Yaping Tu

Mitochondrial dynamics regulates migration and invasion of breast cancer cells

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.

Involvement of Estrogen Receptor Variant ER-α36, Not GPR30, in Nongenomic Estrogen Signaling

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

RGSZ1, a G(z)-selective rgs protein in brain: Structure, membrane association, regulation by Gα(z) phosphorylation, and relationship to a G(z) gtpase-activating protein subfamily

We cloned the cDNA for human RGSZ1, the major Gz-selective GTPase-activating protein (GAP) in brain (Wang, J., Tu, Y., Woodson, J., Song, X., and Ross, E. M. (1997)J. Biol. Chem. 272, 5732–5740) and a member of the RGS family of G protein GAPs. Its sequence is 83% identical to RET-RGS1 (except its N-terminal extension) and 56% identical to GAIP. Purified, recombinant RGSZ1, RET-RGS1, and GAIP each accelerated the hydrolysis of Gαz-GTP over 400-fold withK m values of ∼2 nm. RGSZ1 was 100-fold selective for Gαz over Gαi, unusually specific among RGS proteins. Other enzymological properties of RGSZ1, brain Gz GAP, and RET-RGS1 were identical; GAIP differed only in Mg2+ dependence and in its slightly lower selectivity for Gαz. RGSZ1, RET-RGS1, and GAIP thus define a subfamily of Gz GAPs within the RGS proteins. RGSZ1 has no obvious membrane-spanning region but is tightly membrane-bound in brain. Its regulatory activity in membranes depends on stable bilayer association. When co-reconstituted into phospholipid vesicles with Gz and m2 muscarinic receptors, RGSZ1 increased agonist-stimulated GTPase >15-fold with EC50<12 nm, but RGSZ1 added to the vesicle suspension was <0.1% as active. RGSZ1, RET-RGS1, and GAIP share a cysteine string sequence, perhaps targeting them to secretory vesicles and allowing them to participate in the proposed control of secretion by Gz. Phosphorylation of Gαz by protein kinase C inhibited the GAP activity of RGSZ1 and other RGS proteins, providing a mechanism for potentiation of Gz signaling by protein kinase C.

Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis

Excessive activation of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) pathways has been linked to prostate cancer metastasis. Rac activation by guanine nucleotide exchange factors (GEFs) plays an important role in directional cell migration, a critical step of tumor metastasis cascades. We found that the upregulation of P-Rex1, a Rac-selective GEF synergistically activated by Gβγ freed during GPCR signaling, and PIP3, generated during either RTK or GPCR signaling, strongly correlates with metastatic phenotypes in both prostate cancer cell lines and human prostate cancer specimens. Silencing endogenous P-Rex1 in metastatic prostate cancer PC-3 cells selectively inhibited Rac activity and reduced cell migration and invasion in response to ligands of both epidermal growth factor receptor and G-protein-coupled CXC chemokine receptor 4. Conversely, expression of recombinant P-Rex1, but not its ‘GEF-dead’ mutant, in non-metastatic prostate cancer cells, such as CWR22Rv1, increased cell migration and invasion through Rac-dependent lamellipodia formation. More importantly, using a mouse xenograft model, we showed that the expression of P-Rex1, but not its mutant, induced lymph node metastasis of CWR22Rv1 cells without an effect on primary tumor growth. Thus, by functioning as a coincidence detector of chemotactic signals from both GPCRs and RTKs, P-Rex1-dependent activation of Rac promotes prostate cancer metastasis.

Palmitoylation of a Conserved Cysteine in the Regulator of G Protein Signaling (RGS) Domain Modulates the GTPase-activating Activity of RGS4 and RGS10

RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys95 in RGS4, Cys66 in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys2 or Cys12. The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [3H]palmitate labeling of Cys95. Membrane-bound RGS4 is palmitoylated both at Cys95 and Cys2/12, but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the Gi and Gq families of G proteins. Palmitoylation of Cys95 on RGS4 or Cys66 on RGS10 inhibits GAP activity 80–100% toward either Gαi or Gαzin a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity ≥20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Gαz and increased Gz GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.

