL-1 ratio were detected as being increased in HCC patients. Conclusion: Regorafenib primes tumor cells to BH3-mimetic-induced cell death, allowing BCL-xL inhibition with A-1331852 or other strategies based on BCL-xL degradation to enhance regorafenib efficacy, offering a novel approach for HCC treatment, particularly for tumors with an elevated BCL-xL/MCL-1 ratio

Antiapoptotic BCL-2 proteins determine Sorafenib/regorafenib resistance and BH3-mimetic efficacy in hepatocellular carcinoma

Sorafenib, systemic treatment for advanced hepatocellular carcinoma (HCC), and regorafenib, novel second line treatment after sorafenib failure, have efficacy limited by evasive mechanisms of acquired-drug resistance. BCL-2 proteins participate in the response to tyrosine kinase inhibitors; however, their role in HCC therapy with sorafenib/regorafenib remains uncertain. BH3-mimetic ABT-263 (navitoclax) enhanced sorafenib activity, inducing cell death via a mitochondrial caspase-dependent mechanism, after BCL-xL/BCL-2 inhibition. Sorafenib-resistant hepatoma cells (HepG2R and Hep3BR) exhibited altered mRNA expression of BCL-2 and other anti-apoptotic family members, such as MCL-1, priming drug-resistant cancer cells to death by BH3-mimetics. ABT-263 restored sorafenib efficacy in sorafenib-resistant cell lines and HCC mouse models. Moreover, in mice xenografts from patient-derived BCLC9 cells, better tumor response to sorafenib was associated to higher changes in the BCL-2 mRNA pattern. HCC non-treated patients displayed altered BCL-2, MCL-1 and BCL-xL mRNA levels respect to adjacent non-tumoral biopsies and an increased BCL-2/MCL-1 ratio, predictive of navitoclax efficacy. Moreover, regorafenib administration also modified the BCL-2/MCL-1 ratio and navitoclax sensitized hepatoma cells to regorafenib by a mitochondrial caspase-dependent mechanism. In conclusion, sorafenib/regorafenib response is determined by BCL-2 proteins, while increased BCL-2/MCL-1 ratio in HCC sensitizes drug resistant-tumors against ABT-263 co-administration. Thus, changes in the BCL-2 profile, altered in HCC patients, could help to follow-up sorafenib efficacy, allowing patient selection for combined therapy with BH3-mimetics or early switch them to second line therapy

Regulation of the microenvironment and chemoresistance in liver therapy

Tesis doctoral presentada para lograr el título de Doctor por la Universidad de Barcelona, Facultad de Medicina y Ciencias de la Salud.--2021-11-25.--Excelente Cum Laud

