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Comparative Study
. 2015 Sep 22;6(28):25520-32.
doi: 10.18632/oncotarget.4550.

The PI3K/AKT/mTOR pathway is a potential predictor of distinct invasive and migratory capacities in human ovarian cancer cell lines

Huimin Bai  1   2 , Haixia Li  1 , Weihua Li  1 , Ting Gui  1 , Jiaxin Yang  1 , Dongyan Cao  1 , Keng Shen  1
Affiliations
Comparative Study

The PI3K/AKT/mTOR pathway is a potential predictor of distinct invasive and migratory capacities in human ovarian cancer cell lines

Huimin Bai et al. Oncotarget. .

Abstract

Objectives: 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. VSports手机版.

Methods: 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. V体育安卓版.

Results: 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 V体育ios版. 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. .

Conclusions: Heterogeneous cell subpopulations exhibiting distinct invasive and migratory capacities co-exist within the SKOV3 and A2780 cell lines VSports最新版本. 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. .

Keywords: RNA-Seq; cell subclones; intratumoral heterogeneity; invasion and migration; ovarian cancer. V体育平台登录.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1. Isolation and establishment of subclones exhibiting distinct invasive/migratory capacity
A–B. Isolation and amplification of single-cell subclones derived from human epithelial ovarian cancer cell lines A2780 (A) and SKOV3 (B) using limited dilution methodology. Single adherent A2780/SKOV3 cells were observed as early as 4–5 h after isolation and inoculation (a). Single cells began to divide, and 2–3 cells were observed in some wells of the plates 24 h after inoculation (b). The cell number continued to increase 4 days after inoculation (c). These single-cell subclones significantly differed in cell number of and subclone size ten days after inoculation (d1and d2). C. In the Matrigel invasive/migratory assay, subclone A6 (A-H) demonstrated the highest invasive/migratory activity, and subclone A41 (A-L) displayed the weakest ability to invade and migrate through the membrane (P < 0.001 and = 0.001, respectively). The invasive/migratory abilities of A-H and A-L both were significantly different from those of their parent cell line (A-H vs. A2780: P = 0.001 and 0.002, respectively; A-L vs. A2780: P = 0.044 and 0.001, respectively). Similarly, S15 (S-H) and S4 (S-L) exhibited the most distinct invasive/migratory abilities (P = 0.001 and < 0.001, respectively) among all the SKOV3 subclones. The invasive/migratory capacities of both S-H and S-L were significantly different from those of their parent cell line (S-H vs. SKOV3: P = 0.019 and 0.018, respectively; S-H vs. SKOV3: P = 0.037 and 0.018, respectively). D. The respective invasive/migratory capacities of A-H, A-L, S-H, and S-L were not significantly altered by serial passaging to approximately the thirtieth generation. After passaging to approximately 30 generations, the difference in the invasive/migratory capacities between A-H/S-H and A-L/S-L remained significant (invasion: P = 0.015 and < 0.001, respectively; migration: P < 0.001 and < 0.001, respectively).
Figure 2
Figure 2. Distinct proliferative activities of A-H/A-L and S-H/S-L
A–B. The MTT assay growth curves showed that A-H/S-H cells proliferated faster than A-L/S-L cells, based on a clear reduction in doubling time (P = 0.004 and 0.001, respectively). C. Cell cycle analysis indicated that the A-H/S-H subclones displayed a significantly increased in the percentage of cells in S phase (P = 0.004 and 0.022, respectively) and a concomitant decreased in the percentage of cells in G0/G1 phase (P = 0.012 and 0.007, respectively), suggesting that enhanced proliferation of the A-H/S-H cells might be due to the activation of DNA synthesis.
Figure 3
Figure 3. Heterogeneous biological activities of A-H/A-L and S-H/S-L
