[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-specific phospholipase Cγ1 (PLCγ1) is activated downstream of many receptor tyrosine kinases to promote cell motility. Inhibition of this protein is being explored as a therapeutic strategy for blocking cancer cell invasion and metastasis. The clinical development of such cytostatic therapies requires the implementation of pharmacodynamic biomarkers of target modulation. In this study, we use magnetic resonance spectroscopy to explore metabolic biomarkers of PLCγ1 down-regulation in PC3LN3 prostate cancer cells. We show that inhibition of PLCγ1 via an inducible short hairpin RNA system causes a reduction in phosphocholine levels by up to 50% relative to the control as detected by (1)H and (31)P magnetic resonance spectroscopy analyses. This correlated with a rounded-up morphology and reduced cell migration. Interestingly, the fall in phosphocholine levels was not recorded in cells with constitutive PLCγ1 knockdown where the rounded-up phenotype was no longer apparent. This study reveals alterations in metabolism that accompany the cellular effects of PLCγ1 knockdown and highlights phosphocholine as a potential pharmacodynamic biomarker for monitoring the action of inhibitors targeting PLCγ1 signaling.
Molecular Cancer Therapeutics 06/2009; 8(5):1305-11. DOI:10.1158/1535-7163.MCT-09-0039 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.
[Show abstract][Hide abstract] ABSTRACT: Phospholipase Cgamma1 (PLCgamma1) is activated downstream of a variety of extracellular stimuli and has previously been implicated in the regulation of motility responses central to tumour cell invasion. In this study, we used a novel RNAi vector system to achieve conditional PLCgamma1 knockdown in PC3LN3 human prostate carcinoma cells for further evaluation of PLCgamma1 in tumour cell biology. Using this approach, we revealed a role for PLCgamma1 in the regulation of PC3LN3 cell adhesion that appears to be independent of its effects on tumour cell chemotactic migration and spreading in response to extracellular matrix. Subsequent microarray analysis of PLCgamma1-knockdown cells revealed Rap GEF1 mRNA to be decreased in response to PLCgamma1 loss. This translated into a decrease in Rap GEF1 protein levels and a significant loss of Rap1 activity in PLCgamma1-knockdown cells. Transient knockdown of Rap GEF1 caused a reduction in PC3LN3 adhesion while overexpression of Rap GEF1 rescued the PLCgamma1 knockdown-induced adhesion defect. These data highlight control of the Rap GEF1-Rap1 molecular switch as a specific requirement for PLCgamma1-mediated tumour cell adhesion.
[Show abstract][Hide abstract] ABSTRACT: Cell motility is a critical event in many processes and is underlined by complex signalling interactions. Although many components have been implicated in different forms of cell migration, identification of early key mediators of these events has proved difficult. One potential signalling intermediate, PLCgamma1, has previously been implicated in growth-factor-mediated chemotaxis but its position and roles in more-complex motility events remain poorly understood. This study links PLCgamma1 to early, integrin-regulated changes leading to cell motility. The key role of PLCgamma1 was supported by findings that specific depletion of PLCgamma1 by small interfering (si)RNA, or by pharmacological inhibition, or the absence of this isoform in PLCgamma1(-/-) cells resulted in the failure to form cell protrusions and undergo cell spreading and elongation in response to integrin engagement. This integrin-PLCgamma1 pathway was shown to underlie motility processes involved in morphogenesis of endothelial cells on basement membranes and invasion of cancer cells into such three-dimensional matrices. By combining cellular and biochemical approaches, we have further characterized this signalling pathway. Upstream of PLCgamma1 activity, beta1 integrin and Src kinase are demonstrated to be essential for phosphorylation of PLCgamma1, formation of protein complexes and accumulation of intracellular calcium. Cancer cell invasion and the early morphological changes associated with cell motility were abolished by inhibition of beta1 integrin or Src. Our findings establish PLCgamma1 as a key player in integrin-mediated cell motility processes and identify other critical components of the signalling pathway involved in establishing a motile phenotype. This suggests a more general role for PLCgamma1 in cell motility, functioning as a mediator of both growth factor and integrin-initiated signals.
[Show abstract][Hide abstract] ABSTRACT: Tumor invasion and metastasis are the hallmarks of advanced stage cancer and are associated with poor patient prognosis. EGFR
is overexpressed in a variety of tumor types and this frequently correlates with a more aggressive tumor phenotype. In this
chapter, we discuss the cellular and molecular mechanisms by which EGFR contributes to tumor progression and present evidence
from experimental and clinical observations that reinforce the notion that EGFR actively contributes to the onset of metastatic
disease. EGFR plays a key role in the regulation of processes central to tumor invasion including cell adhesion and motility
through its interactions with molecules such as integrins, cadherins, phospholipase Cγ1 and phosphoinositide 3-kinase. In
addition, EGFR signaling can contribute to both proteolysis and angiogenesis through up-regulated expression of matrix metalloproteinases
(MMPs) and angiogenic cytokines e.g. VEGF-A and IL-8. The significance of these contributions to tumor invasion and metastasis is highlighted by the fact that
a mutant, constitutively active receptor (EGFRvIII) associated with human cancers can induce these behaviors when transfected
into fibroblasts. Finally, we discuss the use of EGFR antagonists to stem metastatic disease and their potential, in combination
with additional novel agents, to improve treatment for cancer patients.