Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis

Abstract

Endometriosis-harboring cancer-associated somatic mutations of PIK3CA and KRAS provides new opportunities for studying the multistep processes responsible for the functional and molecular changes in this disease. We aimed to establish a novel in vitro endometriosis model to clarify the functional behavior and molecular pathogenesis of this disorder. Immortalized HMOsisEC10 human ovarian endometriotic epithelial cell line was used in which KRAS and PIK3CA mutations were introduced. Migration, invasion, proliferation, and microarray analyses were performed using KRAS and PIK3CA mutant cell lines. In vitro assays showed that migration, invasion, and proliferation were significantly increased in KRAS and PIK3CA mutant cell lines, indicating that these mutations played causative roles in the aggressive behavior of endometriosis. Microarray analysis identified a cluster of gene signatures; among them, two significantly upregulated cancer-related genes, lysyl oxidase (LOX) and pentraxin3 (PTX3), were associated with cell proliferation, invasion, and migration capabilities. Furthermore, siRNA knockdown of the two genes markedly reduced the metastatic ability of the cells. These results suggest that endometriosis with KRAS or PIK3CA mutations can significantly enhance cell migration, invasion, and proliferation by upregulating LOX and PTX3. We propose that LOX and PTX3 silencing using small molecules could be an alternative therapeutic regimen for severe endometriosis.

Keywords: KRAS; LOX; PIK3CA; PTX3; cell proliferation; epithelial cell line; invasion; microarray analysis; migration; ovarian endometriosis.

Figure 1

HMOsisEC10 KRAS and HMOsisEC10 PIK3CA cells exhibit increased migration, invasion, and proliferation relative to HMOsisEC10 cells. Cell migration assay (A,B). HMOsisEC10 KRAS and HMOsisEC10 PIK3CA cell lines show increased cell migration in the wound healing assay. Photographs captured immediately after the scratch (0 h, upper left panel) and 24 h post-scratch (upper right panel). The number of migrated cells is significantly higher in the HMOsisEC10 KRAS and HMOsisEC10 PIK3CA mutant cell lines than in the HMOsisEC10 cell line (** p < 0.05). Matrigel invasion assay (C,D). The HMOsisEC10 KRAS and HMOsisEC10 PIK3CA mutant cell lines demonstrate a significantly higher invasion capacity than the control HMOsisEC10 cell line (** p < 0.01 and * p < 0.05, respectively). Cell proliferation assay (E). The proliferation capability of the mutant HMOsisEC10 KRAS and HMOsisEC10 PIK3CA cell lines are significantly higher than that of the HMOsisEC10 cell line (* p < 0.05 and ** p < 0.01, respectively). The error bars indicate standard deviation.

Figure 2

Gene expression analysis. Heat map representing expression pattern for migration (A) and invasion (B)-related gene signatures in the HMOsisEC10 KRAS and HMOsisEC10 PIK3CA mutant cell lines. Red color shows upregulated genes, and green color shows downregulated genes. Arrowhead indicates the genes selected for further analysis.

Figure 3

Targeting KRAS and PIK3CA mutations in endometriosis; KRAS mutation activates RAS, RAF, and MAPK/ERK with the aid of receptor tyrosine kinases through phosphorylation. PI3K is activated by the PIK3CA mutation, which results in phosphorylation of PIP2 to PIP3 and activates AKT and mTOR. MAPK/ERK and mTOR have common effects on RhoA activity, which regulates the serum response elements (SRF) with the help of TNF-α. SRF is a transcription factor that has downstream effects on LOX and PTX3 leading to cell proliferation, migration, and invasion.

Figure 4

mRNA expression levels of LOX (A) and PTX3 (B) are evaluated in mutant cell lines (HMOsisEC10 KRAS and HMOsisEC10 PIK3CA). RT-PCR results revealed that both mutant cell lines showed a significantly higher expression of LOX and PTX3 compared to the HMOsisEC10 cell line (* p < 0.05 and ** p < 0.01). Error bars indicate standard deviation.

Figure 5

Cell migration assay. Migration abilities of mutant cell lines (HMOsisEC10 KRAS and HMOsisEC10 PIK3CA) are measured using a scratch wound healing assay after knockdown with LOX (A1,A2) and PTX3 siRNA (B1,B2). The numbers of migrated cells were significantly lower in siRNA treated cells compared to the control siRNA and untreated cells. Matrigel Invasion assay. siRNA knockdown of LOX (C1,C2) and PTX3 (D1,D2) showed a significantly lower invasion capacity than control siRNA and untreated cells. Cell proliferation assay. Treatment with LOX (E1,E2) and PTX3 siRNA (F1,F2) significantly reduced cell proliferation ability relative to the control siRNA and untreated cells. ** p < 0.01 and * p < 0.05. The error bars indicate standard deviation.

Figure 5

Cell migration assay. Migration abilities of mutant cell lines (HMOsisEC10 KRAS and HMOsisEC10 PIK3CA) are measured using a scratch wound healing assay after knockdown with LOX (A1,A2) and PTX3 siRNA (B1,B2). The numbers of migrated cells were significantly lower in siRNA treated cells compared to the control siRNA and untreated cells. Matrigel Invasion assay. siRNA knockdown of LOX (C1,C2) and PTX3 (D1,D2) showed a significantly lower invasion capacity than control siRNA and untreated cells. Cell proliferation assay. Treatment with LOX (E1,E2) and PTX3 siRNA (F1,F2) significantly reduced cell proliferation ability relative to the control siRNA and untreated cells. ** p < 0.01 and * p < 0.05. The error bars indicate standard deviation.