Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

<div><p>Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 (<em>P</em><0.0001), ERK phosphorylation (<em>P</em><0.0001) and FOXM1 (<em>P</em> = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (<em>P</em><0.0001), ERK phosphorylation (<em>P</em><0.001) and FOXM1 (<em>P</em><0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth <em>in vitro</em> and <em>in vivo</em>. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.</p> </div

Inhibition of ERK phosphorylation or FOXM1 expression reduced tumor growth in a mouse xenograft model.

<p>(A) The GRB7 stably expressing A2780cp cells (Acp-GRB7) were subcutaneously injected into the right flank of nude mice. Mice were divided into 5 groups (5 mice per group) and treated with either DMSO as a control, or U0126 (25 or 50 µM/kg) or Thiostrepton (200 or 300 µM/kg) for every 3-day since on day 6 (Arrows represent the injections). The relative tumor size was calculated relative to those of the first day of treatment (day 0) and are represented as relative mean size (%)±SE for each group (*<i>P</i> = 0. 032, **<i>P</i><0.01, and ***<i>P</i> = 0. 005, are significantly different from the DMSO control group, Student <i>t</i>-test). (B) The representative pictures and bar charts show the average tumor weight of each group taken on day 18. (*<i>P</i> = 0. 043, **<i>P</i> = 0. 001, and ***<i>P</i><0.02, are significantly different from the DMSO control group, Student <i>t</i>-test).</p

A significant stepwise increase in the expressions of GRB7, ERK phosphorylation and FOXM1 along ovarian tumor grade.

<p>(A) The expressions of GRB7, ERK phosphorylation and FOXM1 were evaluated by immunohistochemical analysis using specific antibodies on an ovarian cancer tissue array (OVC1021, Pantomics). (B) Representative pictures show the stepwise increase of GRB7, ERK phosphorylation and FOXM1 expressions from Grade 1 to Grade 3 ovarian cancer (serous subtype) (200× magnifications).</p

Inhibition of either ERK phosphorylation or FOXM1 expression impaired cell growth in ovarian cancer cells.

<p>(A) XTT cell proliferation assays showed that the inhibition of ERK phosphorylation by U0126 (10 µM) significantly abrogated the cell proliferation rate in GRB7 stably expressing OVCA433 cells (<i>P</i> = 0.020, Student <i>t</i>-test) and A2780cp cells (<i>P</i> = 0.030, Student <i>t</i>-test) as compared with their vector controls. (B) XTT cell proliferation assays showed that the suppression of FOXM1 expression by Thiostrepton (20 µM) significantly reduced the cell proliferation rate in GRB7 stably expressing OVCA433 cells (<i>P</i> = 0.015, Student <i>t</i>-test) and A2780cp cells (<i>P</i> = 0.025, Student <i>t</i>-test) as compared with their vector controls.</p

Inhibition of ERK phosphorylation or FOXM1 significantly decreased both migration and invasion of GRB7-overexpressing ovarian cancer cells.

<p>OVCA433 cells with stable expression of GFP/GRB7 (OVCA433-GRB7) were treated with DMSO as control, Thiostrepton (20 µM), PD98059 (20 µM) and U0126 (10 µM) for 6 hours and were analyzed by (A) Transwell cell migration assay. The representative pictures and bar chart showed significant reduction in the number of migratory cells through Matrigel-coated membrane in OVCA433-GRB7 cells treated with Thiostrepton, PD98059 and U0126 than DMSO control (*<i>P</i><0.02, Student <i>t</i>-test) at 8-hour; (B) Transwell cell invasion assay. The representative pictures and bar chart showed significant reduction in the invasion rate in OVCA433-GRB7 cells treated with Thiostrepton, PD98059 and U0126 when compared with DMSO control (*<i>P</i><0.05, Student <i>t</i>-test) at 15-hour.</p

Clinico-pathological analysis of GRB7, ERK phosphorylation and FOXM1 expressions in ovarian cancer tissue array (OVC1021).

<p>Clinico-pathological analysis of GRB7, ERK phosphorylation and FOXM1 expressions in ovarian cancer tissue array (OVC1021).</p

GRB7/ERK/FOXM1 was regulated in the same signaling axis.

<p>(A) Treatment with U0126 (5 µM) showed a significant reduction in the expression of ERK phosphorylation accompanied with FOXM1 in ovarian cancer cells time dependently, whereas no change in GRB7 expression was found in A2780cp cells. (B) Treatment of Thiostrepton (20 µM) remarkably reduced the expression of FOXM1 only but no change in the expressions of GRB7 and ERK phosphorylation in A2780cp cells (<i>left</i>). Depletion of FOXM1 by siRNA knockdown did not alter the expression of GRB7 and ERK phosphorylation (<i>right</i>). C, siRNA scrambled control. si1, si2 and si3 siRNAs targeting three different regions of human FOXM1 and knockdown the expression of FOXM1 by 60%, 45% and 70% respectively. (C) Two out of four GRB7 shRNA constructs (sh1 and sh2) showed ∼70% knockdown of GRB7 accompanied with a reduction of ERK phosphorylation and FOXM1 expressions in OVCA433 cells. The scrambled control (NC) was used as negative control. (D) Enforced expression of GRB7 increased ERK phosphorylation and FOXM1. However, treatment with either U0126 (10 µM) or PD98059 (20 µM) could suppress the induced ERK phosphorylation and FOXM1 in A2780cp and OVCA433 ovarian cancer cells.</p