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Mer receptor tyrosine kinase is frequently overexpressed in human non-small cell lung cancer, confirming resistance to erlotinib.

Xie S, Li Y, Li X, Wang L, Yang N, Wang Y, Wei H - Oncotarget (2015)

Bottom Line: Importantly, Mer overexpression induced resistance to erlotinib (EGFR inhibitor) in otherwise erlotinib-sensitive cells.Furthermore, Mer-specific inhibitor rendered erlotinib-resistant cells sensitive to erlotinib.We conclude that Mer enhances malignant phenotype and pharmacological inhibition of Mer overcomes resistance of NSCLC to EGFR-targeted agents.

View Article: PubMed Central - PubMed

Affiliation: International Joint Cancer Institute, Second Military Medical University, Shanghai, China.

ABSTRACT
Mer is a receptor tyrosine kinase (RTK) with oncogenic properties that is often overexpressed or activated in various malignancies. Using both immunohistochemistry and microarray analyses, we demonstrated that Mer was overexpressed in both tumoral and stromal compartments of about 70% of non-small cell lung cancer (NSCLC) samples relative to surrounding normal lung tissue. This was validated in freshly harvested NSCLC samples; however, no associations were found between Mer expression and patient features. Although Mer overexpression did not render normal lung epithelial cell tumorigenic in vivo, it promoted the in vitro cell proliferation, clonogenic colony formation and migration of normal lung epithelial cells as well as NSCLC cells primarily depending on MAPK and FAK signaling, respectively. Importantly, Mer overexpression induced resistance to erlotinib (EGFR inhibitor) in otherwise erlotinib-sensitive cells. Furthermore, Mer-specific inhibitor rendered erlotinib-resistant cells sensitive to erlotinib. We conclude that Mer enhances malignant phenotype and pharmacological inhibition of Mer overcomes resistance of NSCLC to EGFR-targeted agents.

No MeSH data available.


Related in: MedlinePlus

Mer inhibition rescues the sensitivity of NSCLC cells to erlotinib treatment(A) H1965 cells were treated with Mer-specific inhibitor UNC569 at indicated concentrations for 12 h and Mer phosphorylation was determined by western blotting. (B) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with serially diluted concentrations of erlotinib for 72 h, and cell viability was evaluated by MTT assay. All data were normalized to the control of DMSO treatment. (C and D) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with erlotinib at indicated concentrations for 24 h. Cell apoptosis was determined by Annexin V/7AAD staining (C) and Mer-related signaling pathway and PARP cleavage were evaluated by western blotting (D) Blots representative of three independent experiments were shown. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. *p < 0.05, **p < 0.01, paired student t test (for C).
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Figure 6: Mer inhibition rescues the sensitivity of NSCLC cells to erlotinib treatment(A) H1965 cells were treated with Mer-specific inhibitor UNC569 at indicated concentrations for 12 h and Mer phosphorylation was determined by western blotting. (B) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with serially diluted concentrations of erlotinib for 72 h, and cell viability was evaluated by MTT assay. All data were normalized to the control of DMSO treatment. (C and D) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with erlotinib at indicated concentrations for 24 h. Cell apoptosis was determined by Annexin V/7AAD staining (C) and Mer-related signaling pathway and PARP cleavage were evaluated by western blotting (D) Blots representative of three independent experiments were shown. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. *p < 0.05, **p < 0.01, paired student t test (for C).

Mentions: To define the role of endogenous Mer expression in dictating the sensitivity of NSCLC cells to EGFR inhibitor, we selected H1965 cells harboring EGFR mutation (delE746-A750) which is insensitive to erlotinib treatment and express the high level of endogenous Mer. We pretreated H1965 cells with UNC569, a recently developed small-molecule tyrosine kinase inhibitor specific for Mer, to block the activation of Mer, and then evaluated the sensitivity of these cells to erlotinib treatment by MTT assays. As shown in Fig. 6A, treatment with 500 nmol/L of UNC569 effectively inhibited the phosphorylation of Mer; furthermore, pretreatment of H1965 cells with UNC569 at this concentration significantly improved the sensitivity of H1965 cells to erlotinib, resulting in an approximate 50-fold reductions in the IC50 of erlotinib (13.65 μM vs 0.28 μM for DMSO or UNC569 pretreatment; Fig. 6B). Annexin V/7-AAD staining demonstrated that UNC569-pretreated H1965 cells exhibited a significantly increased apoptosis relative to control pretreatment in response to elrotinib treatment (Fig. 6C), which was also confirmed by increased production of cleaved PARP at the biochemical level (Fig. 6D). Consistent with previous studies [5], we also observed that UNC569 treatment alone decreased H1965 cell viability by inducing their apoptosis (Fig. 6B–6D), suggesting a role of Mer in the survival of Mer-overexpressed NSCLC cells. Treatment with UNC569 and erlotinib simultaneously inhibited the phosphorylation of MAPK and AKT whereas either erlotinib or UNC569 treatment alone merely inhibited the activation of MAPK but not AKT signaling pathway, which at least partially explained the synergistic pro-apoptotic effect of both UNC569 and erlotinib in H1965 cells (Fig. 6D) and was also consistent with above results obtained in PC9 cells (Fig. 5F). The data indicate that abrogating the function of endogenous Mer can improve the therapeutic efficacy of elrotinib treatment in otherwise erlotinib-insensitive NSCLC cells harboring EGFR mutation.


Mer receptor tyrosine kinase is frequently overexpressed in human non-small cell lung cancer, confirming resistance to erlotinib.

