Overcoming chemoresistance of small-cell lung cancer through stepwise HER2-targeted antibody-dependent cell-mediated cytotoxicity and VEGF-targeted antiangiogenesis

Abstract

Small-cell lung cancer (SCLC) easily recurs with a multidrug resistant phenotype. However, standard therapeutic strategies for relapsed SCLC remain unestablished. We found that human epidermal growth factor receptor 2 (HER2) is not only expressed in pretreated human SCLC specimens, but is also upregulated when HER2-positive SCLC cells acquire chemoresistance. Trastuzumab induced differential levels of antibody-dependent cell-mediated cytotoxicity (ADCC) to HER2-positive SCLC cells. Furthermore, as a mechanism of the differential levels of ADCC, we have revealed that coexpression of intracellular adhesion molecule (ICAM)-1 on SCLC cells is essential to facilitate and accelerate the trastuzumab-mediated ADCC. Although SN-38-resistant SCLC cells lacking ICAM-1 expression were still refractory to trastuzumab, their in vivo growth was significantly suppressed by bevacizumab treatment due to dependence on their distinctive and abundant production of vascular endothelial growth factor. Collectively, stepwise treatment with trastuzumab and bevacizumab is promising for the treatment of chemoresistant SCLC.

Figure 1. Development of a highly sensitive IHC system to detect HER2 in SCLC.

(a) Detection of HER2 by IHC in breast cancer (SK-BR-3) and SCLC (H69, SBC-3, and SBC-3/ETP) cell blocks by HercepTest (top panels) or the new system (bottom panels). SK-BR-3 and H69 cells were used as a positive and negative control, respectively. The D8F12 Ab-based new IHC system exhibited improved sensitivity compared with HercepTest without compromising specificity. (b) The new IHC system also works well in human SCLC samples obtained by diagnostic biopsy. Representative histological images of HER2-positive (top panels) and -negative (bottom panels) cases are shown. All scale bars, 50 μm.

Figure 2. Antitumor activity of trastuzumab does not depend on direct inhibition of HER2 signaling but rather on ADCC in HER2-positive SCLC cells.

(a) HER2-positive SCLC cells (SBC-3, SBC-3/CDDP, SBC-3/ETP, and SBC-3/SN-38 cells) and HER2-overexpressing breast carcinoma cells (SK-BR-3 cells) were treated with 10 μg/ml of normal human IgG or trastuzumab (Tzmab) for 72 h. The relative numbers of viable cells were quantified using the CCK-8 assay. Points, mean% viable cells; bars, SD of at least three independent experiments performed in triplicate; *, P = 0.018; **, P = 0.004; ***, P = 0.002. (b) SBC-3/ETP and SK-BR-3 cells were treated with 10 μg/ml of trastuzumab for up to 72 h. Phosphorylation and expression of HER2, Akt, and Erk in whole cell lysates were examined by immunoblotting. PARP was also examined to detect apoptosis. Each lane of SBC-3/ETP and SK-BR-3 cell lysates contains 60 μg and 30 μg of total protein, respectively. Representative blots from three independent experiments with similar results are shown. Blots are cropped in the figure and full-length blots are presented in Supplementary information. (c) Induction of CD16 expression on NK cells. NK cells (YTS, NKL, and NK92MI cells) were treated with or without 10 μg/ml of trastuzumab for 4 h. Then, the cells were labeled with an anti-CD16 monoclonal Ab (black shaded) or isotype-matched control (solid line) and analyzed for cell surface expression of CD16 by FACS. (d), (e), and (f) Evaluation of trastuzumab-mediated ADCC. Target cancer cells and effector NK cells were coincubated at various E/T ratios with 10 μg/ml of normal human IgG or trastuzumab (Tzmab) for 4 h. Cytotoxic activity was determined based on the LDH release assay. Representative data from at least three experiments are shown as the means of triplicate cultures.

Figure 3. Antitumor effects of trastuzumab on HER2-positive SCLC tumor xenografts.

(a) HER2-positive SCLC cells were inoculated subcutaneously into the flanks of athymic nude mice. When the tumor volume reached approximately 200–300 mm³, the mice were randomly assigned to the control (PBS) arm or trastuzumab (Tzmab) treatment arm (n = 5–7 mice per group). Trastuzumab was intraperitoneally administered at a dose of 30 mg/kg twice weekly. Points, mean tumor volumes; bars, SD; *, P = 0.042; **, P < 0.001. (b) Representative histological images of H&E and IHC for HER2, TUNEL, and CD11b. Scale bar, 50 μm. (c) Apoptosis index (%) was determined by calculating the number of TUNEL-positive cells per total number of cells, which consisted of 1000 or more SCLC cells in five randomly selected fields. *, P = 0.003. (d) CD11b-positive cells were quantified by counting at least 1000 cells in five randomly selected fields. *, P = 0.003.