Regulation of the microenvironment and chemoresistance in liver therapy

[eng] Hepatocellular carcinoma (HCC) is the most common liver cancer and currently the fourth cause of cancer-related death. Since its appearance, the multityrosine kinase inhibitor (MKI) sorafenib has been the standard systemic treatment for HCC patients. Recently, novel MKI have become available, such as lenvatinib, regorafenib and cabozantinib, as well as immune-based therapies. Still, the efficacy of MKI should be improved. On the other hand, the BCL-2 family of proteins tightly regulate programmed cell death. Therefore, BH3 mimetics, small molecules which mimic BCL-2 proteins, have been proposed as chemotherapeutic compounds to trigger cell death, although their efficacy against solid tumors has not been widely explored. Firstly, we identified that regorafenib upregulated the mRNA expression of the antiapoptotic BCL-xL in murine liver tumors. Taking these results into account, we proposed that regorafenib anti-tumor action could be enhanced by the addition of the BH3 mimetic A-1331852, which specifically targets the BCL-xL protein. When hepatoma cells were treated with this combination of agents, a potent cytotoxic effect was observed. Contrarily, ABT-199 administration, which blocks BCL-2, did not cooperate with regorafenib to increase cell death in liver cancer cells. The dual treatment of regorafenib and A-1331852 induced a loss in mitochondrial membrane potential, the release of cytochrome c and an increase in caspase-3 activity, indicating that the mitochondrial pathway of apoptosis was activated in liver cancer cells. Furthermore, regorafenib treatment was found to decrease MCL-1 protein levels in hepatoma cells. Hence, a specific inhibitor of MCL-1 together with A-1331852 was administered to HCC cells, resulting in strong cell death. Regorafenib and A-1331852 also reduced tumor liver spheroids growth. In a PDX mouse model, the co-administration of regorafenib and the BCL-xL antagonist A-1331852 reduced tumor volume and decreased its proliferation. This combination also proved to be effective even in regorafenib-resistant cells and animal models. Clinical data showed that the ratio of BCL-xL/MCL-1 was increased in HCC patients, including early and advanced stages of the disease. In short, the dual treatment of regorafenib and the BH3 mimetic A-1331852 was highly effective against HCC preclinical models. MKI therapy has been described to increase reactive oxygen species (ROS) production. We wondered whether the employment of a pro-oxidant compound, like BSO, which provokes an intracellular GSH depletion, could aid in sorafenib, regorafenib and cabozantinib effectiveness, or, on the contrary, the use of antioxidant supplements might counteract its efficacy. We observed that BSO-treated hepatoma cells were much more sensible to the administration of sorafenib and regorafenib. Both MKIs increased mitochondrial ROS generation in HCC cells, and that effect was enhanced with BSO pretreatment. Likewise, the cytotoxic capacity of regorafenib and A-1331852 was found to be ROS-mediated. Hepatoma cells were treated with two different MKIs and BH3 mimetic combinations and the addition of the antioxidants MnTBAP and GSHe increased cell viability, suggesting that their use could considerably interfere with chemotherapy effectiveness. Liver cancer spheroids displayed an increase in mitochondrial ROS production when treated with BSO and sorafenib. Again, the use of MnTBAP and GSHe blocked sorafenib effect on tumor liver spheroids. Finally, an induction of mitophagy was observed with the depletion of GSH and sorafenib/regorafenib treatment in he patoma cells. In conclusion, MKIs exert their cytotoxicity via a mitochondrial ROS generation and antioxidants supplementation may restrain MKI chemotherapeutic effect in liver cancer cells

