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EGFR endocytosis is a novel therapeutic target in lung cancer with wild-type EGFR.

Jo U, Park KH, Whang YM, Sung JS, Won NH, Park JK, Kim YH - Oncotarget (2014)

Bottom Line: However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations.In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells.Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

View Article: PubMed Central - PubMed

Affiliation: BK21 Plus program, Korea University Anam Hospital, Seongbuk-gu, Seoul, Republic of Korea.

ABSTRACT
Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently observed in lung cancer patients with worse differentiation and poor prognosis. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations. Therefore, in this study, we investigated the potential molecular mechanism that contributes to cell viability and the response of gefitinib, one of the EGFR-TKIs, in lung cancer models with wide-type EGFR (wtEGFR). Interestingly, we found that EGF-induced EGFR endocytosis is existed differently between gefitinib-sensitive and -insensitive lung cancer cell lines. Suppressing EGFR endocytos decreased cell viability and increased apoptotic cell death in gefitinib-insensitive lung cancer with wtEGFR in vitro and in vivo. In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells. Rab25 knockdown caused the changed EGFR endocytosis and reverted the gefitinib response in gefitinib-sensitive lung cancer with wtEGFR in vitro and in vivo. Taken together, our findings suggest a novel insight that EGFR endocytosis is a rational therapeutic target in lung cancer with wtEGFR, in which the combined efficacy with gefitinib is expected. Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

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Status of Rab25 expression is associated with EGFR endocytosis and gefitinib response(A) Difference in endosomal localization of EGF-induced EGFR between H358 and H1703 cells was profiled by immunofuorescence staining with the indicated antibodies. The immunofluorescence images were obtained using a fluorescence microscope at a magnification of 400×. Each white arrow indicates co-localization (yellow). (B) Effect of Rab25 expression on EGFR endocytosis was examined in siRab25 transfected H358 cells by flow cytometry. After siRab25 or scrambled (Scr) transfection, the cells were incubated with gefitinib (10 μM) and the percentage of internalized Alexa Fluor 488-conjugated EGF (500 ng/ml) was analyzed. Each bar represents mean values acquired from three experiments with standard error (SE). * p < 0.05. (C) Correlation between Rab25 expression and gefitinib response was determined by comparative analysis. Expression of Rab25 mRNA was evaluated in eight lung cancer cell lines with wtEGFR using quantitative RT-PCR. Relative Rab25 expression values were normalized to values of GAPDH. IC50 of each cell was obtained from MTT assay results. The P value represents statistical comparisons between Rab25 expression and gefitinib response. Red: gefitinib-sensitive cell lines. Blue: gefitinib-insensitive cell lines.
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Figure 5: Status of Rab25 expression is associated with EGFR endocytosis and gefitinib response(A) Difference in endosomal localization of EGF-induced EGFR between H358 and H1703 cells was profiled by immunofuorescence staining with the indicated antibodies. The immunofluorescence images were obtained using a fluorescence microscope at a magnification of 400×. Each white arrow indicates co-localization (yellow). (B) Effect of Rab25 expression on EGFR endocytosis was examined in siRab25 transfected H358 cells by flow cytometry. After siRab25 or scrambled (Scr) transfection, the cells were incubated with gefitinib (10 μM) and the percentage of internalized Alexa Fluor 488-conjugated EGF (500 ng/ml) was analyzed. Each bar represents mean values acquired from three experiments with standard error (SE). * p < 0.05. (C) Correlation between Rab25 expression and gefitinib response was determined by comparative analysis. Expression of Rab25 mRNA was evaluated in eight lung cancer cell lines with wtEGFR using quantitative RT-PCR. Relative Rab25 expression values were normalized to values of GAPDH. IC50 of each cell was obtained from MTT assay results. The P value represents statistical comparisons between Rab25 expression and gefitinib response. Red: gefitinib-sensitive cell lines. Blue: gefitinib-insensitive cell lines.

Mentions: EGFR endocytosis is a complex cellular process which includes many modulating proteins [29]. Therefore, EGFR endocytosis-related effector or regulator proteins can be used as molecular markers to improve the therapeutic efficacy of gefitinib in lung cancer with wtEGFR. To demonstrate the hypothesis, we attempted to identify differentially expressed genes related to EGFR endocytosis using a genome-wide gene expression assay in gefitinib-sensitive H358 and -insensitive H1703 cells. We found six downregulated genes and two upregulated genes between these two lung cancer cell lines (Supplemental Table S1). Different expression statuses of selected genes were validated by real time-polymerase chain reaction analysis (Data not shown). Rab GTPases (Rab25 and Rab17) exhibited the most distinct gene expression profile among the selected genes. Basically, Rab25 are key regulators in endosomal trafficking and recycling back to the plasma membrane of EGFR [34]. Therefore, we subsequently monitored the endosomal distribution status of EGFR in H358 and H1703 cells after EGF stimulation. As shown in Figure 5A, EGFR was localized in early and recycling endosomes after EGF stimulation in H358 cells expressing Rab25. In contrast, EGFR was only localized in early endosomes of H1703 cells with no Rab25 expression. To further investigate whether Rab25 expression affects EGFR endocytosis, we transfected siRab25 or the scrambled (Scr) control into H358 cells with Rab25 expression. The change of EGFR endocytosis was analyzed by monitoring the internalized proportion of fluorescent-conjugated EGF by flow cytometry. As shown in Figure 5B, internalized EGF increased and then decreased following gefitinib treatment in Scr-transfected cells. However, this inhibition of EGF internalization by gefitinib was less prominent in siRab25-transfected cells than that in Scr-transfected cells.