Xie S, Li Y, Li X, Wang L, Yang N, Wang Y, Wei H - Oncotarget (2015)

Mer inhibition rescues the sensitivity of NSCLC cells to erlotinib treatment(A) H1965 cells were treated with Mer-specific inhibitor UNC569 at indicated concentrations for 12 h and Mer phosphorylation was determined by western blotting. (B) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with serially diluted concentrations of erlotinib for 72 h, and cell viability was evaluated by MTT assay. All data were normalized to the control of DMSO treatment. (C and D) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with erlotinib at indicated concentrations for 24 h. Cell apoptosis was determined by Annexin V/7AAD staining (C) and Mer-related signaling pathway and PARP cleavage were evaluated by western blotting (D) Blots representative of three independent experiments were shown. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. *p < 0.05, **p < 0.01, paired student t test (for C).
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Related In: Results  -  Collection

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Figure 6: Mer inhibition rescues the sensitivity of NSCLC cells to erlotinib treatment(A) H1965 cells were treated with Mer-specific inhibitor UNC569 at indicated concentrations for 12 h and Mer phosphorylation was determined by western blotting. (B) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with serially diluted concentrations of erlotinib for 72 h, and cell viability was evaluated by MTT assay. All data were normalized to the control of DMSO treatment. (C and D) H1965 cells were pretreated with Mer-specific inhibitor UNC569 or vehicle control (DMSO) for 12 h followed by incubation with erlotinib at indicated concentrations for 24 h. Cell apoptosis was determined by Annexin V/7AAD staining (C) and Mer-related signaling pathway and PARP cleavage were evaluated by western blotting (D) Blots representative of three independent experiments were shown. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. *p < 0.05, **p < 0.01, paired student t test (for C).
Mentions: To define the role of endogenous Mer expression in dictating the sensitivity of NSCLC cells to EGFR inhibitor, we selected H1965 cells harboring EGFR mutation (delE746-A750) which is insensitive to erlotinib treatment and express the high level of endogenous Mer. We pretreated H1965 cells with UNC569, a recently developed small-molecule tyrosine kinase inhibitor specific for Mer, to block the activation of Mer, and then evaluated the sensitivity of these cells to erlotinib treatment by MTT assays. As shown in Fig. 6A, treatment with 500 nmol/L of UNC569 effectively inhibited the phosphorylation of Mer; furthermore, pretreatment of H1965 cells with UNC569 at this concentration significantly improved the sensitivity of H1965 cells to erlotinib, resulting in an approximate 50-fold reductions in the IC50 of erlotinib (13.65 μM vs 0.28 μM for DMSO or UNC569 pretreatment; Fig. 6B). Annexin V/7-AAD staining demonstrated that UNC569-pretreated H1965 cells exhibited a significantly increased apoptosis relative to control pretreatment in response to elrotinib treatment (Fig. 6C), which was also confirmed by increased production of cleaved PARP at the biochemical level (Fig. 6D). Consistent with previous studies [5], we also observed that UNC569 treatment alone decreased H1965 cell viability by inducing their apoptosis (Fig. 6B–6D), suggesting a role of Mer in the survival of Mer-overexpressed NSCLC cells. Treatment with UNC569 and erlotinib simultaneously inhibited the phosphorylation of MAPK and AKT whereas either erlotinib or UNC569 treatment alone merely inhibited the activation of MAPK but not AKT signaling pathway, which at least partially explained the synergistic pro-apoptotic effect of both UNC569 and erlotinib in H1965 cells (Fig. 6D) and was also consistent with above results obtained in PC9 cells (Fig. 5F). The data indicate that abrogating the function of endogenous Mer can improve the therapeutic efficacy of elrotinib treatment in otherwise erlotinib-insensitive NSCLC cells harboring EGFR mutation.

Bottom Line: Importantly, Mer overexpression induced resistance to erlotinib (EGFR inhibitor) in otherwise erlotinib-sensitive cells.Furthermore, Mer-specific inhibitor rendered erlotinib-resistant cells sensitive to erlotinib.We conclude that Mer enhances malignant phenotype and pharmacological inhibition of Mer overcomes resistance of NSCLC to EGFR-targeted agents.

View Article: PubMed Central - PubMed

Affiliation: International Joint Cancer Institute, Second Military Medical University, Shanghai, China.

ABSTRACT
Mer is a receptor tyrosine kinase (RTK) with oncogenic properties that is often overexpressed or activated in various malignancies. Using both immunohistochemistry and microarray analyses, we demonstrated that Mer was overexpressed in both tumoral and stromal compartments of about 70% of non-small cell lung cancer (NSCLC) samples relative to surrounding normal lung tissue. This was validated in freshly harvested NSCLC samples; however, no associations were found between Mer expression and patient features. Although Mer overexpression did not render normal lung epithelial cell tumorigenic in vivo, it promoted the in vitro cell proliferation, clonogenic colony formation and migration of normal lung epithelial cells as well as NSCLC cells primarily depending on MAPK and FAK signaling, respectively. Importantly, Mer overexpression induced resistance to erlotinib (EGFR inhibitor) in otherwise erlotinib-sensitive cells. Furthermore, Mer-specific inhibitor rendered erlotinib-resistant cells sensitive to erlotinib. We conclude that Mer enhances malignant phenotype and pharmacological inhibition of Mer overcomes resistance of NSCLC to EGFR-targeted agents.

No MeSH data available.


Related in: MedlinePlus