Cell Invasion of Highly Metastatic MTLn3 Cancer Cells Is Dependent on Phospholipase D2 (PLD2) and Janus Kinase 3 (JAK3)

doi:10.1016/j.jmb.2011.03.017

Journal of Molecular Biology

Volume 408, Issue 5, 20 May 2011, Pages 850-862

https://doi.org/10.1016/j.jmb.2011.03.017 Get rights and content

Abstract

MTLn3 cells are highly invasive breast adenoacarcinoma cells. The relative level of the epidermal-growth-factor-stimulated invasion of this cell line is greater than two other breast cancer cell lines (MDA-MB-231 and MCF-7) and one non-small cell lung cancer cell line (H1299). We have determined that the mechanism of cancer cell invasion involves the presence of an enzymatically active phospholipase D (PLD), with the PLD2 isoform being more relevant than PLD1. PLD2 silencing abrogated invasion, whereas ectopic expression of PLD2 augmented cell invasion in all four cell lines, with an efficacy (MTLn3 ± MDA-MB-231 > H1299 ± MCF-7) that correlated well with their abilities to invade Matrigel in vitro. We also report that PLD2 is under the control of Janus kinase 3 (JAK3), with the kinase phosphorylating PLD2 at the Y415 residue, thus enabling its activation. Y415 is located downstream of a PH domain and upstream of the catalytic HKD-1 domain of PLD2. JAK3 knockdown abrogated lipase activity and epidermal-growth-factor-stimulated cell invasion directly. For the purposes of activating PLD2 for cell invasion, JAK3 operates via an alternative pathway that is independent of STAT, at least in MTLn3 cells. We also consistently found that JAK3 and PLD2 pathways are utilized at the maximum efficiency (phosphorylation and activity) in highly invasive MTLn3 cells versus a relatively low utilization in the less invasive MCF-7 cell line. In summary, a high level of cell invasiveness of cancer cells can be explained for the first time by combined high JAK3/PLD2 phosphorylation and activity involving PLD2's Y415 residue, which might constitute a novel target to inhibit cancer cell invasion.

Introduction

Invasion through basement membranes is the first step leading to cancer metastasis. When carcinoma cells reach the stromal compartment, they begin to intravasate in capillaries and lymphatic vessels and transfer to other sites where they can colonize new tissues.1, 2 Invasion is a form of cell migration that enables malignant cells to penetrate into neighboring tissues and to degrade the extracellular matrix with proteases MMP-1, MMP-2, and MMP-9. Cancer cell invasion is thus critical in the overall multistep phenomena of tumor metastasis; however, in spite of this, a clear understanding of the signal pathways relevant to invasiveness is still lacking. Our laboratory and those of others have demonstrated that phospholipase D (PLD) is a key element needed for the cell migration of leukocytes.3, 4, 5, 6, 7, 8 For the present study, we hypothesize that, in a similar fashion, cancer cell invasiveness could be dependent on the presence of PLD at the time of metastatic initiation. For this, we have utilized a panel of breast cancer cells that are responsive to epidermal growth factor (EGF) as chemoattractant, including MTLn3 cells (a highly metastatic rat mammary adenocarcinoma cell line)⁴ and human MDA-MB-231, H1299, and MCF-7 cells. MDA-MB-231 cells possess the largest number of EGF receptors per cell, calculated to be ∼ 700,000, followed in descending order by MTLn3 cells, H1299 cells, and MCF-7 cells, which have the fewest EGF receptors per cell estimated at ∼ 3000–6000 per cell.9, 10, 11, 12

PLD is an enzyme that breaks down phosphatidylcholine to phosphatidic acid (PA) and choline. These second messengers, PA and choline, are involved in many cellular functions ranging from cytoskeletal rearrangement to phagocytosis, vesicle trafficking, exocytosis,13, 14 and, importantly, cell migration.¹⁵ PLD has been implicated in cell proliferation and cancer.16, 17, 18 PLD has two common isoforms: PLD1 and PLD2. PLD1 is a 1072-amino-acid 120-kDa protein that is localized to the long arm (q) of chromosome 3 (3q26) and is found in perinuclear, Golgi, and heavy membrane fractions.16, 19 The PLD1 location (3q26) is a very common chromosomal alteration that is found in many cancers, including ovarian, lung, prostate, esophageal, nasopharyngeal, and breast cancers.20, 21, 22, 23, 24 PLD2 is a 933-amino-acid 106-kDa protein that is located on the short arm (p) of chromosome 17 (17p13) and is predominantly found in the plasma membrane.²⁵ Additionally, the PLD2 location (17p13) is situated in an area of chromosome 17 that, besides being involved in brain and eye development and in somatic growth, is also found to be frequently altered in breast cancers and contributes to high S-phase, higher aggressive tumor phenotype, and possibly metastasis.26, 27, 28

In the present article, we have investigated cell invasion and defined a new mechanism that explains the highly invasive potential of adenocarcinoma cells. It is shown for the first time that Janus kinase 3 (JAK3)-specific lipase phosphorylation of PLD2 directly mediates MTLn3 cell invasion, which implicates a role for JAK3 in EGF-mediated processes and cancer.

