Precise targeting of cancer metastasis using multi-ligand nanoparticles incorporating four different ligands.

Metastasis displays a highly heterogeneous cellular population with cancer cells continuously evolving. As a result, a single-ligand nanoparticle cannot account for the continuously changing expression of targetable biomarkers over time and space. To effectively direct nanoparticles to metastasis, we developed a multi-ligand nanoparticle by using four different types of ligands on the same nanoparticle that target biomarkers on the endothelium associated with metastatic disease. These vascular targets included αvß3 integrin, P-selectin, EGFR and fibronectin. Using terminal and in vivo imaging studies, the targeting performance of the multi-ligand nanoparticles was compared to the single-ligand nanoparticle variants. All four single-ligand nanoparticle variants achieved significant targeting of lung metastasis in the 4T1 mouse model of breast cancer metastasis with about 2.5% of the injected dose being deposited into metastasis. A dual-ligand nanoparticle resulted in a nearly 2-fold higher deposition into lung metastases than its single-ligand counterparts. The multi-ligand nanoparticle significantly outperformed its targeting nanoparticle counterparts achieving a deposition of ∼7% of its injected nanoparticles into lung metastases. Using the high sensitivity of radionuclide imaging, PET imaging showed that a multi-ligand nanoparticle labeled with [18F]fluoride was able to precisely target metastatic disease at its very early stage of development in three different animal models of metastatic breast cancer.

TGF-ß stimulates Pyk2 expression as part of an epithelial-mesenchymal transition program required for metastatic outgrowth of breast cancer.

Epithelial-mesenchymal transition (EMT) programs are essential in promoting breast cancer invasion, systemic dissemination and in arousing proliferative programs in breast cancer micrometastases, a reaction that is partially dependent on focal adhesion kinase (FAK). Many functions of FAK are shared by its homolog, protein tyrosine kinase 2 (Pyk2), raising the question as to whether Pyk2 also participates in driving the metastatic outgrowth of disseminated breast cancer cells. In addressing this question, we observed Pyk2 expression to be (i) significantly upregulated in recurrent human breast cancers; (ii) differentially expressed across clonal isolates of human MDA-MB-231 breast cancer cells in a manner predictive for metastatic outgrowth, but not for invasiveness; and (iii) dramatically elevated in ex vivo cultures of breast cancer cells isolated from metastatic lesions as compared with cells that produced the primary tumor. We further show that metastatic human and murine breast cancer cells robustly upregulate their expression of Pyk2 during EMT programs stimulated by transforming growth factor-ß (TGF-ß). Genetic and pharmacological inhibition of Pyk2 demonstrated that the activity of this protein tyrosine kinase was dispensable for the ability of breast cancer cells to undergo invasion in response to TGF-ß, and to form orthotopic mammary tumors in mice. In stark contrast, Pyk2-deficiency prevented TGF-ß from stimulating the growth of breast cancer cells in 3D-organotypic cultures that recapitulated pulmonary microenvironments, as well as inhibited the metastatic outgrowth of disseminated breast cancer cells in the lungs of mice. Mechanistically, Pyk2 expression was inversely related to that of E-cadherin, such that elevated Pyk2 levels stabilized ß1 integrin expression necessary to initiate the metastatic outgrowth of breast cancer cells. Thus, we have delineated novel functions for Pyk2 in mediating distinct elements of the EMT program and metastatic cascade regulated by TGF-ß, particularly the initiation of secondary tumor outgrowth by disseminated cells.

Transforming growth factor-ß-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression.

Transforming growth factor-ß (TGF-ß) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-ß in altering the response of breast cancer cells to EGF remains unknown. We show in this study that murine metastatic 4T1 breast cancer cells formed compact and dense spheroids when cultured under three-dimensional (3D) conditions, which was in sharp contrast to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, whereas their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-ß was sufficient to manifest dense spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent on increased activation of EGF receptor (EGFR) and p38 mitogen-activated protein kinase, all of which could be abrogated either by pharmacologic (PF-562271) or by genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-ß increased cell-surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with TßR-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-ß stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyperinvasive to EGF and showed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-ß in aggressive and invasive forms of breast cancer.

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling.

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.

Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling.

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF- and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately N-terminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide.

A deletion in the gene for transforming growth factor beta type I receptor abolishes growth regulation by transforming growth factor beta in a cutaneous T-cell lymphoma.

