p70 S6 kinase as a regulator of actin and adhesion dynamics in ovarian cancer

Abstract

Ovarian cancer is a highly metastatic disease having a poor prognosis (<25%). The

factors and underlying mechanisms that regulate ovarian cancer metastasis, however,

are still incompletely understood. p70 S6 kinase (p70S6K), a serine/threonine kinase, is

frequently activated in high-grade malignant human ovarian cancer. The aim of this

study is to investigate the molecular mechanisms by which p70S6K may promote

ovarian cancer metastasis. The results show that p70S6K is a critical regulator of the

actin cytoskeleton, peritoneal adhesion and dissemination, and multicellular

aggregates/spheroids formation in the acquisition of the metastatic phenotype. The

regulation of p70S6K on the actin cytoskeleton is through two important functions: as

an actin cross-linking protein and as a Rho family GTPase-activating protein. Ectopic

expression of constitutively active p70S6K induced a marked reorganization of the

actin cytoskeleton and directional migration of ovarian cancer cells. Actin binding and

immunofluorescence studies showed that p70S6K had a direct interaction with the actin

filaments with no other proteins involved. This interaction did not affect actin

polymerization kinetics but cross-linked the actin filaments to inhibit cofilin-induced

actin depolymerization. In addition, p70S6K mediated the activation of Rac1 and

Cdc42 GTPases and their downstream effector p21-activated kinase 1, but not RhoA.

Peritoneal adhesion and dissemination is regulated by p70S6K through integrin

expression. Expression of p70S6K siRNA efficiently inhibited ovarian cancer cell

adhesion to fibronectin and laminin among different peritoneal extracellular matrix

components, as well as to human primary peritoneal mesothelial cells. These effects

were associated with the expression of alpha5 and beta1 integrin. Studies into the

mechanisms suggest that p70S6K may upregulate alpha5 integrin by a transcriptional

mechanism whereas beta1 integrin is regulated at a post-transcriptional level.

Enhanced expression of alpha5 and beta1 integrin by active p70S6K mediated the

subsequent peritoneal adhesion. In ovarian cancer xenografts, p70S6K and beta1

integrin interference significantly inhibited peritoneal dissemination through

reduction in the number and weight of tumors. Multicellular spheroids are present in

the malignant ascites of ovarian cancer patients. Using a 3-dimensional culture system,

expression of p70S6K siRNA resulted in inhibition of multicellular spheroid formation,

which was mediated by N-cadherin but not E- or P-cadherin. In addition to spheroid

formation, inhibition of p70S6K was associated with reduced growth of spheroids and

disaggregation capabilities on different extracellular matrix components. Taken

together, these findings indicate that p70S6K plays an important role in the biology of

ovarian cancer metastasis through regulation of several critical steps in dissemination

and migration, suggesting that p70S6K could be explored as a potential therapeutic

target in ovarian cancer.