Tuberc Respir Dis.  2013 Sep;75(3):95-103.

Neuroendocrine Differentiation in Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

• Chang Y
• Kim SY
• Choi YJ
• So KS
• Rho JK
• Kim WS
• Lee JC
• Chung JH
• Choi CM

Abstract

BACKGROUND
Small cell lung cancer (SCLC) transformation during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer has been suggested as one of possible resistance mechanisms.
METHODS
We evaluated whether SCLC transformation or neuroendocrine (NE) differentiation can be found in the cell line model. In addition, we also investigated its effect on responses to conventional chemotherapeutic drugs of the SCLC treatment.
RESULTS
Resistant cell lines to various kinds of EGFR-TKIs such as gefitinib, erlotinib, CL-387,785 and ZD6474 with A549, PC-9 and HCC827 lung adenocarcinoma cell lines were established. Among them, two resistant cell lines, A549/GR (resistant to gefitinib) and PC-9/ZDR (resistant to ZD6474) showed increased expressions of CD56 while increased synaptophysin, Rb, p16 and poly(ADP-ribose) polymerase were found only in A549/GR in western blotting, suggesting that NE differentiation occurred in A549/GR. A549/GR cells were more sensitive to etoposide and cisplatin, chemotherapeutic drugs for SCLC, compared to parental cells. Treatment with cAMP and IBMX induced synaptophysin and chromogranin A expression in A549 cells, which also made them more sensitive to etoposide and cisplatin than parental cells. Furthermore, we found a tissue sample from a patient which showed increased expressions of CD56 and synaptophysin after development of resistance to erlotinib.
CONCLUSION
NE differentiation can occur during acquisition of resistance to EGFR-TKI, leading to increased chemosensitivity.

Keyword

Small Cell Lung Carcinoma; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm

MeSH Terms

1-Methyl-3-isobutylxanthine
Adenocarcinoma
Blotting, Western
Cell Line
Cell Transformation, Neoplastic
Chromogranin A
Cisplatin
Drug Resistance, Neoplasm
Epidermal Growth Factor
Etoposide
Humans
Lung
Lung Neoplasms
Parents
Piperidines
Poly(ADP-ribose) Polymerases
Protein-Tyrosine Kinases
Quinazolines
Receptor, Epidermal Growth Factor
Small Cell Lung Carcinoma
Synaptophysin
Erlotinib Hydrochloride
1-Methyl-3-isobutylxanthine
Adenocarcinoma
Chromogranin A
Cisplatin
Epidermal Growth Factor
Etoposide
Lung Neoplasms
Piperidines
Poly(ADP-ribose) Polymerases
Protein-Tyrosine Kinases
Quinazolines
Receptor, Epidermal Growth Factor
Synaptophysin

Figure

• 

  Figure 1 The basal level of neuroendocrine marker proteins was evaluated by western blotting (A, B) and immunocytochemistry (C). The chemosensitivity of gefitinib-resistant cells was determined by a MTT assay (D). The cells were exposed to the indicated concentrations of etoposide or cisplatin for 72 hours (*p<0.01 compared with A549 cells, **p<0.001 compared with A549 cells). Cells stained with H&E showed morphological changes between A549 cells and A549/GR cells (E). SYP: synaptophysin; Chr-A: chromogranin A.

• 

  Figure 2 The expression of synaptophysin (SYP) and CD56 was increased in re-biopsied tumor tissues procured after development of erlotinib-resistance in a patient. (A, C) Tumor obtained before treatment with erlotinib. (B, D) Tumor after acquisition of resistance.

• 

  Figure 3 Neuroendocrine (NE) differentiation was induced by treatment with 0.5 mmol/L cAMP and IBMX for 72 hours. Morphological changes by light microscopy (A), immunocytochemistry (B) and western blots (C) indicated NE differentiation. (D) MTT assay was performed to confirm whether small cell lung cancer can enhance the sensitivity against etoposide or cisplatin. The cells were treated with the indicated doses of etoposide or cisplatin in the presence or absence of cAMP and IBMX for 72 hours. The p-values were calculated to assess the effect of combined treatment of cAMP and IBMX (*p<0.01, **p<0.001). SYP: synaptophysin; Chr-A: chromogranin.

• 

  Figure 4 The poly (ADP-ribose) polymerase (PARP) expression was increased in A549/GR cells, which did not affect chemosensitivity. (A) The basal level of PARP protein was evaluated by western blotting. (B) Response to AZD2281, a PARP inhibitor, was determined by a MTT assay. The cells were exposed to the indicated concentrations of AZD2281 for 72 hours. (C) Cells were treated with indicated doses of etoposide or cisplatin for 72 hours alone, AZD2281 and two drugs in combination. The viability of the cells was determined using the MTT assay.

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