Spontaneous regression of skin lesions is characteristic of lymphomatoid papulosis (LyP), a clonal cutaneous lymphoproliferative disorder. A minority of LyP patients progress to anaplastic large cell lymphoma (ALCL) in which skin lesions no longer regress and extracutaneous dissemination often occurs. In 1 such case, we developed a tumor cell line, JK cells, and show that these cells are resistant to the growth inhibitory effects of transforming growth factor beta (TGF-beta) due to the loss of cell surface expression of the TGF-beta type I receptor (TbetaR-I). Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing of JK cell TbetaR-I cDNA clones identified a deletion that spanned the last 178 bp of exon 1, including the initiating methionine. Hybridization of a radiolabeled fragment internal to the deletion was detected in the genomes of TGF-beta-responsive cells, but not in JK cells, indicating that they contain no wild-type TbetaR-I gene. PCR primers that flanked the deleted TbetaR-I region amplified a single band from JK cell genomic DNA that lacked the last 178 bp of exon 1 and all of the approximately 5 kb of intron 1. This JK cell-specific genomic TbetaR-I PCR product was distinct from products amplified from TGF-beta-responsive cells and was also readily detected in tumor biopsies obtained before the establishment of the JK cell line. Our results identify the first inactivating mutation in TbetaR-I gene in a human lymphoma that renders it insensitive to growth inhibition by TGF-beta.

Raf-1 independent stimulation of mitogen-activated protein kinase by leukemia inhibitory factor in 3T3-L1 cells.

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulated Raf-1 activity in a time- and dose-dependent manner. Although phorbol ester failed to activate Raf-1 directly, a protein kinase C-stimulated signal was found to be necessary, but not sufficient, for LIF-mediated activation of Raf-1. Elevation of intracellular cAMP levels completely blocked Raf-1 activation by LIF, but was without effect on the magnitude of mitogen-activated protein kinase (MAPK) stimulation by the cytokine, suggesting the presence of a Raf-1-independent, cAMP-insensitive MAPK kinase kinase (MAPKKK) pathway in 3T3-L1 cells. Mono Q-fractionation of LIF-stimulated 3T3-L1 extracts identified a single peak of MAPKKK activity that was largely insensitive to elevated intracellular levels of cAMP, and that failed to correlate with stimulation of either Raf-1 or MEKK1 protein kinases. Our results demonstrate that LIF-mediated activation of the MAP kinase cascade in 3T3-L1 cells proceeds through both Raf-1-dependent and -independent pathways which differ in their sensitivity to inhibition by intracellular cAMP.

Box 3-independent signaling mechanisms are involved in leukemia inhibitory factor receptor alpha- and gp130-mediated stimulation of mitogen-activated protein kinase. Evidence for participation of multiple signaling pathways which converge at Ras.

Chimeric receptors containing the entire or various cytoplasmic domains of either gp130 or leukemia inhibitory factor receptor alpha (LIFR) were used to identify signaling molecules and regions of these polypeptides required for the stimulation of mitogen-activated protein kinase (MAPK). Coexpression of dominant-negative Jak2 inhibited chimeric receptor-stimulated MAPK activity by approximately 70%, while expression of dominant-negative Ras completely blocked MAPK activation by either receptor polypeptide. Deletion analysis identified a 24-amino acid region of gp130 that was necessary for maximal stimulation of MAPK, and contained box 3 (positions 120-129) and a consensus tyrosine binding motif (Tyr-118) for the protein-tyrosine phosphatase, SHP2. Expression of receptors lacking this region or of chimeric gp130(Y118F) point mutants inhibited MAPK activity by approximately 55%, suggesting that Tyr-118, but not box 3, was required during activation of MAPK by gp130. Similarly, expression of chimeric LIFR constructs lacking box 3 maximally stimulated MAPK activity, while those lacking Tyr-115, a putative SHP2 binding site, inhibited stimulation of MAPK by this polypeptide. Our results demonstrate that gp130 and LIFR stimulate MAPK activity through box 3-independent mechanisms involving: (i) effects at Tyr-118 and Tyr-115, respectively, for maximal stimulation of MAPK activity and (ii) a Jak/Tyk-dependent pathway that, together with Tyr-118- or Tyr-115-generated signals, converges at the level of Ras during activation of MAPK by cytokine.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Involvement of protein kinase C during activation of the mitogen-activated protein kinase cascade by leukemia inhibitory factor. Evidence for participation of multiple signaling pathways.