Figure 4. Trastuzumab is rapidly internalized, and subsequently undergoes ubiquitination and lysosomal degradation.

(a) After HER2-positive SCLC cells were incubated with 10 μg/ml of trastuzumab for 45 min at 4°C, cells were warmed to 37°C to allow internalization or maintained at 4°C. The residual levels of cell surface trastuzumab were calculated based on the mean fluorescence intensity (MFI) analyzed by FACSort. (b) After HER2-positive SCLC cells were treated with 10 μg/ml of trastuzumab (Tzmab) for up to 240 min, whole cell lysates were immunoprecipitated with an Ab against ubiquitin. The immunoprecipitant was immunoblotted using an Ab against HER2 to detect HER2 ubiquitination. Blots are cropped in the figure and full-length blots are presented in Supplementary information. (c) SBC-3/CDDP cells were treated with 10 μg/ml of Alexa 488–labeled trastuzumab (green) in the presence of LysoTracker for 45 min at 4°C. Thereafter, trastuzumab was allowed to internalize for up to 120 min at 37°C and fluorescence images are shown. Arrows, lysosomal localization of trastuzumab.

Figure 5. CAM-1 facilitates trastuzumab-mediated ADCC.

(a) Parental and chemoresistant SBC-3 cells are labeled with 5 μg/ml of either an anti-CD54 mouse monoclonal Ab (black shaded) or an isotype-matched control Ab (solid line) and presented as histograms. (b) Upregulation of ICAM-1 expression on SBC-3/ETP cells compared to SBC-3/ETP cells was also confirmed by IHC. Scale bar, 50 μm. (c) SCLC cells in biopsy specimens from chemonaïve patients heterogeneously expressing ICAM-1. Scale bar, 50 μm. (d) SBC-3/ETP cells were coincubated with YTS cells in the presence of 10 μg/ml of normal human IgG or trastuzumab (Tzmab) with or without 5 μg/ml of an anti-human ICAM-1 (CD54) Ab. Column, mean relative lysis ratio compared to that of normal IgG; bars, SD of three independent experiments; *, P = 0.038; **, P = 0.026; ***, P = 0.025. (e) Facilitation of trastuzumab-mediated ADCC by ICAM-1 is schematized. NK, natural killer cell; Mono, macrophage and monocyte; FcγR, Fcγ receptor.

Figure 6. Antitumor effects of bevacizumab against SBC-3/SN-38 xenografts.

(a) In vitro proliferation assay of parental SBC-3 and SBC-3–derived chemoresistant cells. Points, mean fold increase in cell number; bars, SD from three experiments performed in triplicate. (b) VEGF-A mRNA expression in parental SBC-3 and SBC-3–derived chemoresistant cells was quantified by real-time PCR. Column, mean relative expression ratio to that of HUV-EC-C; bars, SD. (c) Soluble human VEGF₁₆₅ in the supernatants of each SCLC cell line was measured by ELISA. Column, mean concentration; bars, SD. Both experiments were performed at least thrice with triplicate samples. (d) SBC-3/SN-38 cells were inoculated into the flanks of athymic nude mice. When the tumor volume reached approximately 200–300 mm³, the mice were randomly assigned to the PBS-treated control arm or bevacizumab (BV)-treated arm (n = 7 mice per group). Bevacizumab was intraperitoneally administered at a dose of 10 mg/kg twice weekly. Points, mean tumor volumes; bars, SD; *, P = 0.031; **, P = 0.010; ***, P = 0.002. (e) Representative H&E and IHC images for CD31 and Ki67. Scale bar, 100 μm. (f) MVD was determined based on the ratio of the CD31-positive area to the total observation area in five randomly selected fields. Column, mean; bars, SD; *, P = 0.002. (g) The proliferation index was determined by dividing the number of Ki67-positive cells by the total number of SCLC cells in five randomly selected fields. Column, mean; bars, SD; *, P < 0.001.

Figure 7. Novel therapeutic decision tree to overcome chemoresistant SCLC based on HER2 expression.

After HER2 is detected by IHC at the time of diagnosis, patients are divided into two groups according to the 1^st line chemotherapy. Trastuzumab- or bevacizumab-based 2^nd line chemotherapy is ideal for patients who have acquired resistance to etoposide-based or irinotecan-based 1^st line chemotherapy, respectively. Upregulation of both HER2 and ICAM-1 in etoposide-resistant SCLC cells facilitates trastuzumab-mediated ADCC. Abundant VEGF produced from irinotecan-resistant SCLC cells is the main target of bevacizumab.