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Oncotarget. 2017;8(38):63901-63910.

EGFR or HER2 inhibition modulates the tumor microenvironment by suppression of PD-L1 and cytokines release.

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제1저자 서경진, 성지혜 · 교신저자 김진원

PMID 28969039 PMC5609971 DOI 10.18632/oncotarget.19194 IF 5.168 혈액종양내과
소속 (4)
  1. Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Bundang-gu, Seongnam, Korea.
  2. Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Jongno-gu, Seoul, Korea.
  3. Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Bundang-gu, Seongnam, Korea.
  4. Cancer Research Institute, Seoul National University, Jongno-gu, Seoul, Korea.

Abstract

BACKGROUND Characteristics of tumor microenvironment have been suggested as predictive markers of anti-EGFR or anti-HER2 treatment response. However, the effect of EGFR/HER2 signal blockade on the tumor immune microenvironment is unclear.

METHODS EGFR/HER2 pathway signaling and PD-L1 expression in gastric cancer cell lines were screened by western blot analysis. PD-L1 and HER2 expressions in 251 resected gastric tumors were determined by immunohistochemistry, and changes in EFGR, HER2, and PD-L1 expression in paired specimens between pre- and post-chemotherapy were evaluated. PD-L1 expression in HER2-amplified cell lines was evaluated by western blotting, fluorescence-activated cell sorting, reverse transcription, and real-time quantitative PCR analyses before and after afatinib, lapatinib, pictilisib and trametinib treatment. Changes in cytokines were evaluated by reverse transcription, real-time quantitative PCR, and enzyme-linked immunosorbent assay after EGFR/HER2 inhibition.

RESULTS Cell lines with pEGFR or pHER2 overexpression showed higher PD-L1 expression. In resected gastric tumors, HER2 expression was significantly associated with PD-L1 expression (p=0.030). PD-L1 overexpression accompanied by increased HER2 expression was identified in a post-chemotherapy specimen from a patient with an initial HER2/PD-L1-negative tumor. In HER2-overexpressing cell lines, PD-L1 expression was decreased in a dose- and time-dependent manner after afatinib and lapatinib treatment. PI3K pathway inhibition by pictilisib, but not MEK pathway inhibition by trametinib, resulted in PD-L1 suppression. After lapatinib treatment, the release of CCL2, CCL21, VEGF and CXCL1 decreased in a dose-dependent manner.

CONCLUSIONS Inhibition of the EGFR/HER2 signaling pathway, particularly of downstream PI3K activity, suppressed PD-L1 and release of cytokines, suggesting that EGFR/HER2 inhibition may create a more favorable milieu for tumor immunotherapy.

Keywords

EGFRHER2PD-L1PI3Kcytokine

Figures

Figure 1
Figure 1 Association of PD-L1 expression with the EGFR/HER2 signaling pathway in gastric cancer cell lines Seven gastric cancer cell lines (SNU216, SNU668, SNU719, AGS, NCI-N87, YCC3 and YCC10) were seeded at a density of 1 × 106 cells per well in 6-well plates and incubated in a humidified CO2 incubator at 37°C. The protein expressions of molecules related to the EGFR/HER2 signaling pathway were screened by western blot analysis. PD-L1 expression in cell lines with pEGFR overexpression or pHER2 overexpression (SNU216, SNU668, and NCI-N87) was relatively higher than that in other cell lines. As positive controls, HER2-amplified breast cancer cell line (SKBR3) and EGFR-mutated lung cancer cell line (PC9) showed higher PD-L1 expression.
Figure 2
Figure 2 Immunohistochemistry analysis of the paired tumor specimens of pre-chemotherapy and post-chemotherapy The specimens were stained for (A-C) EGFR, (D-F) HER2, and (G-I) PD-L1 expression. HER2 and PD-L1 were overexpressed in the post-chemotherapy specimen of an initial HER2/PD-L1-negative tumor; original magnification ×400.
Figure 3
Figure 3 Decrease in PD-L1 expression following EGFR/HER2 inhibition in HER2-amplified cell lines HER2-amplified gastric (SNU216, NCI-N87) and breast (SKBR3) cancer cell lines were seeded at a density of 1 × 106 cells per well in 6-well plates and incubated in a humidified CO2 incubator at 37°C. After overnight growth, (A) afatinib (0, 2, 20, and 200 nM) and (B) lapatinib (0, 0.01, 0.1, and 1 μM) were added. Cells were harvested at 24 h, and the expression of PD-L1 was analyzed by western blot analysis. PD-L1 was suppressed in a dose-dependent manner. The mRNA expression of PD-L1 after lapatinib (0, 0.01, 0.1, and 1 μM) treatment was determined by (C) RT-PCR and (D) qPCR. (E) The mean fluorescence intensity of PD-L1 was determined by flow cytometry after lapatinib (0, 0.01, 0.1, and 1 μM) treatment. (F) PD-L1 expression was examined by qPCR after treatment with 0.1 μM lapatinib for different times (0, 4, 24, and 72 h). The PD-L1 expression of three independent experiments is shown as mean ± SD. *0.001 ≤ p < 0.05 and **p < 0.001 in ANOVA.
Figure 4
Figure 4 Involvement of the AKT pathway in PD-L1 expression regulation HER2-amplified cancer cell lines were seeded at a density of 1 × 106 cells per well in 6-well plates and incubated in a humidified CO2 incubator at 37°C. After overnight growth, cells were treated with 0.1 μM pictilisib, 0.1 μM trametinib, 0.1 μM lapatinib, and 0.02 μM afatinib for 24 h. (A) The mean fluorescence intensity (MFI) of PD-L1 was determined by flow cytometry. PD-L1 was expression decreased in pictilisib-treated cell lines. In contrast, PD-L1 expression was not suppressed in trametinib-treated SNU216 and SKBR3 cell lines. To enhance PD-L1 expression, IFNγ (10 ng/mL) was added after 4 h of drug treatment, and incubation was continued for a further 24 h. At 28 h after drug treatment, cells were harvested for evaluation of PD-L1 protein expression by (B) western blotting and (C) RT-PCR analyses. In comparison with trametinib inhibition, pictilisib inhibition of the AKT pathway suppressed IFNγ-mediated PD-L1 protein upregulation. The PD-L1 expression of three independent experiments is shown as mean ± SD. *p < 0.05 in paired Student's t-test (2-tailed).
Figure 5
Figure 5 Effect of HER2 blockade on the cytokine release of tumor cells HER2-amplified cell lines were seeded at a density of 1 × 106 cells per well in 6-well plates and incubated in a humidified CO2 incubator at 37°C. After overnight growth, lapatinib (0, 0.01, 0.1, and 1 μM) was added. Cells were harvested after 24 h. The mRNA expression of CCL2, CCL21, VEGF, and CXCL1 was examined by (A) RT-PCR, (B) qPCR, and (C) ELISA which demonstrated a dose-dependent suppression. The expression of three independent experiments is shown as mean ± SD. *0.001 ≤ p < 0.05 and **p < 0.001 in ANOVA.

그림 출처: PubMed Central 공개 논문 (PMC5609971)

Conflict of interest statement

CONFLICTS OF INTEREST All authors declare no conflicts of interest.

그 밖의 정보
출판 형태
Journal Article
저널
Oncotarget · eISSN 1949-2553
전자 출판
2017-07-12
참고문헌
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