Regulation of the microenvironment and chemoresistance in liver therapy

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut d'Investigacions Biomèdiques de Barcelona (IIBB-CSIC)[eng] Hepatocellular carcinoma (HCC) is the most common liver cancer and currently the fourth cause of cancer-related death. Since its appearance, the multityrosine kinase inhibitor (MKI) sorafenib has been the standard systemic treatment for HCC patients. Recently, novel MKI have become available, such as lenvatinib, regorafenib and cabozantinib, as well as immune-based therapies. Still, the efficacy of MKI should be improved. On the other hand, the BCL-2 family of proteins tightly regulate programmed cell death. Therefore, BH3 mimetics, small molecules which mimic BCL-2 proteins, have been proposed as chemotherapeutic compounds to trigger cell death, although their efficacy against solid tumors has not been widely explored. Firstly, we identified that regorafenib upregulated the mRNA expression of the antiapoptotic BCL-xL in murine liver tumors. Taking these results into account, we proposed that regorafenib anti-tumor action could be enhanced by the addition of the BH3 mimetic A-1331852, which specifically targets the BCL-xL protein. When hepatoma cells were treated with this combination of agents, a potent cytotoxic effect was observed. Contrarily, ABT-199 administration, which blocks BCL-2, did not cooperate with regorafenib to increase cell death in liver cancer cells. The dual treatment of regorafenib and A-1331852 induced a loss in mitochondrial membrane potential, the release of cytochrome c and an increase in caspase-3 activity, indicating that the mitochondrial pathway of apoptosis was activated in liver cancer cells. Furthermore, regorafenib treatment was found to decrease MCL-1 protein levels in hepatoma cells. Hence, a specific inhibitor of MCL-1 together with A-1331852 was administered to HCC cells, resulting in strong cell death. Regorafenib and A-1331852 also reduced tumor liver spheroids growth. In a PDX mouse model, the co-administration of regorafenib and the BCL-xL antagonist A-1331852 reduced tumor volume and decreased its proliferation. This combination also proved to be effective even in regorafenib-resistant cells and animal models. Clinical data showed that the ratio of BCL-xL/MCL-1 was increased in HCC patients, including early and advanced stages of the disease. In short, the dual treatment of regorafenib and the BH3 mimetic A-1331852 was highly effective against HCC preclinical models. MKI therapy has been described to increase reactive oxygen species (ROS) production. We wondered whether the employment of a pro-oxidant compound, like BSO, which provokes an intracellular GSH depletion, could aid in sorafenib, regorafenib and cabozantinib effectiveness, or, on the contrary, the use of antioxidant supplements might counteract its efficacy. We observed that BSO-treated hepatoma cells were much more sensible to the administration of sorafenib and regorafenib. Both MKIs increased mitochondrial ROS generation in HCC cells, and that effect was enhanced with BSO pretreatment. Likewise, the cytotoxic capacity of regorafenib and A-1331852 was found to be ROS-mediated. Hepatoma cells were treated with two different MKIs and BH3 mimetic combinations and the addition of the antioxidants MnTBAP and GSHe increased cell viability, suggesting that their use could considerably interfere with chemotherapy effectiveness. Liver cancer spheroids displayed an increase in mitochondrial ROS production when treated with BSO and sorafenib. Again, the use of MnTBAP and GSHe blocked sorafenib effect on tumor liver spheroids. Finally, an induction of mitophagy was observed with the depletion of GSH and sorafenib/regorafenib treatment in he patoma cells. In conclusion, MKIs exert their cytotoxicity via a mitochondrial ROS generation and antioxidants supplementation may restrain MKI chemotherapeutic effect in liver cancer cells

La proteína BCL-xL regula la resistencia adquirida a regorafenib del carcinoma hepatocellular (HCC) sensibilizando a miméticos de BH3 en terapia experimental

Trabajo presentado en el 44º Congreso Anual de la Asociación Española para el Estudio del Hígado (AEEH), celebrado en Madrid (España), del 20 al 22 de febrero de 2019Introducción: El inhibidor multiquinasa regorafenib tiene una eficacia clínica limitada en el tratamiento del carcinoma hepatocelular avanzado (HCC) debido a la aparición de resistencia celular adquirida. El daño mitocondrial inducido por regorafenib no ha sido estudiado en la terapia del HCC, a diferencia de la toxicidad mitocondrial inducida por sorafenib. La posible efectividad de miméticos de BH3, inhibidores de proteínas BCL-2 específicas, en combinación con regorafenib es desconocida y podría aportar nuevas dianas en el tratamiento del HCC. Métodos: Se trataron líneas celulares de hepatoma HepG2 y Hep3B con regorafenib y miméticos de BH3 (inhibidor BCL- xL: A-1331852 e inhibidor BCL-2: ABT-199). Se realizaron silenciamientos con siRNAs control, siBCL-2 y siBCL-xL, así como Western blots y qPCRs. La funcionalidad mitocondrial, la activación de caspasas y la inducción de apoptosis se analizaron en células de hepatoma tratadas con regorafenib. Se analizó la efectividad antitumoral del regorafenib en terapias con miméticos de BH3 en esferoides tumorales hepáticos (Hep3B/LX2), y en el crecimiento tumoral de xenoinjertos (BCLC9) en ratones. Resultados: Las células Hep3B exhibieron cambios en varias proteínas del sistema de BCL-2, como BCL-xL, MCL-1 o BIM tras exposición a regorafenib indicativos de una adaptación en las células resistentes. El silenciamiento de Hep3B con siRNAs frente a BCL-2 y BCL-xL, principales proteínas antiapoptóticas responsables de la resistencia BCL-2-dependiente, demostró que la reducción de BCL-xL sensibiliza las células de hepatoma frente al regorafenib. Utilizando miméticos de BH3 específicos de BCL-2 (ABT-199) y BCL-xL (A1331852) se observó un claro incremento de la muerte celular inducida por regorafenib (HepG2 y Hep3B) por un mecanismo mitocondrial caspasa-dependiente únicamente tras del inhibición de BCL-xL. Prueba de ello, la coadministración de A-1331852 incrementó la reducción del potencial de membrana mitocondrial inducida por regorafenib, la liberación de citocromo c y la actividad caspasa-3. De forma similar, esferoides tumorales hepáticos tratados con regorafenib manifestaron reducción de volumen, pérdida de potencial de membrana e incremento de la muerte celular tras la adición del inhibidor de BCL-xL A-1331852. Además, xenoinjertos de ratones de células BCLC9 derivadas de pacientes mostraron una regulación al alza de BCL-xL y BIM tras tratamiento con regorafenib evidenciando la relevancia de esta adaptación mitocondrial y mostrando mecanismos de muerte celular tras análisis transcriptómico diferenciados a los inducidos por sorafenib. La coadministración de A-1331852 potenció los efectos del regorafenib disminuyendo el crecimiento tumoral. Conclusiones: El regorafenib induce cambios en los niveles de proteínas de la familia de BCL-2, promoviendo sensibilización mitocondrial utilizable por miméticos de BH3 como A-1331852 para incrementar la eficacia del tratamiento del HCC.Trabajo de investigación financiado por Fondo de Investigaciones Sanitarias, Gobierno de España, confinanciado por FEDER (SAF2015-66515-R para A.M. y PI16/00930 para M.M.

