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