Chemotherapy failure is one of most common and crucial problem in the treatment of tumors. Indeed, acquired mutations and alterations can lead to cell death resistance for the cancer cells. Leiomyosarcomas (LMS) are rare but aggressive smooth muscle tumors, characterized by complex karyotypes. Limited therapeutic options are available for LMS. Beside chirurgical resection, the treatment with the genotoxic compound Doxorubicin is commonly adopted to limit the dissemination and progression of the disease. However, the acquired resistance of LMS to Doxorubicin treatment, due to the accumulations of mutations, leads to relapse and correlates with bad prognosis. Hence, new therapeutic strategies need to be found. The small molecule 2c is a dienone derivative with two sterically accessible electrophilic β-carbons, which can act as Michael acceptors to target nucleophiles, such as cysteines. 2c triggers multiple stresses, which converge in the activation of the proteotoxic stress. Bioinformatic analysis of a signature of genes upregulated after 2c treatment, involving several elements of the proteotoxic response, correlates negatively with the survival of LMS patients. From this observation, we hypothesize that aggressive LMS coexist with high levels of proteotoxic stress, and they could be under crisis when challenged with further proteotoxic stress making them more vulnerable. We show that 2c can induce proteotoxic stress in LMS cells before leading them to enter in cell death programs. Indeed, the chaperones HSPA6 and HSPA1A show a dramatic increase in mRNA levels in these cells after treatment with 2c. Moreover, 2c triggers mitochondrial dysfunction and by STED technique microscopy we unveil that this small molecule can reorganize the sub-mitochondrial clusters of DIABLO/SMAC. In order to improve its efficiency in vivo, 2c was engineered through a conjugation with PEG and a small peptide, generating a pro-drug version of the compound called 2cPP. This new molecule can release 2c through the action of secreted proteases present in the tumor microenvironment. 2cPP induces a similar level of cell death in LMS cells as 2c, but unlike 2c, is unable to induce cell death in normal smooth muscle cells. When assessed for anti-tumoral activities in vivo, using different xenograft models of LMS, 2cPP showed a strong anti-tumor effect. The cell death mechanism and the genes involved in it induced by 2c is still not elucidated. To better understand and dissect this pathway, RNA-Seq experiment was performed by comparing the results between tumoral cell lines and its normal counterpart treated with 2c. The results reveal that normal cells modulate by both upregulation and downregulation more genes than the tumoral one after 2c treatment. Furthermore, while tumoral cells generally upregulate pro-apoptotic genes and downregulate anti-apoptotic genes, normal cells could have a more balance response in order to keep the cells alive, demonstrating the fact that normal cells show less cell death than the tumoral ones after 2c treatment. Regarding proteotoxic stress, both the cells activate similar pathways when they are treated with 2c. Cotreatments of 2c with other common small molecules in LMS cells reveal that two compounds can induce an additive effect of 2c in terms of cell death rate: MKC3946, inhibitor of IRE1, and YKL-06-061 inhibitor of SIKs. Despite the final role is to increase the cell death induced by 2c for both the inhibitors, they we show that they can act in a different way to achieve this result. Indeed, MKC3946 augment the levels of cell death through blocking survival pathways induced by the activation of the UPR response, instead YKL-06-061 potentiate these pathways in an uncontrolled way leading to a switch from pro-survival to a pro-death effect. In conclusion all our data seem promising for LMS treatment and highlight that proteotoxic stress may be an alternative strategy in anti-cancer therapy.