Breast Cancer Migration and Invasion Depend on Proteasome Degradation of Regulator of G-Protein Signaling 4

Aberrant signaling through G-protein coupled receptors promotes metastasis, the major cause of breast cancer death. We identified regulator of G-protein signaling 4 (RGS4) as a novel suppressor of breast cancer migration and invasion, important steps of metastatic cascades. By blocking signals initiated through G(i)-coupled receptors, such as protease-activated receptor 1 and CXC chemokine receptor 4, RGS4 disrupted Rac1-dependent lamellipodia formation, a key step involved in cancer migration and invasion. RGS4 has GTPase-activating protein (GAP) activity, which inhibits G-protein coupled receptor signaling by deactivating G-proteins. An RGS4 GAP-deficient mutant failed to inhibit migration and invasion of breast cancer cells in both in vitro assays and a mouse xenograft model. Interestingly, both established breast cancer cell lines and human breast cancer specimens showed that the highest levels of RGS4 protein were expressed in normal breast epithelia and that RGS4 down-regulation by proteasome degradation is an index of breast cancer invasiveness. Proteasome blockade increased endogenous RGS4 protein to levels that markedly inhibit breast cancer cell migration and invasion, which was reversed by an RGS4-targeted short hairpin RNA. Our findings point to the existence of a mechanism for posttranslational regulation of RGS4 function, which may have important implications for the acquisition of a metastatic phenotype by breast cancer cells. Preventing degradation of RGS4 protein should attenuate aberrant signal inputs from multiple G(i)-coupled receptors, thereby retarding the spread of breast cancer cells and making them targets for surgery, radiation, and immune treatment.

Binding of Regulator of G Protein Signaling (RGS) Proteins to Phospholipid Bilayers CONTRIBUTION OF LOCATION AND/OR ORIENTATION TO GTPase-ACTIVATING PROTEIN ACTIVITY

Regulator of G protein signaling (RGS) proteins must bind membranes in an orientation that permits the protein-protein interactions necessary for regulatory activity. RGS4 binds to phospholipid surfaces in a slow, multistep process that leads to maximal GTPase-activating protein (GAP) activity. When RGS4 is added to phospholipid vesicles that contain m2 or m1 muscarinic receptor and Gi, Gz, or Gq, GAP activity increases ∼3-fold over 4 h at 30 °C and more slowly at 20 °C. This increase in GAP activity is preceded by several other events that suggest that, after binding, optimal interaction with G protein and receptor requires reorientation of RGS4 on the membrane surface, a conformational change, or both. Binding of RGS4 is initially reversible but becomes irreversible within 5 min. Onset of irreversibility parallels initial quenching of tryptophan fluorescence (t 1 2 ∼ 30 s). Further quenching occurs after binding has become irreversible (t 1 2 ∼ 6 min) but is complete well before maximal GAP activity is attained. These processes all appear to be energetically driven by the amphipathic N-terminal domain of RGS4 and are accelerated by palmitoylation of cysteine residues in this region. The RGS4 N-terminal domain confers similar membrane binding behavior on the RGS domains of either RGS10 or RGSZ1.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G protein-coupled receptors (GPCRs) promotes breast cancer metastasis. G proteins convey GPCR signals by dissociating into Gα and Gβγ subunits. The aim of the present study was to determine whether blockade of Gβγ signaling suppresses breast cancer cell migration and invasion, which are critical components of metastasis. Conditioned media (CM) of NIH-3T3 fibroblasts are widely used as chemoattractants in in vitro cancer metastasis studies. Expression of a Gβγ scavenger peptide attenuated NIH-3T3 CM-induced migration and invasion of both metastatic breast cancer MDA-MB-231 and MDA-MB-436 cells by 40 to 50% without effects on cell viability. Migration and invasion of cells in response to NIH-3T3 CM were also blocked by 8-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-1-naph-thalene-carboxylic acid) (M119K), a Gβγ inhibitor, with maximum inhibition exceeding 80% and half-maximal inhibitory concentration (IC50) values of 1 to 2 μM. M119K also attenuated Rac-dependent formation of lamellipodia, a key structure required for metastasis. Constitutively active Rac1 rescued Gβγ blockade-mediated inhibition of breast cancer cell migration, whereas dominant negative Rac1 inhibited cell migration similar to Gβγ blockade. Furthermore, M119K suppressed Gi protein-coupled CXC chemokine receptor 4 (CXCR4)-dependent MDA-MB-231 cell migration by 80% with an IC50 value of 1 μM, whereas tyrosine kinase receptor-dependent cell migration was significantly less inhibited. However, CXCR4-dependent inhibition of adenylyl cyclase, a Giα-mediated response in MDA-MB-231 cells, was not blocked by M119K but was blocked by pertussis toxin, which selectively inactivates Giα. This report is the first to directly demonstrate the role of Gβγ in cancer cell migration and invasion and suggests that targeting Gβγ signaling pathways may provide a novel strategy for suppressing breast cancer metastasis.

