Impact of ERBB2 mutations on in vitro sensitivity of bladder cancer to lapatinib

Abstract

Lapatinib, a dual tyrosine kinase inhibitor of ErbB1 and ErbB2, shows a clinical benefit in a subset of patients with advanced urothelial bladder cancer (UBC). We hypothesized that the corresponding gene, ERBB2, is affected by mutations in a subset of UBC and that these mutations impact ErbB2 function, signaling, UBC proliferation, gene expression, and predict response to lapatinib. We found ERBB2 mutations in 5 of 33 UBC cell lines (15%), all of which were derived from invasive or high grade tumors. Phosphorylation and activation of ErbB2 and its downstream pathways were markedly enhanced in mutated cell lines compared with the ERBB2 wild-type. In addition, the gene expression profile was distinct, specifically for genes encoding for proteins of the extracellular matrix. RT112 cells infected with ERBB2 mutants showed a particular growth pattern ("mini-foci"). Upon treatment with lapatinib, 93% of these "mini-foci" were reversed. The sensitivity to lapatinib was greatest among cell lines with ERBB2 mutations. In conclusion, ERBB2 mutations occur in a subset of UBC and impact proliferation, signaling, gene expression and predict a greater response to lapatinib. If confirmed in the clinical setting, this may lead the way toward personalized treatment of a subset of UBC.

Keywords: ERBB2; bladder cancer; epidermal growth factor; lapatinib; personalized medicine.

Figure 1. Knockdown of ERBB2 in five cell lines with and in four cell lines without ERBB2 mutations. Five days after infection with shRNA, cells were fixed and stained by crystal violet (A). (B) Normalized (sh-Ctrl) relative quantification of cells. (C) Western blot for ErbB2 and β-tubulin. Data show the essential role of ErbB2 in some cell lines.

Figure 2. (A) Activation/phosphorylation of ErbB2 according to mutational status. Western blot for the protein phosphorylation in RT112 cells infected with a vector, wild-type or mutant ERBB2. (B) Activation/phosphorylation of ErbB2 according to mutational status. Formation of “mini-foci” of infected RT112 cells: 1, vector; 2, ERBB2-wild type; 3, ERBB2-L15F; 4, R143Q; 5, D277H; 6, S310F; 7, S653C; 8, R678Q; 9, D277+S310F. Arrows point to “mini-foci”.

Figure 3. (A) Effects of 0.75uM lapatinib on “mini-foci” formation (arrows). (B) Effects of AZD6244, lapatinib and afatinib on the density of “mini-foci”.

Figure 4. Gene expression by ERBB2 mutational status. (A) Array tree for the gene hierarchical clustering of the gene altered in RT112 cells with either vector, wild type (WT), or mutant ERBB2 (S310F, S653C). (B, C, D, and F) Association of the clusters with the gene expression. The most significant changes were observed in extracellular proteins, with the Gene Oncology (GO) terms “extracellular region” or “extracellular space” (group I). They were further clustered into extracellular matrix (group II; GO terms “extracellular matrix”, “proteinaceous extracellular matrix”, and “basement membrane”), cell adhesion (group III; GO terms “cell adhesion”, biological adhesion”, and “cell–cell adhesion”), secreted active molecules (group IV; GO terms “growth factor activity”, “cytokine activity”, and “hormone activity”) and enzyme inhibitors (group V; GO terms “enzyme inhibitor activity”, “peptidase inhibitor activity”, “endopeptidase inhibitor activity”, and “serine-type endopeptidase inhibitor activity”). The transport, secretion and localization of extracellular proteins is supported by regulators of protein secretion (group VI; GO terms “regulation of protein transport”, “regulation of protein secretion”, “regulation of establishment of protein localization”, “regulation of protein localization”, “regulation of cellular localization”, and “regulation of secretion.” Another two gene clusters among the most altered ones were related to epithelial differentiation (C) When the differentially expressed genes between mutants and wild-type were analyzed, the cluster was more significantly altered (D). (E and G) Heat map of gene expression levels from RNA sequencing. TF, transcription factor; SF, signaling factor.

Figure 5. Response to lapatinib by ERBB2 mutational status. (A) Cluster according to the sensitivity to lapatinib (upper panel) and IC₅₀ for the cell lines as well as the relative expression levels of EGFR and ErbB2 (lower panel). The gray IC₅₀ bars refer to cell lines with ERBB2 mutation. DSH1 containing three ERBB2 mutations was found to be the most sensitive cell line among all (group IIIB). (B and C) Correlation slope and contingency table of the numbers of ERBB2 mutations and the sensitivity of cell lines to lapatinib, showing that the sensitivity was significantly increased in cell lines with ERBB2 mutations.