Chemotherapy failure is one of most common and crucial problem in the treatment of tumors. Indeed, acquired mutations and alterations can lead to cell death resistance for the cancer cells. Leiomyosarcomas (LMS) are rare but aggressive smooth muscle tumors, characterized by complex karyotypes. Limited therapeutic options are available for LMS. Beside chirurgical resection, the treatment with the genotoxic compound Doxorubicin is commonly adopted to limit the dissemination and progression of the disease. However, the acquired resistance of LMS to Doxorubicin treatment, due to the accumulations of mutations, leads to relapse and correlates with bad prognosis. Hence, new therapeutic strategies need to be found. The small molecule 2c is a dienone derivative with two sterically accessible electrophilic β-carbons, which can act as Michael acceptors to target nucleophiles, such as cysteines. 2c triggers multiple stresses, which converge in the activation of the proteotoxic stress. Bioinformatic analysis of a signature of genes upregulated after 2c treatment, involving several elements of the proteotoxic response, correlates negatively with the survival of LMS patients. From this observation, we hypothesize that aggressive LMS coexist with high levels of proteotoxic stress, and they could be under crisis when challenged with further proteotoxic stress making them more vulnerable. We show that 2c can induce proteotoxic stress in LMS cells before leading them to enter in cell death programs. Indeed, the chaperones HSPA6 and HSPA1A show a dramatic increase in mRNA levels in these cells after treatment with 2c. Moreover, 2c triggers mitochondrial dysfunction and by STED technique microscopy we unveil that this small molecule can reorganize the sub-mitochondrial clusters of DIABLO/SMAC. In order to improve its efficiency in vivo, 2c was engineered through a conjugation with PEG and a small peptide, generating a pro-drug version of the compound called 2cPP. This new molecule can release 2c through the action of secreted proteases present in the tumor microenvironment. 2cPP induces a similar level of cell death in LMS cells as 2c, but unlike 2c, is unable to induce cell death in normal smooth muscle cells. When assessed for anti-tumoral activities in vivo, using different xenograft models of LMS, 2cPP showed a strong anti-tumor effect. The cell death mechanism and the genes involved in it induced by 2c is still not elucidated. To better understand and dissect this pathway, RNA-Seq experiment was performed by comparing the results between tumoral cell lines and its normal counterpart treated with 2c. The results reveal that normal cells modulate by both upregulation and downregulation more genes than the tumoral one after 2c treatment. Furthermore, while tumoral cells generally upregulate pro-apoptotic genes and downregulate anti-apoptotic genes, normal cells could have a more balance response in order to keep the cells alive, demonstrating the fact that normal cells show less cell death than the tumoral ones after 2c treatment. Regarding proteotoxic stress, both the cells activate similar pathways when they are treated with 2c. Cotreatments of 2c with other common small molecules in LMS cells reveal that two compounds can induce an additive effect of 2c in terms of cell death rate: MKC3946, inhibitor of IRE1, and YKL-06-061 inhibitor of SIKs. Despite the final role is to increase the cell death induced by 2c for both the inhibitors, they we show that they can act in a different way to achieve this result. Indeed, MKC3946 augment the levels of cell death through blocking survival pathways induced by the activation of the UPR response, instead YKL-06-061 potentiate these pathways in an uncontrolled way leading to a switch from pro-survival to a pro-death effect. In conclusion all our data seem promising for LMS treatment and highlight that proteotoxic stress may be an alternative strategy in anti-cancer therapy.

Alternative targets in anticancer therapy: the emerging role of proteotoxic stress / Luca Iuliano , 2022 Jul 13. 34. ciclo, Anno Accademico 2020/2021.

Alternative targets in anticancer therapy: the emerging role of proteotoxic stress