EGFR endocytosis is a novel therapeutic target in lung cancer with wild-type EGFR.

Jo U, Park KH, Whang YM, Sung JS, Won NH, Park JK, Kim YH - Oncotarget (2014)

Status of Rab25 expression is associated with EGFR endocytosis and gefitinib response(A) Difference in endosomal localization of EGF-induced EGFR between H358 and H1703 cells was profiled by immunofuorescence staining with the indicated antibodies. The immunofluorescence images were obtained using a fluorescence microscope at a magnification of 400×. Each white arrow indicates co-localization (yellow). (B) Effect of Rab25 expression on EGFR endocytosis was examined in siRab25 transfected H358 cells by flow cytometry. After siRab25 or scrambled (Scr) transfection, the cells were incubated with gefitinib (10 μM) and the percentage of internalized Alexa Fluor 488-conjugated EGF (500 ng/ml) was analyzed. Each bar represents mean values acquired from three experiments with standard error (SE). * p < 0.05. (C) Correlation between Rab25 expression and gefitinib response was determined by comparative analysis. Expression of Rab25 mRNA was evaluated in eight lung cancer cell lines with wtEGFR using quantitative RT-PCR. Relative Rab25 expression values were normalized to values of GAPDH. IC50 of each cell was obtained from MTT assay results. The P value represents statistical comparisons between Rab25 expression and gefitinib response. Red: gefitinib-sensitive cell lines. Blue: gefitinib-insensitive cell lines.
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Figure 5: Status of Rab25 expression is associated with EGFR endocytosis and gefitinib response(A) Difference in endosomal localization of EGF-induced EGFR between H358 and H1703 cells was profiled by immunofuorescence staining with the indicated antibodies. The immunofluorescence images were obtained using a fluorescence microscope at a magnification of 400×. Each white arrow indicates co-localization (yellow). (B) Effect of Rab25 expression on EGFR endocytosis was examined in siRab25 transfected H358 cells by flow cytometry. After siRab25 or scrambled (Scr) transfection, the cells were incubated with gefitinib (10 μM) and the percentage of internalized Alexa Fluor 488-conjugated EGF (500 ng/ml) was analyzed. Each bar represents mean values acquired from three experiments with standard error (SE). * p < 0.05. (C) Correlation between Rab25 expression and gefitinib response was determined by comparative analysis. Expression of Rab25 mRNA was evaluated in eight lung cancer cell lines with wtEGFR using quantitative RT-PCR. Relative Rab25 expression values were normalized to values of GAPDH. IC50 of each cell was obtained from MTT assay results. The P value represents statistical comparisons between Rab25 expression and gefitinib response. Red: gefitinib-sensitive cell lines. Blue: gefitinib-insensitive cell lines.
Mentions: EGFR endocytosis is a complex cellular process which includes many modulating proteins [29]. Therefore, EGFR endocytosis-related effector or regulator proteins can be used as molecular markers to improve the therapeutic efficacy of gefitinib in lung cancer with wtEGFR. To demonstrate the hypothesis, we attempted to identify differentially expressed genes related to EGFR endocytosis using a genome-wide gene expression assay in gefitinib-sensitive H358 and -insensitive H1703 cells. We found six downregulated genes and two upregulated genes between these two lung cancer cell lines (Supplemental Table S1). Different expression statuses of selected genes were validated by real time-polymerase chain reaction analysis (Data not shown). Rab GTPases (Rab25 and Rab17) exhibited the most distinct gene expression profile among the selected genes. Basically, Rab25 are key regulators in endosomal trafficking and recycling back to the plasma membrane of EGFR [34]. Therefore, we subsequently monitored the endosomal distribution status of EGFR in H358 and H1703 cells after EGF stimulation. As shown in Figure 5A, EGFR was localized in early and recycling endosomes after EGF stimulation in H358 cells expressing Rab25. In contrast, EGFR was only localized in early endosomes of H1703 cells with no Rab25 expression. To further investigate whether Rab25 expression affects EGFR endocytosis, we transfected siRab25 or the scrambled (Scr) control into H358 cells with Rab25 expression. The change of EGFR endocytosis was analyzed by monitoring the internalized proportion of fluorescent-conjugated EGF by flow cytometry. As shown in Figure 5B, internalized EGF increased and then decreased following gefitinib treatment in Scr-transfected cells. However, this inhibition of EGF internalization by gefitinib was less prominent in siRab25-transfected cells than that in Scr-transfected cells.

Bottom Line: However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations.In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells.Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

View Article: PubMed Central - PubMed

Affiliation: BK21 Plus program, Korea University Anam Hospital, Seongbuk-gu, Seoul, Republic of Korea.

ABSTRACT
Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently observed in lung cancer patients with worse differentiation and poor prognosis. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations. Therefore, in this study, we investigated the potential molecular mechanism that contributes to cell viability and the response of gefitinib, one of the EGFR-TKIs, in lung cancer models with wide-type EGFR (wtEGFR). Interestingly, we found that EGF-induced EGFR endocytosis is existed differently between gefitinib-sensitive and -insensitive lung cancer cell lines. Suppressing EGFR endocytos decreased cell viability and increased apoptotic cell death in gefitinib-insensitive lung cancer with wtEGFR in vitro and in vivo. In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells. Rab25 knockdown caused the changed EGFR endocytosis and reverted the gefitinib response in gefitinib-sensitive lung cancer with wtEGFR in vitro and in vivo. Taken together, our findings suggest a novel insight that EGFR endocytosis is a rational therapeutic target in lung cancer with wtEGFR, in which the combined efficacy with gefitinib is expected. Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

Show MeSH
Related in: MedlinePlus