Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway.

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110alpha) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110alpha. The expression of p110alpha siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110alpha siRNA induced CDK inhibitor p27(KIP1) levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110alpha siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110alpha and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27(KIP1) levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.

[1]  Mario Mellado,et al.  Role of the Pi3k Regulatory Subunit in the Control of Actin Organization and Cell Migration , 2000, The Journal of cell biology.

[2]  M. Crouch,et al.  Cellular function of p70S6K: A role in regulating cell motility , 2000, Immunology and cell biology.

[3]  M. Waterfield,et al.  Signaling by distinct classes of phosphoinositide 3-kinases. , 1999, Experimental cell research.

[4]  A. Okamoto,et al.  Allelic imbalance and mutations of the PTEN gene in ovarian cancer , 2000, International journal of cancer.

[5]  M. Crouch,et al.  Nitric oxide induces polarization of actin in encephalitogenic T cells and inhibits their in vitro transendothelial migration in a p70S6 kinase‐independent manner , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  D. Roymans,et al.  Phosphatidylinositol 3-kinases in tumor progression. , 2001, European journal of biochemistry.

[7]  A. Toker,et al.  Signalling through the lipid products of phosphoinositide-3-OH kinase , 1997, Nature.

[8]  Taylor Murray,et al.  Cancer Statistics, 2001 , 2001, CA: a cancer journal for clinicians.

[9]  G. Macino,et al.  Post-transcriptional gene silencing across kingdoms. , 2000, Current opinion in genetics & development.

[10]  D. Engelke,et al.  Effective expression of small interfering RNA in human cells , 2002, Nature Biotechnology.

[11]  J. Woodgett,et al.  Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. , 1998, The Biochemical journal.

[12]  J. Cheng,et al.  Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas , 1995, International journal of cancer.

[13]  J. Slingerland,et al.  The prognostic significance of altered cyclin-dependent kinase inhibitors in human cancer. , 1999, Annual review of medicine.

[14]  Andrew K Godwin,et al.  AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth , 2004, Oncogene.

[15]  G. Mills,et al.  Inhibition of phosphatidylinositol 3'-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. , 2002, Cancer research.

[16]  R. Pearson,et al.  Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k , 1997, The EMBO journal.

[17]  S. Rudikoff,et al.  Insulin-like growth factor I induces migration and invasion of human multiple myeloma cells. , 2004, Blood.

[18]  D. Lauffenburger,et al.  Cell Migration: A Physically Integrated Molecular Process , 1996, Cell.

[19]  U. Puistola,et al.  Ovarian cancer cell invasion is inhibited by paclitaxel , 1997, Clinical & Experimental Metastasis.

[20]  G. Mills,et al.  In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[21]  Jiahuai Han,et al.  Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP kinase. , 2000, Cellular signalling.

[22]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[23]  R. Ozols,et al.  Ovarian cancer biology. , 1991, Seminars in oncology.

[24]  M. Andjelkovic,et al.  Phosphorylation and activation of p70s6k by PDK1. , 1998, Science.

[25]  D. Tindall,et al.  Mutation analysis of the putative tumor suppressor PTEN/MMAC1 in human ovarian cancer. , 1998, International journal of oncology.

[26]  Amy A. Caudy,et al.  Post-transcriptional gene silencing by double-stranded RNA , 2001, Nature Reviews Genetics.

[27]  J. Hauser,et al.  Autocrine Transforming Growth Factor α Regulates Cell Adhesion by Multiple Signaling via Specific Phosphorylation Sites of p70S6 Kinase in Colon Cancer Cells* , 2004, Journal of Biological Chemistry.

[28]  J. Ptak,et al.  High Frequency of Mutations of the PIK3CA Gene in Human Cancers , 2004, Science.

[29]  C. Sawyers,et al.  The phosphatidylinositol 3-Kinase–AKT pathway in human cancer , 2002, Nature Reviews Cancer.

[30]  K. Taira,et al.  U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells , 2002, Nature Biotechnology.

[31]  Joe W. Gray,et al.  PIK3CA is implicated as an oncogene in ovarian cancer , 1999, Nature Genetics.

[32]  J. Blenis,et al.  Nuclear localization and regulation of erk- and rsk-encoded protein kinases , 1992, Molecular and cellular biology.

[33]  Yan Shi,et al.  Rapamycin inhibits Akt-mediated oncogenic transformation and tumor growth. , 2004, Anticancer research.

[34]  J E Paciga,et al.  AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. , 2001, The American journal of pathology.

[35]  J. Blenis,et al.  The p70S6K signalling pathway: a novel signalling system involved in growth regulation. , 1996, Cancer surveys.

[36]  G. Mills,et al.  MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines. , 1997, Cancer research.