Section snippets

Cell invasion of MTLn3 and MCF-7 cells is negatively affected by PLD2 silencing

MTLn3 and MDA-MB-231 cancer cells are two highly invasive breast adenocarcinoma cell lines. Figure 1a represents hematoxylin-stained MTLn3 cells that invade through the Matrigel medium in response to a chemoattractant and are shown as an example of the morphology of the invading cell. In the Matrigel setting, we have determined the relative levels of the EGF-mediated cell invasion of three breast adenocarcinoma cell lines and one non-small cell lung cancer cell line in response to 3 nM EGF (

Discussion

Our laboratory and those of others have reported that PLD is a key element needed for the cell migration of leukocytes.3, 4, 5, 6, 7, 8 In the present study, we have demonstrated that cancer cell invasiveness is dependent on the presence of PLD (particularly the PLD2 isoform) at the time of metastatic initiation. For this, we have utilized a panel of breast cancer cells, including MTLn3 cells (a highly metastatic rat mammary adenocarcinoma cell line)⁴ and human MDA-MB-231 and MCF-7 cells, as

Reagents

Dulbecco's modified Eagle's medium (DMEM) was obtained from Cellgro. α-MEM, Lipofectamine, Plus reagent and Lipofectamine 2000 were obtained from Invitrogen (polyethylene terephthalate). Matrigels were obtained from BD Biosciences. Hematoxylin was obtained from Ricca Chemical Co. Purified recombinant human JAK3 and JAK3tide synthetic peptide substrate were obtained from Millipore. [³H]butanol was obtained from American Radiolabeled Chemicals. [³²P]γ-ATP was obtained from Perkin-Elmer. ECL

Acknowledgements

We thank Jennalee Post and Stephen Short for technical assistance, and Erin Lintz for her expertise with the immunofluorescence microscopy of MTLn3 cells. This work was supported by grant HL056653 (J.G.-C.) from the National Institutes of Health and by Research Challenge Grant 667131 from Wright State University.

