Study of the resistance's mechanisms to Irinotecan in colorectal cancer : p38 MAPK's involvement.

Malgré les récentes avancées réalisées dans le traitement du cancer du côlon, la résistance des tumeurs est une cause fréquente de l'échec des chimiothérapies. Cette thèse a pour objectif d'identifier les mécanismes moléculaires impliqués dans la résistance à l'Irinotécan, un agent couramment utilisé dans le traitement des cancers colorectaux. Nous avons montré l'implication de la MAPK p38 dans la résistance à l'Irinotécan et en particulier avons démontré que les isoformes α et β étaient impliquées dans cette résistance. De plus, nous avons corrélé la faible phosphorylation de p38 dans des tumeurs coliques primaires de patient sensibles au traitement à l'Irinotécan par rapport aux patients non répondeurs. Dans la suite du projet nous avons étudié le rôle de p38 dans les processus autophagiques et leur impact dans la réponse à l'Irinotécan. Nous avons démontré que p38 induisait une autophagie qui mène à la survie des cellules cancéreuses déficientes pour p53, et que l'inhibition de l'autophagie sensibilisait ces cellules au traitement au SN38, métabolite actif de l'Irinotécan. Enfin de manière préliminaire, nous avons étudié le rôle de p38 dans l'augmentation du métabolisme lipidique dans des cellules déficiente pour p53. Ces travaux ouvrent de nouvelles voies de recherche pour l'identification des mécanismes impliqués dans la résistance aux traitements anticancéreux et pour le développement d'approches pharmacologiques pour contourner la résistance.Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. This work was aimed to determine the molecular mechanisms involved in the resistance to Irinotecan, an anticancer agent widely used in colorectal cancer treatment. We have demonstrated that the α and β forms of p38 MAPK were involved in this resistance. Moreover, we have correlated less phospho-p38 in colon cancer primary tumor of patients sensitive to Irinotecan-based treatment, compared to non responder patients. During the project, we aimed to determine the role of p38 MAPK in the processes of autophagy in colorectal cancer cells, and their impact in Irinotecan cytotoxicity. We have shown that p38 induced survival autophagy in p53 deficient cells. Then, we have shown that autophagy inhibition increased the SN38 cytotoxicity (active metabolite of Irinotecan) in p53 deficient cell lines. Finally, we have studied the role of p38 MAPK in lipid metabolism in p53 deficient cells. All these findings highlight new ways of research to identify the molecular mechanisms involved in chemoresistance as well as new pharmacological approaches to overcome the resistance

Étude des mécanismes de résistance à l'Irinotécan dans le cancer colorectal (implication de la MAPK p38)

Malgré les récentes avancées réalisées dans le traitement du cancer du côlon, la résistance des tumeurs est une cause fréquente de l'échec des chimiothérapies. Cette thèse a pour objectif d'identifier les mécanismes moléculaires impliqués dans la résistance à l'Irinotécan, un agent couramment utilisé dans le traitement des cancers colorectaux. Nous avons montré l'implication de la MAPK p38 dans la résistance à l'Irinotécan et en particulier avons démontré que les isoformes a et b étaient impliquées dans cette résistance. De plus, nous avons corrélé la faible phosphorylation de p38 dans des tumeurs coliques primaires de patient sensibles au traitement à l'Irinotécan par rapport aux patients non répondeurs. Dans la suite du projet nous avons étudié le rôle de p38 dans les processus autophagiques et leur impact dans la réponse à l'Irinotécan. Nous avons démontré que p38 induisait une autophagie qui mène à la survie des cellules cancéreuses déficientes pour p53, et que l'inhibition de l'autophagie sensibilisait ces cellules au traitement au SN38, métabolite actif de l'Irinotécan. Enfin de manière préliminaire, nous avons étudié le rôle de p38 dans l'augmentation du métabolisme lipidique dans des cellules déficiente pour p53. Ces travaux ouvrent de nouvelles voies de recherche pour l'identification des mécanismes impliqués dans la résistance aux traitements anticancéreux et pour le développement d'approches pharmacologiques pour contourner la résistance.Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. This work was aimed to determine the molecular mechanisms involved in the resistance to Irinotecan, an anticancer agent widely used in colorectal cancer treatment. We have demonstrated that the a and b forms of p38 MAPK were involved in this resistance. Moreover, we have correlated less phospho-p38 in colon cancer primary tumor of patients sensitive to Irinotecan-based treatment, compared to non responder patients. During the project, we aimed to determine the role of p38 MAPK in the processes of autophagy in colorectal cancer cells, and their impact in Irinotecan cytotoxicity. We have shown that p38 induced survival autophagy in p53 deficient cells. Then, we have shown that autophagy inhibition increased the SN38 cytotoxicity (active metabolite of Irinotecan) in p53 deficient cell lines. Finally, we have studied the role of p38 MAPK in lipid metabolism in p53 deficient cells. All these findings highlight new ways of research to identify the molecular mechanisms involved in chemoresistance as well as new pharmacological approaches to overcome the resistance.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

