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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

(A) Antiproliferative activity and impact on loss of potential mitochondrial membrane with TMRM fluorescent assay (cell viability) (B) of RG7388 in parental and secondary resistant IB111, IB115 and IB128 STS cell lines. (C) Apoptosis induction analysis using FITC annexin-V and propidium iodide assay. Sensitive (IB111 and IB115) and resistant cells (IB111P4 and IB115P4) were treated 72h by RG-7388 at 1μM.
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pone.0137794.g003: (A) Antiproliferative activity and impact on loss of potential mitochondrial membrane with TMRM fluorescent assay (cell viability) (B) of RG7388 in parental and secondary resistant IB111, IB115 and IB128 STS cell lines. (C) Apoptosis induction analysis using FITC annexin-V and propidium iodide assay. Sensitive (IB111 and IB115) and resistant cells (IB111P4 and IB115P4) were treated 72h by RG-7388 at 1μM.

Mentions: Among the entire panel of STS cell lines tested, RG7388 displayed the highest cell growth inhibition effects in IB111, IB115 and IB128. In order to investigate mechanisms of secondary resistance to RG7388, IB111, IB115 and IB128 cells were treated with RG7388 for 3 days. The cells were then rinsed to remove RG7388, and the remaining cells were expanded in normal medium (minus RG7388). We repeated the process four times, and we obtained populations that survived 1–4 rounds of RG7388 treatment (P1 for one round—P4 for four rounds). We compared the extent to which IB111, IB115 and IB128 underwent apoptosis and cell cycle inhibition when treated for 3 days with RG7388. Results indicated that the selected populations became progressively more resistant to cell growth inhibition (Fig 3A) to apoptosis (Fig 3B) and cell cycle inhibition (data not shown). For instance, whereas 1 μM of RG7388 induced significant apoptosis in parental IB115 cells (80.7% apoptosis) after 3 days of treatment with RG7388, this effect was significantly reduced in IB115P2 (36.7% apoptosis) and IB111P4 (8.8% apoptosis) cells. Beside, we confirmed resistance to apoptosis at 1μM of RG7388 in resistant cell lines IB115P4 (69.4% apoptosis) and IB111P4 (28.4% apoptosis) versus parental cell lines IB115 (98.7% apoptosis) and IB111 (76.7% apoptosis) by FITC annexin-V and propidium iodide assay (Fig 3C).


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)

(A) Antiproliferative activity and impact on loss of potential mitochondrial membrane with TMRM fluorescent assay (cell viability) (B) of RG7388 in parental and secondary resistant IB111, IB115 and IB128 STS cell lines. (C) Apoptosis induction analysis using FITC annexin-V and propidium iodide assay. Sensitive (IB111 and IB115) and resistant cells (IB111P4 and IB115P4) were treated 72h by RG-7388 at 1μM.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0137794.g003: (A) Antiproliferative activity and impact on loss of potential mitochondrial membrane with TMRM fluorescent assay (cell viability) (B) of RG7388 in parental and secondary resistant IB111, IB115 and IB128 STS cell lines. (C) Apoptosis induction analysis using FITC annexin-V and propidium iodide assay. Sensitive (IB111 and IB115) and resistant cells (IB111P4 and IB115P4) were treated 72h by RG-7388 at 1μM.
Mentions: Among the entire panel of STS cell lines tested, RG7388 displayed the highest cell growth inhibition effects in IB111, IB115 and IB128. In order to investigate mechanisms of secondary resistance to RG7388, IB111, IB115 and IB128 cells were treated with RG7388 for 3 days. The cells were then rinsed to remove RG7388, and the remaining cells were expanded in normal medium (minus RG7388). We repeated the process four times, and we obtained populations that survived 1–4 rounds of RG7388 treatment (P1 for one round—P4 for four rounds). We compared the extent to which IB111, IB115 and IB128 underwent apoptosis and cell cycle inhibition when treated for 3 days with RG7388. Results indicated that the selected populations became progressively more resistant to cell growth inhibition (Fig 3A) to apoptosis (Fig 3B) and cell cycle inhibition (data not shown). For instance, whereas 1 μM of RG7388 induced significant apoptosis in parental IB115 cells (80.7% apoptosis) after 3 days of treatment with RG7388, this effect was significantly reduced in IB115P2 (36.7% apoptosis) and IB111P4 (8.8% apoptosis) cells. Beside, we confirmed resistance to apoptosis at 1μM of RG7388 in resistant cell lines IB115P4 (69.4% apoptosis) and IB111P4 (28.4% apoptosis) versus parental cell lines IB115 (98.7% apoptosis) and IB111 (76.7% apoptosis) by FITC annexin-V and propidium iodide assay (Fig 3C).

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