Reduced tumor burden through increased oxidative stress in lung adenocarcinoma cells of PARP-1 and PARP-2 knockout mice.

Lung cancer (LC) is currently a major leading cause of cancer deaths worldwide. Poly(ADP-ribose) polymerases (PARP)-1 and -2 play important roles in DNA repair and other cell functions. Oxidative stress triggers autophagy and apoptosis. PARP inhibitors are currently used as anticancer strategies including LC. We hypothesized that inhibition of either PARP-1 or -2 expressions in the host animals influences tumor burden through several biological mechanisms, mainly redox imbalance (enhanced oxidative stress and/or decreased antioxidants, and cell regulators) in wild type (WT) lung adenocarcinoma cells. Compared to WT control tumors, in those of Parp-1(-/-) and Parp-2(-/-) mice: 1) tumor burden, as measured by weight, and cell proliferation rates were decreased, 2) oxidative stress levels were greater, whereas those of the major antioxidant enzymes were lower especially catalase, 3) tumor apoptosis and autophagy levels were significantly increased, and 4) miR-223 and nuclear factor of activated T-cells (NFAT)c-2 expression was decreased (the latter only in Parp-1(-/-) mice). Furthermore, whole body weight gain at the end of the study period also improved in Parp-1(-/-) and Parp-2(-/-) mice compared to WT animals. We conclude that PARP-1 and -2 genetic deletions in the host mice induced a significant reduction in tumor burden most likely through alterations in redox balance (downregulation of antioxidants, NFATc-2 and miR223, and increased oxidative stress), which in turn led to increased apoptosis and autophagy. Furthermore, tumor progression was also reduced probably as a result of cell cycle arrest induced by PARP-1 and -2 inhibition in the host mice. These results highlight the relevance of the host status in tumor biology, at least in this experimental model of lung adenocarcinoma in mice. Future research will shed light on the effects of selective pharmacological inhibitors of PARP-1 and PARP-1 in the host and tumor burden, which could eventually be applied in actual clinical settings.The study has been funded by Instituto de Salud Carlos-III: CIBERES, FIS 11/02029, FIS 14/ 00713 and Fundació La Marató de TV3: 2013 e4130

Reduced tumor burden through increased oxidative stress in lung adenocarcinoma cells of PARP-1 and PARP-2 knockout mice.