IULIANO, LUCA
2022-07-13

Abstract

Chemotherapy failure is one of most common and crucial problem in the treatment of tumors. Indeed, acquired mutations and alterations can lead to cell death resistance for the cancer cells. Leiomyosarcomas (LMS) are rare but aggressive smooth muscle tumors, characterized by complex karyotypes. Limited therapeutic options are available for LMS. Beside chirurgical resection, the treatment with the genotoxic compound Doxorubicin is commonly adopted to limit the dissemination and progression of the disease. However, the acquired resistance of LMS to Doxorubicin treatment, due to the accumulations of mutations, leads to relapse and correlates with bad prognosis. Hence, new therapeutic strategies need to be found. The small molecule 2c is a dienone derivative with two sterically accessible electrophilic β-carbons, which can act as Michael acceptors to target nucleophiles, such as cysteines. 2c triggers multiple stresses, which converge in the activation of the proteotoxic stress. Bioinformatic analysis of a signature of genes upregulated after 2c treatment, involving several elements of the proteotoxic response, correlates negatively with the survival of LMS patients. From this observation, we hypothesize that aggressive LMS coexist with high levels of proteotoxic stress, and they could be under crisis when challenged with further proteotoxic stress making them more vulnerable. We show that 2c can induce proteotoxic stress in LMS cells before leading them to enter in cell death programs. Indeed, the chaperones HSPA6 and HSPA1A show a dramatic increase in mRNA levels in these cells after treatment with 2c. Moreover, 2c triggers mitochondrial dysfunction and by STED technique microscopy we unveil that this small molecule can reorganize the sub-mitochondrial clusters of DIABLO/SMAC. In order to improve its efficiency in vivo, 2c was engineered through a conjugation with PEG and a small peptide, generating a pro-drug version of the compound called 2cPP. This new molecule can release 2c through the action of secreted proteases present in the tumor microenvironment. 2cPP induces a similar level of cell death in LMS cells as 2c, but unlike 2c, is unable to induce cell death in normal smooth muscle cells. When assessed for anti-tumoral activities in vivo, using different xenograft models of LMS, 2cPP showed a strong anti-tumor effect. The cell death mechanism and the genes involved in it induced by 2c is still not elucidated. To better understand and dissect this pathway, RNA-Seq experiment was performed by comparing the results between tumoral cell lines and its normal counterpart treated with 2c. The results reveal that normal cells modulate by both upregulation and downregulation more genes than the tumoral one after 2c treatment. Furthermore, while tumoral cells generally upregulate pro-apoptotic genes and downregulate anti-apoptotic genes, normal cells could have a more balance response in order to keep the cells alive, demonstrating the fact that normal cells show less cell death than the tumoral ones after 2c treatment. Regarding proteotoxic stress, both the cells activate similar pathways when they are treated with 2c. Cotreatments of 2c with other common small molecules in LMS cells reveal that two compounds can induce an additive effect of 2c in terms of cell death rate: MKC3946, inhibitor of IRE1, and YKL-06-061 inhibitor of SIKs. Despite the final role is to increase the cell death induced by 2c for both the inhibitors, they we show that they can act in a different way to achieve this result. Indeed, MKC3946 augment the levels of cell death through blocking survival pathways induced by the activation of the UPR response, instead YKL-06-061 potentiate these pathways in an uncontrolled way leading to a switch from pro-survival to a pro-death effect. In conclusion all our data seem promising for LMS treatment and highlight that proteotoxic stress may be an alternative strategy in anti-cancer therapy.
13-lug-2022
Chemotherapy failure is one of most common and crucial problem in the treatment of tumors. Indeed, acquired mutations and alterations can lead to cell death resistance for the cancer cells. Leiomyosarcomas (LMS) are rare but aggressive smooth muscle tumors, characterized by complex karyotypes. Limited therapeutic options are available for LMS. Beside chirurgical resection, the treatment with the genotoxic compound Doxorubicin is commonly adopted to limit the dissemination and progression of the disease. However, the acquired resistance of LMS to Doxorubicin treatment, due to the accumulations of mutations, leads to relapse and correlates with bad prognosis. Hence, new therapeutic strategies need to be found. The small molecule 2c is a dienone derivative with two sterically accessible electrophilic β-carbons, which can act as Michael acceptors to target nucleophiles, such as cysteines. 2c triggers multiple stresses, which converge in the activation of the proteotoxic stress. Bioinformatic analysis of a signature of genes upregulated after 2c treatment, involving several elements of the proteotoxic response, correlates negatively with the survival of LMS patients. From this observation, we hypothesize that aggressive LMS coexist with high levels of proteotoxic stress, and they could be under crisis when challenged with further proteotoxic stress making them more vulnerable. We show that 2c can induce proteotoxic stress in LMS cells before leading them to enter in cell death programs. Indeed, the chaperones HSPA6 and HSPA1A show a dramatic increase in mRNA levels in these cells after treatment with 2c. Moreover, 2c triggers mitochondrial dysfunction and by STED technique microscopy we unveil that this small molecule can reorganize the sub-mitochondrial clusters of DIABLO/SMAC. In order to improve its efficiency in vivo, 2c was engineered through a conjugation with PEG and a small peptide, generating a pro-drug version of the compound called 2cPP. This new molecule can release 2c through the action of secreted proteases present in the tumor microenvironment. 2cPP induces a similar level of cell death in LMS cells as 2c, but unlike 2c, is unable to induce cell death in normal smooth muscle cells. When assessed for anti-tumoral activities in vivo, using different xenograft models of LMS, 2cPP showed a strong anti-tumor effect. The cell death mechanism and the genes involved in it induced by 2c is still not elucidated. To better understand and dissect this pathway, RNA-Seq experiment was performed by comparing the results between tumoral cell lines and its normal counterpart treated with 2c. The results reveal that normal cells modulate by both upregulation and downregulation more genes than the tumoral one after 2c treatment. Furthermore, while tumoral cells generally upregulate pro-apoptotic genes and downregulate anti-apoptotic genes, normal cells could have a more balance response in order to keep the cells alive, demonstrating the fact that normal cells show less cell death than the tumoral ones after 2c treatment. Regarding proteotoxic stress, both the cells activate similar pathways when they are treated with 2c. Cotreatments of 2c with other common small molecules in LMS cells reveal that two compounds can induce an additive effect of 2c in terms of cell death rate: MKC3946, inhibitor of IRE1, and YKL-06-061 inhibitor of SIKs. Despite the final role is to increase the cell death induced by 2c for both the inhibitors, they we show that they can act in a different way to achieve this result. Indeed, MKC3946 augment the levels of cell death through blocking survival pathways induced by the activation of the UPR response, instead YKL-06-061 potentiate these pathways in an uncontrolled way leading to a switch from pro-survival to a pro-death effect. In conclusion all our data seem promising for LMS treatment and highlight that proteotoxic stress may be an alternative strategy in anti-cancer therapy.
Proteotoxic stress; Anticancer therapy; Cell death; ER stress; UPS inhibition
Proteotoxic stress; Anticancer therapy; Cell death; ER stress; UPS inhibition
Alternative targets in anticancer therapy: the emerging role of proteotoxic stress / Luca Iuliano , 2022 Jul 13. 34. ciclo, Anno Accademico 2020/2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11390/1231009
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