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G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

ABSTRACT: Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

SUBMITTER: Billard MJ

PROVIDER: S-EPMC4822790 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

Billard Matthew J MJ Fitzhugh David J DJ Parker Joel S JS Brozowski Jaime M JM McGinnis Marcus W MW Timoshchenko Roman G RG Serafin D Stephen DS Lininger Ruth R Klauber-Demore Nancy N Sahagian Gary G Truong Young K YK Sassano Maria F MF Serody Jonathan S JS Tarrant Teresa K TK

PloS one 20160406 4

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a ne ...[more]

PMID: 27049755

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Project description:The biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising.Human tissue samples of invasive breast cancer and normal breast, as well as breast cancer cell lines, were evaluated for protein kinase D (PKD) expression, to test if altered expression could serve as a marker for invasive breast cancer. We further utilised specific PKD1-shRNA and a system to inducibly-express PKD1 to analyse the role of PKD1 in the invasive behaviour of breast cancer cell lines in two-dimensional (2D) and three-dimensional (3D) culture. Invasive behaviour in breast cancer cell lines has been linked to matrix metalloproteinases (MMPs), so we also determined if PKD1 regulates the expression and activity of these enzymes.We found that the serine/threonine kinase, PKD1, is highly expressed in ductal epithelial cells of normal human breast tissue, but is reduced in its expression in more than 95% of all analysed samples of human invasive breast tumours. Additionally, PKD1 is not expressed in highly invasive breast cancer cell lines, whereas non-invasive or very low-invasive breast cancer cell lines express PKD1. Our results further implicate that in MDA-MB-231 cells PKD1 expression is blocked by epigenetic silencing via DNA methylation. The re-expression of constitutively-active PKD1 in MDA-MB-231 cells drastically reduced their ability to invade in 2D and 3D cell culture. Moreover, MCF-7 cells acquired the ability to invade in 2D and 3D cell culture when PKD1 expression was knocked-down by shRNA. PKD1 also regulated the expression of breast cancer cell MMPs, MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, providing a potential mechanism for PKD1 mediation of the invasive phenotype.Our results identify decreased expression of the PKD1 as a marker for invasive breast cancer. They further suggest that the loss of PKD1 expression increases the malignant potential of breast cancer cells. This may be due to the function of PKD1 as a negative regulator of MMP expression. Our data suggest re-expression of PKD1 as a potential therapeutic strategy.

Project description:Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in tumorigenesis, little is known about the roles of mitochondrial dynamics in metastasis, the major cause of cancer death. In the present study, we found a marked upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1) expression in human invasive breast carcinoma and metastases to lymph nodes. Compared with non-metastatic breast cancer cells, mitochondria also were more fragmented in metastatic breast cancer cells that express higher levels of total and active Drp1 and less mitochondrial fusion protein 1 (Mfn1). Silencing Drp1 or overexpression of Mfn1 resulted in mitochondria elongation or clusters, respectively, and significantly suppressed metastatic abilities of breast cancer cells. In contrast, silencing Mfn proteins led to mitochondrial fragmentation and enhanced metastatic abilities of breast cancer cells. Interestingly, these manipulations of mitochondrial dynamics altered the subcellular distribution of mitochondria in breast cancer cells. For example, silencing Drp1 or overexpression of Mfn1 inhibited lamellipodia formation, a key step for cancer metastasis, and suppressed chemoattractant-induced recruitment of mitochondria to lamellipodial regions. Conversely, silencing Mfn proteins resulted in more cell spreading and lamellipodia formation, causing accumulation of more mitochondria in lamellipodia regions. More importantly, treatment with a mitochondrial uncoupling agent or adenosine triphosphate synthesis inhibitor reduced lamellipodia formation and decreased breast cancer cell migration and invasion, suggesting a functional importance of mitochondria in breast cancer metastasis. Together, our findings show a new role and mechanism for regulation of cancer cell migration and invasion by mitochondrial dynamics. Thus targeting dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing breast cancer metastasis.

Project description:PurposeClinical studies have shown that breast cancer risk is increased in hypertensive women. The underlying molecular mechanism remains undetermined. The current study tests our hypothesis that G protein coupled receptor kinase 4 (GRK4) is a molecule that links hypertension and breast cancer. GRK4 plays an important role in regulation of renal sodium excretion. Sustained activation of GRK4 as in the circumstances of single nucleotide polymorphism (SNPs) causes hypertension. Expression of GRK4 in the kidney is regulated by cMyc, an oncogene that is amplified in breast cancer.MethodsWestern analysis was used to evaluate GRK4 protein expression in seven breast cancer cell lines. GRK4 gene single nucleotide polymorphism in breast cancer cell lines and in breast cancer cDNA arrays were determined using TaqMan Genotyping qPRC. The function of GRK4 was evaluated in MCF-7 cells with cMyc knock-down and GRK4 re-expression and in MDA-MB-468 cells expressing inducible GRK4 shRNA lentivirus constructs. Nuclei counting and 5-Bromo-2'-deoxy-uridine (BrdU) labeling were used to evaluate cell growth and proliferation.ResultsGenotyping of GRK4 SNPs in breast cancer cDNA arrays (n = 94) revealed that the frequency of carrying two hypertension related SNPs A142 V or R65 L is threefold higher in breast cancer patients than in healthy people (P = 7.53E-11). GRK4 protein is expressed in seven breast cancer cell lines but not the benign mammary epithelial cell line, MCF-10A. Three hypertension related SNPs in the GRK4 gene were identified in the breast cancer cell lines. Except for BT20, all other breast cancer lines have 1-3 GRK4 SNPs of which A142 V occurs in all 6 lines. MDA-MB-468 cells contain homozygous A142 V and R65 L SNPs. Knocking down cMyc in MCF-7 cells significantly reduced the growth rate, which was rescued by re-expression of GRK4. We then generated three stable GRK4 knock-down MDA-MB-468 lines using inducible lentiviral shRNA vectors. Doxycycline (Dox) induced GRK4 silencing significantly reduced GRK4 mRNA and protein levels, growth rates, and proliferation. As a marker of cell proliferation, the percentage of BrdU-labeled cells decreased from 45 ± 3% in the cells without Dox to 32 ± 5% in the cells treated with 0.1 µg/mL Dox.ConclusionsGRK4 acts as an independent proliferation promotor in breast cancer. Our results suggest that targeted inhibition of GRK4 could be a new therapy for both hypertension and breast cancer.

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G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

Publications

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

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