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Phospholipase D is a key enzyme, which possesses domains for responding to extracellular growth factors and docking with receptor tyrosine kinases, G-protein coupled receptors, and adaptors, which transduce the signaling of these molecules. PLD hydrolyzes phosphatidyl choline to form its mutagenic lipid mediator, phosphatidic acid, and also releases choline. PLD activation leads to pleiotropic signals, which are instrumental in the maintenance of normal cellular physiology and cellular structural integrity. Dysregulated PLD signaling in response to stress and pro-oncogenic hormonal messengers leads to cancer cell proliferation and survival. This chapter attempts to collate the structure-function aspects of this key and relatively underexplored protein and promote new research into the chemistry of this enzyme and the signal transduction pathways controlled by it. Moreover, we encapsulate the structure-function aspects of PLD inhibitors in conventional use, which are either in the drug discovery pipeline, or on the market. Another facet explored herein is the availability of plant metabolites, which are known to possess PLD-modulating functions. New perspectives on or insights into PLD action can lead to discovery of better therapeutic strategies to combat diseases such as cancer, heart disease, and neurological disorders, where PLD dysregulation has been implicated.
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Phospholipase D (PLD) enzymes belong to a superfamily of phospholipases that are indispensable to intracellular and extracellular signaling. Human PLD enzymes have emerged as drug targets for various diseases such as cardiovascular disease, cancer, infectious diseases, and neurodegenerative conditions such as Alzheimer's and Parkinson’s. Growing links of dysregulation of PLD to human disease have prompted interest in therapeutics that target its function. Thus, it is vital to understand how the signaling network of PLD expression is regulated and contributes to the progression of these diseases. In this review, we have attempted to summarize the recent scientific developments regarding the role of PLD in cancer progression and therapeutics.
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It has been known for some decades that the expression of phospholipase D (PLD) isoforms becomes altered in the transition from normal mammary epithelium to breast carcinoma. The products of PLD activity, choline and phosphatidic, have been implicated in the transformation process, and PLD itself has a scaffold function in the assembly of signaling complexes. PLD expression is also responsive to environmental stressors, and this has raised questions about its role in the adaptive response of malignant cells under conditions of hypoxia and nutrient depletion in the developing tumor. PLD is involved in epithelial mesenchymal transition and is particularly important in mTOR and ERBB1/EGFR and ERBB2/HER2 coupled signaling process. PLD has not only been implicated in cell survival signaling but also in the process of metastasis and critically in changes in the signaling profile that develop as breast cancer cells become more invasive.
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In mammals, phospholipase D (PLD) enzymes involve 6 isoforms, of which only three have established lipase activity to produce the signaling lipid phosphatidic acid (PA). This phospholipase activity has been postulated to contribute to cancer progression for over three decades now, but the exact mechanisms involved have yet to be uncovered. Indeed, using various models, an altered PLD activity has been proposed altogether to increase cell survival rate, promote angiogenesis, boost rapamycin resistance, and favor metastasis. Although for some part, the molecular pathways by which this increase in PA is pro-oncogenic are partially known, the pleiotropic functions of PA make it quite difficult to distinguish which among these simple signaling pathways is responsible for each of these PLD facets. In this review, we will describe an additional potential contribution of PA generated by PLD1 and PLD2 in the biogenesis, secretion, and uptake of exosomes. Those extracellular vesicles are now viewed as membrane vehicles that carry informative molecules able to modify the fate of receiving cells at distance from the original tumor to favor homing of metastasis. The perspectives for a better understanding of these complex role of PLDs will be discussed.
The peculiarities of cancer cell metabolism: A route to metastasization and a target for therapy
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In breast and lung cancer, silencing PLD2 affected negatively the ability of invasiveness, which indicates some dependency of cell invasion on PLD2 [163]. Moreover, overexpression of PLD2 or PLD1 enhanced cell invasion in Matrigel [163,164]. Ablation of PLD1 in the tumour environment suppressed the activation of Akt and MAPK signalling pathways by VEGF, resulting in integrin-dependent cell adhesion, and migration on extracellular matrices attenuated the neovascularization and tumour progression [165].
The last decade has witnessed the peculiarities of metabolic reprogramming in tumour onset and progression, and their relevance in cancer therapy. Also, it has been indicated that the metastatic process may depend on the metabolic rewiring and adaptation of cancer cells to the pressure of tumour microenvironment and limiting nutrient availability. The present review gatherers the existent knowledge on the influence of tumour microenvironment and metabolic routes driving metastasis. A focus will be given to glycolysis, fatty acid metabolism, glutaminolysis, and amino acid handling. In addition, the role of metabolic waste driving metastasization will be explored. Finally, we discuss the status of cancer treatment approaches targeting metabolism. This knowledge revision will highlight the critical metabolic targets in metastasis and the chemicals already used in preclinical studies and clinical trials, providing clues that would be further exploited in medicinal chemistry research.
EZH2 phosphorylation by JAK3 mediates a switch to noncanonical function in natural killer/T-cell lymphoma
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Deregulation of the epigenetic machinery and gene regulation may therefore represent an additional key mechanism by which JAK3 contributes to cancer. Of importance is that JAK3 is aberrant in several hematologic malignancies and solid tumors, such as colon32,33 and breast carcinoma,33-35 and seems to be of functional relevance in these cancers. Because tumors in which JAK3 is activated overlaps with those with reduced H3K27me3 and EZH2 overexpression, the findings in our study could have broader utility and implications outside NKTL.
The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.

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Journal of Molecular Biology

Cell Invasion of Highly Metastatic MTLn3 Cancer Cells Is Dependent on Phospholipase D2 (PLD2) and Janus Kinase 3 (JAK3)

Abstract

Introduction

Section snippets

Cell invasion of MTLn3 and MCF-7 cells is negatively affected by PLD2 silencing

Discussion

Reagents

Acknowledgements

Curr. Opin. Genet. Dev.

Mol. Immunol.

Blood

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

FEBS Lett.

J. Biol. Chem.

Cancer Lett.

Cytogenet. Cell Genet.

Eur. J. Med. Genet.

Cytokine

J. Biol. Chem.

J. Biol. Chem.

Vitam. Horm.

J. Biol. Chem.

Cell. Signalling

Tumour-cell invasion and migration: diversity and escape mechanisms

Nat. Rev. Cancer

Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors

Cancer Res.

Inhibitory effects of phytopolyphenols on TPA-induced transformation, PKC activation, and c-jun expression in mouse fibroblast cells

Nutr. Cancer

Epidermal growth factor binding by breast tumor biopsies and relationship to estrogen receptor and progestin receptor levels

Cancer Res.

Ligand mediated activation of ectopic EGF receptor promotes matrix protein adhesion and lung colonization of rat mammary adenocarcinoma cells

Oncogene

Epidermal growth factor receptors and effect of epidermal growth factor on growth of human breast cancer cells in long-term tissue culture

Cancer Res.