Generation and Characterization of Trastuzumab/Pertuzumab-Resistant HER2-Positive Breast Cancer Cell Lines

Abstract

The combination of trastuzumab and pertuzumab as first-line therapy in patients with HER2-positive breast cancer has shown significant clinical benefits compared to trastuzumab alone. However, despite initial therapeutic success, most patients eventually progress, and tumors develop acquired resistance and invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance in order to develop targeted therapeutic strategies with improved efficacy. We generated four novel HER2-positive cell lines via prolonged exposure to trastuzumab and pertuzumab and determined their resistance rates. Long-term resistance was confirmed by a significant increase in the colony-forming capacity of the derived cells. We authenticated the molecular identity of the new lines via both immunohistochemistry for the clinical phenotype and molecular profiling of point mutations. HER2 overexpression was confirmed in all resistant cell lines, and acquisition of resistance to trastuzumab and pertuzumab did not translate into differences in ER, PR, and HER2 receptor expression. In contrast, changes in the expression and activity of other HER family members, particularly HER4, were observed. In the same vein, analyses of the receptor and effector kinase status of different cellular pathways revealed that the MAPK pathway may be involved in the acquisition of resistance to trastuzumab and pertuzumab. Finally, proteomic analysis confirmed a significant change in the abundance patterns of more than 600 proteins with implications in key biological processes, such as ribosome formation, mitochondrial activity, and metabolism, which could be relevant mechanisms in the generation of resistance in HER2-positive breast cancer. We concluded that these resistant BCCLs may be a valuable tool to better understand the mechanisms of acquisition of resistance to trastuzumab and pertuzumab-based anti-HER2 therapy.

Keywords: HER2 positive; breast cancer; label-free proteomics; pertuzumab; resistance; targeted therapy; trastuzumab.

Figure 1

Generation of cell lines resistant to dual anti-HER2 therapy, trastuzumab and pertuzumab. Effect of treatment with 15 µg/mL trastuzumab (T), 20 µg/mL pertuzumab (P), or a combination of T + P for 7 days in a panel of sensitive and acquired-resistant cell lines, after a period of several months of generation of acquired resistance. Viable cells were counted via trypan blue exclusion. Viability was presented as a percentage of the respective DMSO-treated control group (C). (A) AU-565-sensitive (AU-565) and trastuzumab and pertuzumab-resistant (AU-565.rTP) cells. (B) BT-474-sensitive (BT-474) and -resistant (BT-474.rTP) cells. (C) EFM-192A-sensitive (EFM-192A) and -resistant (EFM-192A.rTP) cells. (D) SK-BR-3-sensitive (SK-BR-3) and -resistant (SK-BR-3.rTP) cells. Error bars represent the standard deviation between replicates (n ≥ 3). ** denotes p ≤ 0.01; and *** denotes p ≤ 0.001.

Figure 2

Effect of long-term treatment with trastuzumab and pertuzumab in sensitive and resistant cell lines. Clonogenic capacity of sensitive vs. resistant cell lines was compared after 21 days of treatment with T + P. (A–D). Representative images of colony formation after staining with 1% crystal violet. (A) AU-565 and AU-565.rTP. (B) BT-474 and BT-474.rTP. (C) EFM-192A and EFM-192A.rTP. (D) SK-BR-3 and SK-BR-3.rTP. (E–H) Relative number of colonies presented as a percentage of the DMSO-treated control colony number. (E) AU-565 and AU-565.rTP. (F) BT-474 and BT-474.rTP. (G) EFM-192A and EFM-192A.rTP. (H) SK-BR-3 and SK-BR-3.rTP. Error bars indicate the standard deviation between replicates (n ≥ 3). ** denotes p ≤ 0.01; *** denotes p ≤ 0.001.

Figure 3

Characterization of sensitive and resistant cell lines according to their IHC profiles of ER, PR, and HER2 expression. The cells were prepared as FFPE pellets. Resistant populations showed no change in their molecular profile compared to their sensitive counterparts. (A) AU-565 and AU-565.rTP. (B) BT-474 and BT-474.rTP. (C) EFM-192A and EFM-192A.rTP. (D) SK-BR-3 and SK-BR-3.rTP. BT-474- and EFM-192A-sensitive and resistant cell lines were confirmed as luminal (ER positive, PR positive, and HER2 positive). AU-565- and SK-BR-3-sensitive and resistant lines were determined to be HER2 positive and hormonal receptor-negative. The black line represents a length of 50 µm. Magnification: ×200.

Figure 4

Evaluation of the phosphorylation pattern of HER family receptors via WB. Sensitive and resistant HER2-positive BCCLs were treated with T + P for 24 h. (A) AU-565 and AU-565.rTP. (B) BT-474 and BT-474.rTP. (C) EFM-192A and EFM-192A.rTP. (D) SK-BR-3 and SK-BR-3.rTP. Representative images of three replicates are depicted. Relative abundance levels of proteins were determined via densitometric analyses of the images.

Figure 5

Analysis of the protein phosphorylation patterns of the PI3K/AKT and MAPK pathways was assessed via WB in the sensitive and resistant cell lines. In the eight models, AKT and S6 (the PI3K/AKT pathway), as well as the ERK and P38 (the MAPK pathway) proteins, were examined under control conditions and with T + P treatment for 6 h or 24 h. (A) AU-565 and AU-565.rTP. (B) BT-474 and BT-474.rTP. (C) EFM-192A and EFM-192A.rTP. (D) SK-BR-3 and SK-BR-3.rTP. Representative images of three replicates are depicted. The relative abundance levels of these proteins were determined via densitometric analyses of these images.