Limits...
Heterogeneous Mechanisms of Secondary Resistance and Clonal Selection in Sarcoma during Treatment with Nutlin.

Laroche A, Tran-Cong K, Chaire V, Lagarde P, Hostein I, Coindre JM, Chibon F, Neuville A, Lesluyes T, Lucchesi C, Italiano A - PLoS ONE (2015)

Bottom Line: Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death.Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways.Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.

View Article: PubMed Central - PubMed

Affiliation: INSERM U916, Institut Bergonié, Bordeaux, France; Sarcoma Unit, Institut Bergonié, Bordeaux, France.

ABSTRACT
Nutlin inhibits TP53-MDM2 interaction and is under investigation in soft-tissue sarcomas (STS) and other malignancies. Molecular mechanisms of secondary resistance to nutlin in STS are unknown. We performed whole-transcriptome sequencing (RNA-seq) on three pretreatment and secondary resistant STS cell lines selected based on their high primary sensitivity to nutlin. Our data identified a subset of cancer gene mutations and ploidy variations that were positively selected following treatment, including TP53 mutations in 2 out of 3 resistant cell lines. Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death. Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways. Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.

No MeSH data available.


Related in: MedlinePlus

Secondary resistance to RG7388 is associated with autophagy induction.(A) parental and secondary resistant cells were incubated with 20μM of chloroquine for 6h, or RG7388 alone for 72h or RG7388 (over 72h) and chloroquine for 6h before protein extraction, and immunoblot. (B) Densitometry of the immunoblot, the graph represents the percentage of LC3-II /GAPDH relative to the untreated control (C) IB111 and IB111P4 were treated with 1μM and 10 μM of RG-7388 respectively with or without chloroquine and fixed for immuno-staining with LC3 antibody (D) Quantification of autophagy induction (number of GFP-LC3 punctuae) in IB111 and IB111P4 treated with RG7388, chloroquine and RG7388+chloroquine (E) IC50 fold-change related to antiproliferative activity of RG7388+chloroquine versus RG7388 alone in secondary resistant IB111, and sensitive cells IB111.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591276&req=5

pone.0137794.g005: Secondary resistance to RG7388 is associated with autophagy induction.(A) parental and secondary resistant cells were incubated with 20μM of chloroquine for 6h, or RG7388 alone for 72h or RG7388 (over 72h) and chloroquine for 6h before protein extraction, and immunoblot. (B) Densitometry of the immunoblot, the graph represents the percentage of LC3-II /GAPDH relative to the untreated control (C) IB111 and IB111P4 were treated with 1μM and 10 μM of RG-7388 respectively with or without chloroquine and fixed for immuno-staining with LC3 antibody (D) Quantification of autophagy induction (number of GFP-LC3 punctuae) in IB111 and IB111P4 treated with RG7388, chloroquine and RG7388+chloroquine (E) IC50 fold-change related to antiproliferative activity of RG7388+chloroquine versus RG7388 alone in secondary resistant IB111, and sensitive cells IB111.

Mentions: We also analyzed the sequencing data to identify differences in gene expression between secondary resistant and parental cell lines. We found 196, 105 and 370 genes with 2.5X or more fold changes (adjusted p-value < 0.01) between the parental and secondary resistant cell lines in IB111, IB115 and IB128 respectively (S2 Table). We then applied these genes to Ingenuity Pathway Analysis software (IPA®,QIAGEN Redwood City, www.qiagen.com/ingenuity). The results showed that these genes were mainly enriched in proliferative, growth and movement networks (Table 3). We focus particularly on the genes differentially expressed between the resistant and parental IB111 cells in order to identify some genetic and transcriptomic alterations that may compensate for the lack of TP53 mutations observed in the IB111 resistant cells. Strikingly, in the resistant IB111 cells, we found a significant alteration of genes involved in the regulation of apoptosis and more particularly, a down-regulation of pro-apoptotic activators such as BMF, BIM and PUMA (S3 Fig). Among them, the most strongly down-regulated was BMF with a fold change of 17. BMF also has a role as a regulator of autophagy. Since autophagy has been associated with resistance to radiation, chemotherapy, and targeted agents [30–39], we decided to investigate whether secondary resistance to RG7388 was associated with autophagy induction in STS cell lines. When autophagy is not activated, LC3 is localized homogeneously in the cytoplasm, while upon initiation of autophagy, it associates with the membrane of autophagosomes. Since an increase in LC3-II levels or GFP-LC3 vesicles can occur not only due to increased autophagosome synthesis but also due to impaired autophagosome-lysosome fusion, we assessed LC3-II levels also in the presence of chloroquine, a blocker of LC3-II degradation. Analysis of LC3-II levels by western blotting and by fluorescence microscopy allowed us to detect autophagy induction in the resistant IB111 cells but not in the IB115 and the IB128 resistant cells (Fig 5A and 5B). We therefore wondered whether blocking the induction of autophagy with chloroquine could restore sensitivity to RG7388 in IB111 resistant cells. The RG7388 IC50's were significantly reduced (divided by 6) in the secondary resistant IB111 cells when they were treated with chloroquine whereas RG7388 IC50 was almost unchanged in parental IB111 cells treated with chloroquine in the same conditions. (Fig 5C).