Epidermal Growth Factor–Ferritin H‐Chain Protein Nanoparticles for Tumor Active Targeting

Human ferritin H-chain protein (FTH1)-based nanoparticles possess a precisely assembled nanometer-scale structure and high safety. However, their applications for imaging and drug delivery towards cancer cells remain limited due to a lack of target specificity. Epidermal growth factor receptor (EGFR) is overexpressed in many malignant tissues including breast cancer, and has been used as a therapeutic target for cancer treatment. Herein, a genetic method is shown to generate EGF-FTH1 chimeric proteins. EGF-FTH1 nanoparticles with EGF on the surface are then produced. The data demonstrate that EGF-FTH1 nanoparticles, with a small size (11.8 ± 1.8 nm), narrow size distribution, and high biosafety, can specifically bind to and then be taken up by breast cancer MCF-7 cells and MDA-MB-231 cells, but not normal breast epithelial MCF-10A cells. In contrast, binding and absorption of nontargeted ferritin-based nanoparticles to breast cancer cells are negligible. In vivo studies show that EGF-FTH1 nanoparticles are accumulated in breast tumors in a mouse xenograft model. Interestingly, the concentration of EGF-FTH1 nanoparticles in the tumor site is significantly reduced when mice are pretreated with an excess of free EGF. These results imply that EGF-EGFR interaction plays an important role in regulating the tumor retention of EGF-FTH1 nanoparticles.

Energy metabolism analysis reveals the mechanism of inhibition of breast cancer cell metastasis by PEG-modified graphene oxide nanosheets

Recent advances in nanomedicine provide promising alternatives for cancer treatment that may improve the survival of patients with metastatic disease. The goal of the present study was to evaluate graphene oxide (GO) as a potential anti-metastatic agent. For this purpose, GO was modified with polyethylene glycol (PEG) to form PEG-modified GO (PEG-GO), which improves its aqueous stability and biocompatibility. We show here that PEG-GO exhibited no apparent effects on the viability of breast cancer cells (MDA-MB-231, MDA-MB-436, and SK-BR-3) or non-cancerous cells (MCF-10A), but inhibited cancer cell migration in vitro and in vivo. Analysis of cellular energy metabolism revealed that PEG-GO significantly impaired mitochondrial oxidative phosphorylation (OXPHOS) in breast cancer cells; however, PEG-GO showed no effect on OXPHOS in non-cancerous cells. To explore the underlying mechanisms, a SILAC (Stable Isotope Labeling by Amino acids in Cell culture) labeling strategy was used to quantify protein expression in PEG-GO-exposed breast cancer versus non-cancerous cells. The results indicated that PEG-GO selectively down-regulated PGC-1α in breast cancer cells and thus modified the expression of diverse energy generation-related proteins, which accounts for the inhibition of OXPHOS. The inhibition of OXPHOS by PEG-GO significantly reduced ATP production and impaired assembly of the F-actin cytoskeleton in breast cancer cells, which is required for the migratory and invasive phenotype of cancer cells. Taken together, these effects of PEG-GO on cancer cell metastasis may allow the development of a new approach to treat metastatic breast cancer.