In vitro assays for investigating the FLASH effect

FLASH radiotherapy is a novel technique that has been shown in numerous preclinical in vivo studies to have the potential to be the next important improvement in cancer treatment. However, the biological mechanisms responsible for the selective FLASH sparing effect of normal tissues are not yet known. An optimal translation of FLASH radiotherapy into the clinic would require a good understanding of the specific beam parameters that induces a FLASH effect, environmental conditions affecting the response, and the radiobiological mechanisms involved. Even though the FLASH effect has generally been considered as an in vivo effect, studies finding these answers would be difficult and ethically challenging to carry out solely in animals. Hence, suitable in vitro studies aimed towards finding these answers are needed. In this review, we describe and summarise several in vitro assays that have been used or could be used to finally elucidate the mechanisms behind the FLASH effect

DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy.: Signaling pathways induced by 125I-RIT

International audienceINTRODUCTION: Low dose-rate radioimmunotherapy (RIT) using (125)I-labelled monoclonal antibodies ((125)I-mAbs) is associated with unexpected high cytotoxicity per Gy. METHODS: We investigated whether this hypersensitivity was due to lack of detection of DNA damage by the targeted cells. DNA damage was measured with the alkaline comet assay, gamma-H2AX foci and the micronucleus test in p53(-/-) and p53(+/+) HCT116 cells exposed to increasing activities of internalizing anti-HER1 (125)I-mAbs or non-internalizing anti-CEA (125)I-mAbs. The expression of proteins involved in radiation response and progression of cells through the cycle were determined. RESULTS: Cell hypersensitivity to low absorbed doses of anti-CEA (125)I-mAbs was not due to defect in DNA damage detection, since ATM (ataxia telangiectasia mutated gene), gamma-H2AX, p53 and p21 were activated in RIT-treated HCT116 cells and G2/M cell cycle arrest was observed. Moreover, the alkaline comet assay showed that DNA breaks accumulated when cells were placed at 4°C during exposure but were repaired under standard RIT conditions (37°C), suggesting that lesions detected under alkaline conditions (mostly DNA single strand breaks and alkali-labile sites) are efficiently repaired in treated cells. The level of gamma-H2AX protein corroborated by the level of foci measured in nuclei of treated cells was shown to accumulate with time thereby suggesting the continuous presence of DNA double strand breaks. This was accompanied by the formation of micronuclei. CONCLUSION: Hypersensitivity to non-internalizing (125)I-mAbs is not due to lack of detection of DNA damage after low absorbed dose-rates. However, DNA double strand breaks accumulate in cells exposed both to internalizing and non-internalizing (125)I-mAbs and lead to micronuclei formation. These results suggest impairment in DNA double strand breaks repair after low absorbed doses of (125)I-mAbs

Apoptosis and p53 are not involved in the anti-tumor efficacy of (125)I-labeled monoclonal antibodies targeting the cell membrane.: Running title: 125I-RIT-induced cell death mechanisms

International audienceINTRODUCTION: (125)I-labeled monoclonal antibodies ((125)I-mAbs) can efficiently treat small solid tumors. Here, we investigated the role of apoptosis, autophagy and mitotic catastrophe in (125)I-mAb toxicity in p53(-/-) and p53(+/+) cancer cells. METHODS: We exposed p53(-/-) and p53(+/+) HCT116 cells to increasing activities of internalizing (cytoplasmic location) anti-HER1 (125)I-mAbs, or non-internalizing (cell surface location) anti-CEA (125)I-mAbs. For each targeting model we established the relationship between survival and mean nucleus absorbed dose using the MIRD formalism. RESULTS: In both p53(-/-) and p53(+/+) HCT116 cells, anti-CEA (125)I-mAbs were more cytotoxic per Gy than anti-HER1 (125)I-mAbs. Sensitivity to anti-CEA (125)I-mAbs was p53-independent, while sensitivity to anti-HER1 (125)I-mAbs was higher in p53(-/-) HCT 116 cells, suggesting that they act through different signaling pathways. Apoptosis was only induced in p53(+/+) HCT116 cells and could not explain cell membrane radiation sensitivity. Inhibition of autophagy did not modify the cell response to (125)I-mAbs. By contrast, mitotic death was similarly induced in both p53(-/-) and p53(+/+) HCT116 cells by the two types of (125)I-mAbs. We also showed using medium transfer experiments that γ-H2AX foci were produced in bystander cells. CONCLUSION: Cell membrane sensitivity to (125)I-mAbs is not mediated by apoptosis and is p53-independent. Bystander effects-mediated mitotic death could be involved in the efficacy of (125)I-mAbs binding cell surface receptors

