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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 overexpression induces the resistance of NSCLC cells to erlotinib(A) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with serially diluted concentrations of erlotinib for 72 h and cell viability was determined by MTT assay. (B) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then activation of EGFR-related signaling pathway and PARP cleavage were evaluated by western blotting. Blots representative of three independent experiments were shown. (C and D) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then cell apoptosis were determined by Annexin V/7AAD staining. The representative dotplots of three experiments were shown in D. (E) PC9 cells stably expressing Mer (PC9-Mer) were pretreated with the corresponding inhibitors for MAPK (SB203580, 1 μM), FAK (PF-562271, 1 μM)) and AKT (MK2206, 4 μM)) signals with DMSO treatment as controls for 4 h followed by addition of erlotinib for 20 h and cell apoptosis were determined by Annexin V/7AAD staining. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. **p < 0.01, paired student t test (for C) or ANOVA followed by Tukey's multiple comparisons test (for E).
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Figure 5: Mer overexpression induces the resistance of NSCLC cells to erlotinib(A) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with serially diluted concentrations of erlotinib for 72 h and cell viability was determined by MTT assay. (B) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then activation of EGFR-related signaling pathway and PARP cleavage were evaluated by western blotting. Blots representative of three independent experiments were shown. (C and D) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then cell apoptosis were determined by Annexin V/7AAD staining. The representative dotplots of three experiments were shown in D. (E) PC9 cells stably expressing Mer (PC9-Mer) were pretreated with the corresponding inhibitors for MAPK (SB203580, 1 μM), FAK (PF-562271, 1 μM)) and AKT (MK2206, 4 μM)) signals with DMSO treatment as controls for 4 h followed by addition of erlotinib for 20 h and cell apoptosis were determined by Annexin V/7AAD staining. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. **p < 0.01, paired student t test (for C) or ANOVA followed by Tukey's multiple comparisons test (for E).

Mentions: Previous study shows that Mer knockdown improves in vitro sensitivity of NSCLC cells to chemotherapeutic drugs by increased apoptosis induction, however, whether it is the case for EGFR inhibitor remains unexplored. To do this, we determined the sensitivity of EGFR mutation-harbored PC9 cells (delE746-A750) with or without stable Mer overexpression to EGFR inhibitor erlotinib by MTT assay. Mer overexpression induced a notable resistance to erlotinib treatment in otherwise elrotinib-sensitive PC9 cells, resulting in an approximate 24-fold increase in the half-maximal inhibitory concentrations (IC50) of erlotinib (0.34 μM vs 8.22 μM for control or Mer overexpression; Fig. 5A). Analysis of EGFR-related signaling pathways demonstrated that erlotinib treatment did not block the AKT phosphorylation in PC9-Mer cells though it inhibited the EGFR and MAPK phosphorylation in both PC9-pCDH and PC9-Mer cells (Fig. 5B). We further applied Annexin V/7-AAD staining to analyze the apoptosis of PC9-pCDH and PC9-Mer cells in response to erlotinib treatment. PC9-Mer cells exhibited a significant decrease in apoptosis relative to control PC9-pCDH cells following erlotinib treatment (Fig. 5C). The representative dotplots were shown in Fig. 5D. In addition, compared with control PC9-pCDH cells, PC9-Mer cells displayed a significantly decreased PARP cleavage in response to erlotinib treatment indicative of decreased apoptotic pathway activation at the biochemical level (Fig. 5B). Furthermore, blockade of AKT signaling pathway by MK2206 recovered the sensitivity of PC9-Mer cells to erlotinib treatment (Fig. 5E), suggesting that Mer expression could be an advantage for cancer cells by enhancing drug resistance through sustained AKT activation.


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 overexpression induces the resistance of NSCLC cells to erlotinib(A) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with serially diluted concentrations of erlotinib for 72 h and cell viability was determined by MTT assay. (B) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then activation of EGFR-related signaling pathway and PARP cleavage were evaluated by western blotting. Blots representative of three independent experiments were shown. (C and D) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then cell apoptosis were determined by Annexin V/7AAD staining. The representative dotplots of three experiments were shown in D. (E) PC9 cells stably expressing Mer (PC9-Mer) were pretreated with the corresponding inhibitors for MAPK (SB203580, 1 μM), FAK (PF-562271, 1 μM)) and AKT (MK2206, 4 μM)) signals with DMSO treatment as controls for 4 h followed by addition of erlotinib for 20 h and cell apoptosis were determined by Annexin V/7AAD staining. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. **p < 0.01, paired student t test (for C) or ANOVA followed by Tukey's multiple comparisons test (for E).
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Figure 5: Mer overexpression induces the resistance of NSCLC cells to erlotinib(A) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with serially diluted concentrations of erlotinib for 72 h and cell viability was determined by MTT assay. (B) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then activation of EGFR-related signaling pathway and PARP cleavage were evaluated by western blotting. Blots representative of three independent experiments were shown. (C and D) PC9 cells stably expressing control (PC9-pCDH) or Mer (PC9-Mer) were treated with erlotinib at indicated concentrations for 24 h and then cell apoptosis were determined by Annexin V/7AAD staining. The representative dotplots of three experiments were shown in D. (E) PC9 cells stably expressing Mer (PC9-Mer) were pretreated with the corresponding inhibitors for MAPK (SB203580, 1 μM), FAK (PF-562271, 1 μM)) and AKT (MK2206, 4 μM)) signals with DMSO treatment as controls for 4 h followed by addition of erlotinib for 20 h and cell apoptosis were determined by Annexin V/7AAD staining. All data are expressed as mean ± SD of triplicates and representative of three independent experiments. **p < 0.01, paired student t test (for C) or ANOVA followed by Tukey's multiple comparisons test (for E).
Mentions: Previous study shows that Mer knockdown improves in vitro sensitivity of NSCLC cells to chemotherapeutic drugs by increased apoptosis induction, however, whether it is the case for EGFR inhibitor remains unexplored. To do this, we determined the sensitivity of EGFR mutation-harbored PC9 cells (delE746-A750) with or without stable Mer overexpression to EGFR inhibitor erlotinib by MTT assay. Mer overexpression induced a notable resistance to erlotinib treatment in otherwise elrotinib-sensitive PC9 cells, resulting in an approximate 24-fold increase in the half-maximal inhibitory concentrations (IC50) of erlotinib (0.34 μM vs 8.22 μM for control or Mer overexpression; Fig. 5A). Analysis of EGFR-related signaling pathways demonstrated that erlotinib treatment did not block the AKT phosphorylation in PC9-Mer cells though it inhibited the EGFR and MAPK phosphorylation in both PC9-pCDH and PC9-Mer cells (Fig. 5B). We further applied Annexin V/7-AAD staining to analyze the apoptosis of PC9-pCDH and PC9-Mer cells in response to erlotinib treatment. PC9-Mer cells exhibited a significant decrease in apoptosis relative to control PC9-pCDH cells following erlotinib treatment (Fig. 5C). The representative dotplots were shown in Fig. 5D. In addition, compared with control PC9-pCDH cells, PC9-Mer cells displayed a significantly decreased PARP cleavage in response to erlotinib treatment indicative of decreased apoptotic pathway activation at the biochemical level (Fig. 5B). Furthermore, blockade of AKT signaling pathway by MK2206 recovered the sensitivity of PC9-Mer cells to erlotinib treatment (Fig. 5E), suggesting that Mer expression could be an advantage for cancer cells by enhancing drug resistance through sustained AKT activation.

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