A–B. The apoptotic rates of A-H/S-H cell were significantly lower than those of A-L/S-L cells (P = 0.002 and 0.026, respectively; A). At both the mRNA (a) and protein levels (b), caspase-3 and caspase-7 expression was reduced, but Bcl-2 expression was increased in A-H/S-H cells (B) Additionally, the TrkB and Beclin1 levels were consistently high and low, respectively, in both A-H and S-H cells. Compared with their corresponding counterparts, the A-H and S-H cells exhibited significantly greater proliferative, anti-apoptotic and anti-anoikis activities. Autophagic activity was suppressed in the A-H and S-H subclones. C. Cell viability after cisplatin or taxol treatment was significantly inhibited in A-L/S-L cells compared with A-H/S-H cells, as demonstrated by drug cytotoxicity assays. The sublones showing higher invasive/migratory capabilities (A-H/S-H) displayed markedly higher cisplatin IC50 values (P < 0.001 and < 0.0001, respectively). Similarly, the respective taxol IC50 values of A-H and S-H cells respectively were significantly higher than those of A-L cells and S-L cells (P = 0.002 and 0.002, respectively). The sublones exhibiting higher invasive/migratory capacities were more resistant to chemotherapy. D. Effect of heterogeneous invasive/migratory capacities for tumor formation in vivo. (a): After subcutaneous injection, A-H/S-H tumors were observed much earlier than A-L/S-L tumors (5th day vs. 19th day; 7th day vs. 20th day). (b-d): On the study end date (30 days after inoculation), the tumor formation rates of both the A-H/S-H cells were 100%, and these were significantly higher than those of the A-L and S-L cells, respectively (both 20%, P = 0.008) (b). Additionally, the A-H/S-H tumors were significantly larger than the A-L/S-L tumors (A: 2.34 ± 0.23 cm vs. 0.17 ± 0.33 cm, P < 0.001; S: 2.04 ± 0.61cm vs. 0.12 ± 0.27cm, P < 0.001). The A-H/S-H cells exhibited higher capacities for in vivo tumor formation.
Figure 4
Figure 4. Transcriptome expression profiling of the A-H, A-L, S-H and S-L subclones
A. RNA-Seq was conducted on the A-H, A-L, S-H and S-L cells. In total, 612 genes were significantly differentially expressed in A-H cells compared with A-L cells, and 594 genes were differentially expressed genes were identified in S-H cells compared with S-L cells (P < 0.05 and fold change > 2.0). B. The PI3K pathway plays a pivotal role in many human malignancies [34, 35]. The extracellular matrix (ECM) activates focal adhesion kinases (FAKs), and the PI3K pathway can be activated by FAKs [37]. High ECM1 expression has been correlated with poor prognosis and metastatic potential in human carcinomas [38, 39]. The most studied type I PI3Ks include the α, β, γ and δ subtypes; mutations or abnormal expression of PI3Kα(PIK3CA) are the most commonly reported in cancer [41, 42]. PI3Kγ(PIK3CD) is activated by only stimulated G-protein coupled receptor (GPCR) [43]. The survival mechanism initiated via PI3K is primarily activated through AKT. Increased AKT activation exerts anti-apoptotic effect, antagonizes cell cycle arrest, modulates angiogenesis, and activates mRNA translation via mTOR signaling [50]. P-TEN inhibits PI3K pathway activity [51, 52]. Under the regulation of AKT, Nur77 induces a conformational change in Bcl-2 and promotes apoptosis [54]. Additionally, PKC is located in downstream of mTOR. The β isoform of PKC (PRKCB) might play significant roles in promoting human breast cancers growth [55, 56].
Figure 5
Figure 5. The PI3K/AKT/mTOR pathway is a potential predictor of distinct invasive and migratory capabilities in human ovarian cancer cell lines
RT-PCR A. and Western blot analyses B. consistently confirmed that PIK3CA, PIK3CD, AKT3, ECM1, GPCR, mTOR and PRKCB expression was increased but that Nur77 and PTEN expression was decreased in A-H/S-H cells compared with A-L/S-L cells. PI3K/AKT/mTOR pathway Activation was associated with enhanced invasive and migratory capacities in human ovarian cancer cell lines.
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