Heterogeneous Mechanisms of Secondary Resistance and Clonal Selection in Sarcoma during Treatment with Nutlin.

Laroche A, Tran-Cong K, Chaire V, Lagarde P, Hostein I, Coindre JM, Chibon F, Neuville A, Lesluyes T, Lucchesi C, Italiano A - PLoS ONE (2015)

Secondary resistance to RG7388 is associated with autophagy induction.(A) parental and secondary resistant cells were incubated with 20μM of chloroquine for 6h, or RG7388 alone for 72h or RG7388 (over 72h) and chloroquine for 6h before protein extraction, and immunoblot. (B) Densitometry of the immunoblot, the graph represents the percentage of LC3-II /GAPDH relative to the untreated control (C) IB111 and IB111P4 were treated with 1μM and 10 μM of RG-7388 respectively with or without chloroquine and fixed for immuno-staining with LC3 antibody (D) Quantification of autophagy induction (number of GFP-LC3 punctuae) in IB111 and IB111P4 treated with RG7388, chloroquine and RG7388+chloroquine (E) IC50 fold-change related to antiproliferative activity of RG7388+chloroquine versus RG7388 alone in secondary resistant IB111, and sensitive cells IB111.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591276&req=5

pone.0137794.g005: Secondary resistance to RG7388 is associated with autophagy induction.(A) parental and secondary resistant cells were incubated with 20μM of chloroquine for 6h, or RG7388 alone for 72h or RG7388 (over 72h) and chloroquine for 6h before protein extraction, and immunoblot. (B) Densitometry of the immunoblot, the graph represents the percentage of LC3-II /GAPDH relative to the untreated control (C) IB111 and IB111P4 were treated with 1μM and 10 μM of RG-7388 respectively with or without chloroquine and fixed for immuno-staining with LC3 antibody (D) Quantification of autophagy induction (number of GFP-LC3 punctuae) in IB111 and IB111P4 treated with RG7388, chloroquine and RG7388+chloroquine (E) IC50 fold-change related to antiproliferative activity of RG7388+chloroquine versus RG7388 alone in secondary resistant IB111, and sensitive cells IB111.
Mentions: We also analyzed the sequencing data to identify differences in gene expression between secondary resistant and parental cell lines. We found 196, 105 and 370 genes with 2.5X or more fold changes (adjusted p-value < 0.01) between the parental and secondary resistant cell lines in IB111, IB115 and IB128 respectively (S2 Table). We then applied these genes to Ingenuity Pathway Analysis software (IPA®,QIAGEN Redwood City, www.qiagen.com/ingenuity). The results showed that these genes were mainly enriched in proliferative, growth and movement networks (Table 3). We focus particularly on the genes differentially expressed between the resistant and parental IB111 cells in order to identify some genetic and transcriptomic alterations that may compensate for the lack of TP53 mutations observed in the IB111 resistant cells. Strikingly, in the resistant IB111 cells, we found a significant alteration of genes involved in the regulation of apoptosis and more particularly, a down-regulation of pro-apoptotic activators such as BMF, BIM and PUMA (S3 Fig). Among them, the most strongly down-regulated was BMF with a fold change of 17. BMF also has a role as a regulator of autophagy. Since autophagy has been associated with resistance to radiation, chemotherapy, and targeted agents [30–39], we decided to investigate whether secondary resistance to RG7388 was associated with autophagy induction in STS cell lines. When autophagy is not activated, LC3 is localized homogeneously in the cytoplasm, while upon initiation of autophagy, it associates with the membrane of autophagosomes. Since an increase in LC3-II levels or GFP-LC3 vesicles can occur not only due to increased autophagosome synthesis but also due to impaired autophagosome-lysosome fusion, we assessed LC3-II levels also in the presence of chloroquine, a blocker of LC3-II degradation. Analysis of LC3-II levels by western blotting and by fluorescence microscopy allowed us to detect autophagy induction in the resistant IB111 cells but not in the IB115 and the IB128 resistant cells (Fig 5A and 5B). We therefore wondered whether blocking the induction of autophagy with chloroquine could restore sensitivity to RG7388 in IB111 resistant cells. The RG7388 IC50's were significantly reduced (divided by 6) in the secondary resistant IB111 cells when they were treated with chloroquine whereas RG7388 IC50 was almost unchanged in parental IB111 cells treated with chloroquine in the same conditions. (Fig 5C).

Bottom Line: Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death.Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways.Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.

View Article: PubMed Central - PubMed

Affiliation: INSERM U916, Institut Bergonié, Bordeaux, France; Sarcoma Unit, Institut Bergonié, Bordeaux, France.

ABSTRACT
Nutlin inhibits TP53-MDM2 interaction and is under investigation in soft-tissue sarcomas (STS) and other malignancies. Molecular mechanisms of secondary resistance to nutlin in STS are unknown. We performed whole-transcriptome sequencing (RNA-seq) on three pretreatment and secondary resistant STS cell lines selected based on their high primary sensitivity to nutlin. Our data identified a subset of cancer gene mutations and ploidy variations that were positively selected following treatment, including TP53 mutations in 2 out of 3 resistant cell lines. Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death. Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways. Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.

No MeSH data available.


Related in: MedlinePlus