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulates the activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase (MAPK), and S6 protein kinase (S6K) activities both in a time- and dose-dependent manner. A single peak of MAPKK activity, four peaks of activity against the S6 synthetic peptide, RRLSSLRA (S6 peptide), and three distinct peaks toward myelin basic protein (MBP) were observed after Mono-Q chromatography of LIF-stimulated cell extracts. Two of the MBP kinase activities correlated with the stimulation of extracellular signal-regulated kinases 1 and 2. Interestingly, down-regulation of protein kinase C (PKC) by chronic treatment of 3T3-L1 cells with phorbol ester was found to attenuate, but not block, the LIF-mediated stimulation of MAPKK, MAPK, and S6K activities in 3T3-L1 cells. Treatment of 3T3-L1 cells with epidermal growth factor increased MAPKK, MAPK, and S6K activities to a similar extent as LIF, but this activation was not attenuated by down-regulation of PKC. Our results suggest that the full activation of the MAPK cascade by LIF may require inputs from multiple signaling pathways, one of which is dependent upon the presence of functional PKC.

Adenosine A1-receptor coupling to phosphoinositide metabolism in pregnant guinea pig myometrium.

Adenosine contracts pregnant and nonpregnant guinea pig myometrial smooth muscle (MSM). We have 1) described dissociation of A1-adenosine receptors from adenylate cyclase inhibition in nonpregnant MSM (M. A. Smith, J. L. Silverstein, D. P. Westfall, and I. L. O. Buxton, Cell. Signal. 1: 357-365, 1989); 2) described appearance of such inhibitory coupling in pregnant MSM [W. P. Schiemann, D. P. Westfall, and I. L. O. Buxton, Am. J. Physiol. 261 (Endocrinol. Metab. 24): E141-E150, 1991]; and 3) demonstrated a role for myometrial A1 receptors in the rapid formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in nonpregnant MSM and the cyclooxygenase dependence of this effect (W. P. Schiemann, K. O. Doggwiller, and I. L. O. Buxton. J. Pharmacol. Exp. Ther. 258: 429-437, 1991). To further characterize adenosine action in pregnant tissue, we explored A1 coupling to increased phosphoinositide hydrolysis in near-term pregnant MSM. The A1-receptor agonist (+)-N6-(2-phenylisopropyl)adenosine stimulates the rapid dose-dependent formation of Ins(1,4,5)P3 and stimulates rapid degradation of uterine inositol monophosphates (InsP) in a manner paralleling increases in inositol polyphosphates. Both A1-mediated responses were blocked by the A1 antagonist 8-(p-sulfophenyl)theophylline, and, unlike the effect observed in nonpregnant MSM, treatment of pregnant MSM with either meclofenamate or indomethacin failed to block A1-mediated increases in Ins(1,4,5)P3. Pretreatment of MSM with either Li+ or pertussis toxin failed to alter either Ins(1,4,5)P3 formation or InsP degradation. Furthermore, assay of inositol phosphomonoesterase (InsPase) activity in the presence or absence of Li+ confirmed the existence of an MSM Li(+)-insensitive InsPase enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of adenosine in estrogen-primed nonpregnant guinea pig myometrium: characterization of the smooth muscle receptor and coupling to phosphoinositide metabolism.

The smooth muscle of guinea pig uterus is contracted by adenosine in a manner consistent with the presence of a purine nucleoside receptor of the P1-A1 subtype that is uncoupled from adenylate cyclase. Here we investigate the signal transduction mechanism responsible for adenosine's ability to contract uterine smooth muscle. The A1 adenosine receptor antagonist [3H]-8-cyclopentyl-1.3- dipropyl xanthine ([3H]CPX) bound reversibly to a large number (172 +/- 25 fmol/mg of protein) of receptors in myometrial smooth muscle membranes from estrogen-primed virgin guinea pigs with an affinity (KD = 1.77 +/- 0.21 nM) similar to that expected of [3H]CPX binding to both central and peripheral A1 receptors. In the absence of the stable GTP analog, guanosine-5'-O-[3-thiotriphosphate], agonist competition of [3H]CPX binding resulted in a biphasic curve that was best fit assuming the presence of equal populations of two affinity states of the receptor. Addition of guanosine-5'-O-[3-thiotriphosphate] (10 microM) resulted in a monophasic competition curve of low affinity suggesting coupling of this A1 receptor to effector via a GTP binding protein. In [3H]myo-inositol labeled strips of myometrial smooth muscle, the adenosine agonist R-phenylisopropyl adenosine (R-PIA) stimulated the rapid formation of inositol-1,4,5-trisphosphate (InsP3) that was antagonized by addition of the nucleoside receptor antagonist 8-sulfophenyl theophylline. Prostaglandin stimulation of myometrial strips also increased InsP3 formation. Furthermore, R-PIA stimulated the disappearance of inositol phosphate (InsP) in a fashion consistent with agonist stimulation of an inositol phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Smooth muscle adenosine A1 receptors couple to disparate effectors by distinct G proteins in pregnant myometrium.