Lung cancer (LC) is currently a major leading cause of cancer deaths worldwide. Poly(ADP-ribose) polymerases (PARP)-1 and -2 play important roles in DNA repair and other cell functions. Oxidative stress triggers autophagy and apoptosis. PARP inhibitors are currently used as anticancer strategies including LC. We hypothesized that inhibition of either PARP-1 or -2 expressions in the host animals influences tumor burden through several biological mechanisms, mainly redox imbalance (enhanced oxidative stress and/or decreased antioxidants, and cell regulators) in wild type (WT) lung adenocarcinoma cells. Compared to WT control tumors, in those of Parp-1(-/-) and Parp-2(-/-) mice: 1) tumor burden, as measured by weight, and cell proliferation rates were decreased, 2) oxidative stress levels were greater, whereas those of the major antioxidant enzymes were lower especially catalase, 3) tumor apoptosis and autophagy levels were significantly increased, and 4) miR-223 and nuclear factor of activated T-cells (NFAT)c-2 expression was decreased (the latter only in Parp-1(-/-) mice). Furthermore, whole body weight gain at the end of the study period also improved in Parp-1(-/-) and Parp-2(-/-) mice compared to WT animals. We conclude that PARP-1 and -2 genetic deletions in the host mice induced a significant reduction in tumor burden most likely through alterations in redox balance (downregulation of antioxidants, NFATc-2 and miR223, and increased oxidative stress), which in turn led to increased apoptosis and autophagy. Furthermore, tumor progression was also reduced probably as a result of cell cycle arrest induced by PARP-1 and -2 inhibition in the host mice. These results highlight the relevance of the host status in tumor biology, at least in this experimental model of lung adenocarcinoma in mice. Future research will shed light on the effects of selective pharmacological inhibitors of PARP-1 and PARP-1 in the host and tumor burden, which could eventually be applied in actual clinical settings.The study has been funded by Instituto de Salud Carlos-III: CIBERES, FIS 11/02029, FIS 14/ 00713 and Fundació La Marató de TV3: 2013 e4130

Antioxidants threaten multikinase inhibitor efficacy against liver cancer by blocking mitochondrial reactive oxygen species

Sorafenib and regorafenib, multikinase inhibitors (MKIs) used as standard chemotherapeutic agents for hepatocellular carcinoma (HCC), generate reactive oxygen species (ROS) during cancer treatment. Antioxidant supplements are becoming popular additions to our diet, particularly glutathione derivatives and mitochondrial-directed compounds. To address their possible interference during HCC chemotherapy, we analyzed the effect of common antioxidants using hepatoma cell lines and tumor spheroids. In liver cancer cell lines, sorafenib and regorafenib induced mitochondrial ROS production and potent cell death after glutathione depletion. In contrast, cabozantinib only exhibited oxidative cell death in specific HCC cell lines. After sorafenib and regorafenib administration, antioxidants such as glutathione methyl ester and the superoxide scavenger MnTBAP decreased cell death and ROS production, precluding the MKI activity against hepatoma cells. Interestingly, sorafenib-induced mitochondrial damage caused PINK/Parkin-dependent mitophagy stimulation, altered by increased ROS production. Finally, in sorafenib-treated tumor spheroids, while ROS induction reduced tumor growth, antioxidant treatments favored tumor development. In conclusion, the anti-tumor activity of specific MKIs, such as regorafenib and sorafenib, is altered by the cellular redox status, suggesting that uncontrolled antioxidant intake during HCC treatment should be avoided or only endorsed to diminish chemotherapy-induced side effects, always under medical scrutiny.Study funded by grants from Instituto de Salud Carlos III (PI19/01410 to M.M., and PI19/00358 to M.R.), CIBEREHD and CIBERNED; Ministerio de Ciencia, Innovación y Universidades (RTI2018-095672-B-I00 to A.M. and P.G.F., and RTI2018-095572-B-100 to A.C.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea); AGAUR (2017_SGR_177 to A.M.) and CERCA Programme/Generalitat de Catalunya