Comparison between internalizing anti-HER2 mAbs and non-internalizing anti-CEA mAbs in alpha-radioimmunotherapy of small volume peritoneal carcinomatosis using 212Pb.

BACKGROUND AND PURPOSE: We assessed the contribution of antibody internalization in the efficacy and toxicity of intraperitoneal α-radioimmunotherapy (RIT) of small volume carcinomatosis using (212)Pb-labeled monoclonal antibodies (mAbs) that target HER2 (internalizing) or CEA (non-internalizing) receptors. MATERIALS AND METHODS: Athymic nude mice bearing 2-3 mm intraperitoneal tumor xenografts were intraperitoneally injected with similar activities (370, 740 and 1480 kBq; 37 MBq/mg) of (212)Pb-labeled 35A7 (anti-CEA), trastuzumab (anti-HER2) or PX (non-specific) mAbs, or with equivalent amounts of unlabeled mAbs, or with NaCl. Tumor volume was monitored by bioluminescence and survival was reported. Hematologic toxicity and body weight were assessed. Biodistribution of (212)Pb-labeled mAbs and absorbed dose-effect relationships using MIRD formalism were established. RESULTS: Transient hematological toxicity, as revealed by white blood cells and platelets numbering, was reported in mice treated with the highest activities of (212)Pb-labeled mAbs. The median survival (MS) was significantly higher in mice injected with 1.48 MBq of (212)Pb-35A7 (non-internalizing mAbs) (MS = 94 days) than in animals treated with the same activity of (212)Pb-PX mAbs or with NaCl (MS = 18 days). MS was even not reached after 130 days when follow-up was discontinued in mice treated with 1.48 MBq of (212)Pb-trastuzumab. The later efficacy was unexpected since final absorbed dose resulting from injection of 1.48 MBq, was higher for (212)Pb-35A7 (35.5 Gy) than for (212)Pb-trastuzumab (27.6 Gy). These results also highlight the lack of absorbed dose-effect relationship when mean absorbed dose was calculated using MIRD formalism and the requirement to perform small-scale dosimetry. CONCLUSIONS: These data indicate that it might be an advantage of using internalizing anti-HER2 compared with non-internalizing anti-CEA (212)Pb-labeled mAbs in the therapy of small volume xenograft tumors. They support clinical investigations of (212)Pb-mAbs RIT as an adjuvant treatment after cytoreductive surgery in patients with peritoneal carcinomatosis

Evaluation of two I-125-radiolabeled acridine derivatives for Auger-electron radionuclide therapy of melanoma

International audienceWe previously selected two melanin-targeting radioligands [I-125]ICF01035 and [I-125]ICF01040 for melanoma-targeted I-125 radionuclide therapy according to their pharmacological profile in mice bearing B16F0 tumors. Here we demonstrate in vitro that these compounds present different radiotoxicities in relation to melanin and acidic vesicle contents in B16F0, B16F0 PTU and A375 cell lines. ICF01035 is effectively observed in nuclei of achromic (A375) melanoma or in melanosomes of melanized melanoma (B16F0), while ICF01040 stays in cytoplasmic vesicles in both cells. [I-125]ICF01035 induced a similar survival fraction (A(50)) in all cell lines and led to a significant decrease in S-phase cells in amelanotic cell lines. [I-125]ICF01040 induced a higher A(50) in B16 cell lines compared to [I-125]ICF01035 ones. [I-125]ICF01040 induced a G2/M blockade in both A375 and B16F0 PTU, associated with its presence in cytoplasmic acidic vesicles. These results suggest that the radiotoxicity of [I-125]ICF01035 and [I-125]ICF01040 are not exclusively reliant on DNA alterations compatible with gamma rays but likely result from local dose deposition (Auger electrons) leading to toxic compound leaks from acidic vesicles. In vivo, [I-125]ICF01035 significantly reduced the number of B16F0 lung colonies, enabling a significant increase in survival of the treated mice. Targeting melanosomes or acidic vesicles is thus an option for future melanoma therapy