We have previously shown that adenosine, acting at an A1 receptor, contracts the smooth muscle of virgin guinea pig uterus (M. A. Smith, I. L. O. Buxton, and D. P. Westfall. J. Pharmacol, Exp. Ther. 247: 1059-1063, 1988) and is not coupled to the expected inhibition of adenylate cyclase (M. A. Smith, J. L. Silverstein, D. P. Westfall, and I. L. O. Buxton. Cell. Signal. 1: 357-365, 1989). To probe the importance of contractile actions of adenosine in uterine smooth muscle and to further characterize the signal transduction pathway involved in A1-receptor action, we have studied the adenosine receptor and its coupling in pregnant guinea pig myometrium. Adenosine agonist and antagonist radioligands bind to saturable sites of the A1 subtype homogeneously distributed in the smooth muscle of pregnant guinea pig uterus. Agonist competition of antagonist radioligand binding in both the absence and presence of guanine nucleotide reveals high and low agonist affinity states of the receptor. Pretreatment of tissues with pertussis toxin (PTx) shifts the high-affinity sites to a lower affinity but does not affect low-affinity sites, whereas agonist competition in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is indistinguishable from the control, which is consistent with coupling of A1 receptors to both PTx-sensitive and PTx-insensitive GTP-binding proteins. Adenosine receptor inhibition of adenylate cyclase activity is prevented after pretreatment of the tissue with PTx, whereas increased inositol phosphate production is not. The data presented here are consistent with coupling of the A1 receptor to dual effectors in the pregnant state of the smooth muscle. The unique action of an A1 receptor to contract mammalian smooth muscle and the appearance, only in the pregnant state, of coupling to adenylate cyclase inhibition suggest a role for adenosine in parturition biology.

On the ability of endogenous adenosine to regulate purine nucleoside receptor binding of antagonists in smooth muscle membranes.

Adenosine, acting at A1 and A2 purine nucleoside receptors, regulates the physiology of many tissues. Myometrial smooth muscle from pregnant guinea pigs, which is contracted by the actions of adenosine, possesses an A1 receptor whose agonist affinity is regulated by guanine nucleotides. In addition to its expected effect on the affinity of the A1 receptor for agonist, the addition of guanine nucleotide also dramatically increases antagonist binding by as much as 62%. This action of guanine nucleotides on adenosine A1 receptors is common to many smooth muscle preparations and suggests the possibility that GTP-binding proteins might alter the conformation of the adenosine receptor in such a way that receptors not previously able to bind ligands are recruited by the guanine nucleotide. Such an action of guanine nucleotides would alter our general view of the interaction of antagonists with GTP-binding protein coupled receptors, as well as bear significantly on the interpretation of experimental data designed to characterize purinergic receptors. Thus, we have investigated the actions of guanosine-5'-O-[3-thiotriphosphate] on A1 adenosine receptor binding in membranes prepared from pregnant guinea pig myometrium containing 61% right-side-out vesicles. We show that guanosine-5'-O-[3-thiotriphosphate] lowers the affinity of adenosine A1 receptors for agonist in vesicles leading to increased competition of antagonist radioligand for receptor. We suggest that the endogenous adenosine we measure originates from breakdown of significant amounts of adenine nucleotides present in membranes vesicles. Furthermore, we demonstrate that opening membrane vesicles to remove trapped adenosine yields maximal antagonist radioligand binding without subsequent effects of guanosine-5'-O-[3-thiotriphosphate]. We conclude that the presence of endogenous adenosine, unavailable to the actions of adenosine deaminase, is responsible for the effect of guanine nucleotides to increase antagonist binding to adenosine A1 receptors.