Lapatinib antitumor effect is associated with PI3K and MAPK pathway: An analysis in human and canine prostate cancer cells

Abstract

The aberrant activation of HER2 has a pivotal role in bone metastasis implantation and progression in several tumor types, including prostate cancer (PC). Trastuzumab and other anti-HER2 therapies, such as lapatinib, have been used in human breast cancer HER2 positive. Although HER2 overexpression has been reported in PC, anti-HER2 therapy response has revealed conflicting results. We investigated the potential of lapatinib in inhibiting cell migration and inducing apoptosis in two human (LNCaP and PC3) and two canine PC cell lines (PC1 and PC2). Cell migration and apoptosis were evaluated by Annexin V/PI analysis after lapatinib treatment. The transcriptome analysis of all cell lines before and after treatment with lapatinib was also performed. We found increased apoptosis and migration inhibition in LNCaP cells (androgen-sensitive cell line), while PC1, PC2, and PC3 cells showed no alterations after the treatment. The transcriptome analysis of LNCaP and PC3 cell lines showed 158 dysregulated transcripts in common, while PC1 and PC2 cell lines presented 82. At the doses of lapatinib used, we observed transcriptional modifications in all cell lines. PI3K/AKT/mTOR pathway were enriched in human PC cells, while canine PC cells showed enrichment of tyrosine kinase antitumor response and HER2-related pathways. In canine PC cells, the apoptosis failed after lapatinib treatment, possibly due to the downregulation of MAPK genes. Prostate cancer cells insensitive to androgens may be resistant to lapatinib through PI3K gene dysregulation. The association of lapatinib with PI3K inhibitors may provide a more effective antitumor response and clinical benefits to PC patients.

Fig 1. Apoptosis and migration assays performed in human (LNCaP and PC3) and canine (PC1 and PC2) cell lines.

No significant difference was observed in the migration of LNCaP (A), PC3 (D), PC1 (G), and PC2 (J) cells. LNCaP cells treated with lapatinib showed a significant difference in apoptosis and necrosis (B) while no difference was observed in PC3 (E), PC1 (H), and PC2 (K) cell lines. Cell lines treated with DMSO (control) presented viability of 87.9% for LNCaP (C), 90.4% for PC3 (F), 76.1% for the canine PC1, and 83% for the canine PC2. PI: Propidium iodide; AN: Annexin; (+): Positive, (-): Negative; PI-/AN-: Viable cells (alive); PI-/AN+: Early apoptosis; PI+/AN+: Late apoptosis; PI+/AN-: Necrotic cells.

Fig 2. Graphic representation of the apoptotic assay.

A: The LNCaP cell line presented more than 80% of early apoptosis. The human PC3 (B), canine PC1 (C), and PC2 (D) cell lines showed over 70% of viable cells and a low apoptotic rate.

Fig 3

Transcriptomic analysis comparing human (A) and canine (B) prostate cancer cells treated and non-treated with lapatinib. Hierarchical clustering analysis of the differentially expressed genes between treated and non-treated cell lines with lapatinib. Red and blue colors indicate up or downregulated genes, respectively. The mean signals were corrected and transformed to the log2 scale, and genes with at least 1.5-fold changes with p < 0.05 at the 95% confidence level were considered significant. C: Protein-to-protein integration network of LNCaP and PC3 cells treated and non-treated with lapatinib. Several complex interactions among proteins were observed, with CDK1, PLK1, and CCNB1 proteins in the middle of the interaction map with the highest number of interactions. D: Protein-to-protein integration network of canine PC1 and PC2 cells treated and non-treated with lapatinib showing fewer interactions than those observed in human prostate cancer cells. However, the proteins with the highest number of interactions are similar in human and canine cells (PDK1 and PLK1).

Fig 4

A: TOP 10 biological and molecular ontology processes, respectively, of the LNCaP and PC3 cell lines. Different terms were found for each cell line. LNCaP cell line showed terms associated with modification of epithelial cell morphogenesis, including cell migration. PC3 cell line presented processes related to oxidative stress and positive regulation of chemotaxis. B: TOP 10 biological and molecular ontology processes for canine PC1 and PC2 cell lines. PC1 cells presented several processes related to cell division and cell cycle alterations. PC3 cell line showed different processes related to cell binding and heterodimerization, which were related to EGFR tyrosine kinase inhibitor regulation.