Elsevier

Biochemical Pharmacology

Volume 85, Issue 10, 15 May 2013, Pages 1454-1462
Biochemical Pharmacology

Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion

https://doi.org/10.1016/j.bcp.2013.03.001Get rights and content

Abstract

Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, β and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kβ. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis.

Introduction

Recent preclinical and clinical studies have demonstrated that specific G-protein coupled receptor (GPCR) systems are excessively activated in malignant breast cancer due to over-expression of receptors [1], [2], [3], [4], abnormally elevated levels of ligands for GPCRs [4], [5], [6] and/or down-regulation of their regulators [7], which contributes to the progression and spread of breast cancer [8]. For example, signaling initiated by CXC chemokine receptor 4 (CXCR4) and protease-activated receptors (PARs) on breast cancer cells drives cancer cells to migrate and invade through surrounding tissues and spread to distant organs [5], [9]. Unfortunately, clinical trials with drugs inhibiting specific GPCR activation show limited efficacy, presumably because metastasis could be driven by several different classes of GPCR simultaneously, thereby generating metastatic signal redundancy.

GPCRs convey signals via heterotrimeric G-proteins (classified into Gs, Gi, Gq, G12) in the form of activated Gα-GTP and Gβγ subunits. We recently demonstrated that Gβγ released from Gi-proteins promotes migration and invasion of metastatic breast cancer cells by generating the lamellipodia protrusions at the leading edge of migrating cancer cells [10], suggesting that blockade of Gβγ could attenuate breast cancer metastasis. However, Gβγ cannot be blocked indiscriminately because of its diverse physiological roles. Therefore, the challenge is to target Gβγ effectors that are vital to breast cancer metastasis but inconsequential for physiologically normal cells.

The most studied type I phosphatidylinositol 3-kinases (PI3Ks), including α, β, γ and δ, play a pivotal role in numerous cellular functions [11]. PI3Kγ is especially intriguing because it is normally expressed primarily in hematopoietic cells [12], which have a physiological need to migrate. In addition, PI3Kγ is only activated by Gβγ following stimulation of GPCRs, whereas PI3Kα, β and δ are stimulated by receptor tyrosine kinases [11], [13]. In fact, GPCR-dependent activation of PI3Kγ in neutrophils causes its accumulation at the leading edge of migrating cells, which is a critical determinant of cell migration [12], [14]. Although somatic mutations of PI3Kα are very common in cancers [15], [16], and may promote cancer cell growth and invasion in colorectal and breast cancer [17], [18], the contribution of PI3Kγ to human cancer is much less clear with different studies showing conflicting results [19], [20], [21], [22]. Brazzatti et al. [22] recently reported that knockdown of PI3Kγ inhibited lung colonization of human breast cancer MDA-MB-231 cells in xenografts and suppressed primary tumor growth, metastases and lung colonization caused by mouse 4T1.2 mammary carcinoma allografts. While this suggests an important role for PI3Kγ in breast cancer tumor growth and metastasis, these studies did not explore the molecular mechanisms associated with PI3Kγ signaling or whether PI3Kγ protein levels were correlated with the metastatic potential of various human breast cancer cell lines or human breast cancer specimens.

In the present study, we show that PI3Kγ is aberrantly expressed in invasive human breast carcinoma and its expression level correlates with the metastatic potential of established breast cancer cell lines. Most importantly, we show that silencing PI3Kγ with its siRNA or treatment with a PI3Kγ-selective inhibitor, but not with inhibitors or siRNAs of PI3Kα and β, attenuates lamellipodia formation and suppresses migration and invasion of breast cancer cells. Thus, targeting dysregulated PI3Kγ may provide a novel strategy for development of chemotherapeutic agents to suppress breast cancer metastasis.

Section snippets

Cell lines, reagents and plasmids

MCF-10A, MCF-7, T47D, MDA-MB-231 and MDA-MB-436 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). MDA-MB-231 and -436 cells were cultured in DMEM with 10% fetal bovine serum (FBS). MCF-7 cells were cultured in IMEM and 10% FBS with 10 μg/ml insulin. MCF-10A cells were grown in MEBM with additives (ATCC), and T47D cells were maintained in RPMI 1640 with 10%

PI3Kγ is upregulated in human breast tumors

We first performed immunohistochemistry staining for PI3Kγ protein in histologically benign and neoplastic cells of 40 archival human breast tissue blocks using a PI3Kγ specific antibody. PI3Kγ protein expression was elevated in ductal carcinoma in situ and invasive breast carcinoma when compared to adjacent benign mammary tissue (Fig. 1). Expression levels of PI3Kγ were graded from 0 to 3 based on overall staining intensity. Table 1 shows that average PI3Kγ staining intensities in ductal

Discussion

Although several lines of evidence indicate a role of PI3Kγ in pancreatic cancer and breast cancer [19], [20], [21], [22], our results provide the first detailed evidence that expression levels of PI3Kγ are correlated with the metastatic potential of established human breast cancer cell lines. In contrast, PI3Kα, β and δ were ubiquitously expressed in these breast cell lines. More importantly, our data showed that PI3Kγ protein expression was modestly increased in noninvasive ductal carcinoma

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We gratefully acknowledge Dr. Bernd Nürnberg from University of Tübingen for providing the pEYFP-PI3Kγ-CAAX plasmid. The authors thank Chuu-Yun A. Wong for technical support. This work was supported by the National Institutes of Health (CA125661 and P20-RR018759), Nebraska State LB595 and LB692 research program.

References (46)

  • M. Monterrubio et al.

    PI3Kgamma activation by CXCL12 regulates tumor cell adhesion and invasion

    Biochem Biophys Res Commun

    (2009)
  • C. Weiss-Haljiti et al.

    Involvement of phosphoinositide 3-kinase, Rac, and PAK signaling in chemokine-induced macrophage migration

    J Biol Chem

    (2004)
  • P.T. Hawkins et al.

    PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase

    Curr Biol

    (1995)
  • A. Müller et al.

    Involvement of chemokinereceptors in breast cancer metastasis

    Nature

    (2001)
  • P. Wülfing et al.

    Endothelin-1-, endothelin-A- and endothelin-B-receptor expression is correlated with vascular endothelial growth factor expression and angiogenesis in breast cancer

    Clin Cancer Res

    (2004)
  • R.P. Hoey et al.

    The parathyroid hormone- related protein receptor is expressed in breast cancer bone metastases and promotes autocrine proliferation in breast carcinoma cells

    Br J Cancer

    (2003)
  • S. Even-Ram et al.

    Thrombin receptor overexpression in malignant and physiological invasion processes

    Nat Med

    (1998)
  • T.A. Guise et al.

    Role of endothelin-1 in osteoblastic bone metastases

    Cancer

    (2003)
  • Y. Xie et al.

    Breast cancer migration and invasion depends on proteasome degradation of regulator of G protein signaling 4 (RGS4)

    Cancer Res

    (2009)
  • R.T. Dorsam et al.

    G-protein-coupled receptors and cancer

    Nat Rev Cancer

    (2007)
  • J.K. Kirui et al.

    Gβγ signaling promotes breast cancer cell migration and invasion

    J Pharmacol Exp Ther

    (2010)
  • T. Sasaki et al.

    Function of P13Kγ in thymocyte development, T cell activation, and neutrophil migration

    Science

    (2000)
  • G.J. Ferguson et al.

    PI3Kγ has an important context- dependent role in neutrophil chemokinesis

    Nat Cell Biol

    (2007)
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