Abstract
To explore the genetic and molecular events that control subclones exhibiting distinct invasive/migratory capacities derived from human epithelial ovarian cancer (EOC) cell line A2780 and SKOV3. Single-cell subclones were isolated and established that were derived from the SKOV3 and A2780 cell lines through limiting dilution methodology. Transwell insert assays and MTT assays were performed to screen and identify the subclones exhibiting the highest and the lowest invasive/migratory capacities, and the selected subclones were renamed as A-H (A2780 high), A-L (A2780 low), S-H (SKOV3 high), and S-L (SKOV3 low). Their biological characteristics were evaluated. RNA-Seq was conducted on the targeted subclones. Compared with their corresponding counterparts, A-H/S-H cells exhibited significantly higher invasive/migratory capacities (P < 0.001 and = 0.001, respectively). A-H/S-H cells displayed a clear reduction in doubling time (P = 0.004 and 0.001, respectively), and a significant increase in the percentage of cells in S phase (P = 0.004 and 0.022, respectively). Additionally, the apoptotic rates of A-H/S-H cells were significantly lower than those of A-L/S-L cells (P = 0.002 and 0.026, respectively). At both mRNA and protein levels, caspase-3 and caspase-7 expression were reduced but Bcl-2 expression was increased in A-H/S-H cells. The TrkB (anoikis-related) and Beclin1 (autophagy-related) levels were consistently high and low, respectively, in both A-H/S-H cells. Resistance to chemotherapy in vitro and higher capacities on tumor formation in vivo was presented in both A-H/S-H cells. PI3K/AKT/mTOR pathway components, PIK3CA, PIK3CD, AKT3, ECM1, GPCR, mTOR and PRKCB were increased but that the Nur77 and PTEN were decreased in A-H/S-H cells, identified by RNA-Seq and consistently confirmed by RT-PCR and Western blot analyses. Heterogeneous cell subpopulations exhibiting distinct invasive and migratory capacities co-exist within the SKOV3 and A2780 cell lines. PI3K/AKT/mTOR pathway activation is associated with higher invasive and migratory capacities in subpopulations of human ovarian cancer cell lines. Inhibiting this pathway may be useful for the chemoprevention or treatment of EOC.
Highlights
Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy [1]
Heterogeneity is a common phenomenon of parental tumor cell lines, and highly metastatic tumor cell variants pre-exist in parental tumor cell populations [7,8,9]
We isolated and established two pairs of cell subclones exhibiting distinct invasive/migratory capacities that were derived from the SKOV3 and A2780 cell lines
Summary
Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy [1]. Intratumoral heterogeneity (ITH) has been attributed to treatment failure in many human malignancies [2,3,4]. A2780 cells, which were derived from an adenocarcinoma patient prior to treatment, are sensitive to cisplatin [11]. Both of these two cell lines are popular research models for human EOC. We isolated and established two pairs of cell subclones exhibiting distinct invasive/migratory capacities that were derived from the SKOV3 and A2780 cell lines. The present study aimed to investigate the disparities in gene expression between the targeted subclones using RNA-Seq. Determining the genetic and molecular events that control the distinct invasive/migratory capacities of these subclones will facilitate our understanding of EOC treatment failure and will lead to the development of more effective therapeutics
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