Quantitative Longitudinal Imaging Reveals that Inhibiting Hedgehog Activity Alleviates the Hypoxic Tumor Landscape.

Metastases account for the majority of mortalities related to breast cancer. The onset and sustained presence of hypoxia strongly correlates with increased incidence of metastasis and unfavorable prognosis in patients with breast cancer. The Hedgehog (Hh) signaling pathway is dysregulated in breast cancer, and its abnormal activity enables tumor progression and metastasis. In addition to programming tumor cell behavior, Hh activity enables tumor cells to craft a metastasis-conducive microenvironment. Hypoxia is a prominent feature of growing tumors that impacts multiple signaling circuits that converge upon malignant progression. We investigated the role of Hh activity in crafting a hypoxic environment of breast cancer. We used radioactive tracer [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) to image tumor hypoxia. We show that tumors competent for Hh activity are able to establish a hypoxic milieu; pharmacologic inhibition of Hh signaling in a syngeneic mammary tumor model mitigates tumor hypoxia. Furthermore, in hypoxia, Hh activity is robustly activated in tumor cells and institutes increased HIF signaling in a VHL-dependent manner. The findings establish a novel perspective on Hh activity in crafting a hypoxic tumor landscape and molecularly navigating the tumor cells to adapt to hypoxic conditions. IMPLICATIONS: Importantly, we present a translational strategy of utilizing longitudinal hypoxia imaging to measure the efficacy of vismodegib in a preclinical model of triple-negative breast cancer.

Gold Nanoparticles sensitize pancreatic cancer cells to gemcitabine.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid cancers with dismal prognosis. Several mechanisms that are mainly responsible for aggressiveness and therapy resistance of PDAC cells include epithelial to mesenchymal transition (EMT), stemness and Mitogen Activated Protein Kinase (MAPK) signaling. Strategies that inhibit these mechanisms are critically important to improve therapeutic outcome in PDAC. In the current study, we wanted to investigate whether gold nanoparticles (AuNPs) could sensitize pancreatic cancer cells to the chemotherapeutic agent gemcitabine. We demonstrated that treatment with AuNPs of 20 nm diameter inhibited migration and colony forming ability of pancreatic cancer cells. Pre-treatment with AuNPs sensitized pancreatic cancer cells to gemcitabine in both viability and colony forming assays. Mechanistically, pre-treatment of pancreatic cancer cells with AuNPs decreased gemcitabine induced EMT, stemness and MAPK activation. Taken together, these findings suggest that AuNPs could be considered as a potential agent to sensitize pancreatic cancer cells to gemcitabine.

O-GlcNAcylation of GLI transcription factors in hyperglycemic conditions augments Hedgehog activity.

Modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) promotes tumor cell survival, proliferation, epigenetic changes, angiogenesis, invasion, and metastasis. Here we demonstrate that in conditions of elevated glucose, there is increased expression of key drug resistance proteins (ABCB1, ABCG2, ERCC1, and XRCC1), all of which are regulated by the Hedgehog pathway. In elevated glucose conditions, we determined that the Hedgehog pathway transcription factors, GLI1 and GLI2, are modified by O-GlcNAcylation. This modification functionally enhanced their transcriptional activity. The activity of GLI was enhanced when O-GlcNAcase was inhibited, while inhibiting O-GlcNAc transferase caused a decrease in GLI activity. The metabolic impact of hyperglycemic conditions impinges on maintaining PKM2 in the less active state that facilitates the availability of glycolytic intermediates for biosynthetic pathways. Interestingly, under elevated glucose conditions, PKM2 directly influenced GLI activity. Specifically, abrogating PKM2 expression caused a significant decline in GLI activity and expression of drug resistance proteins. Cumulatively, our results suggest that elevated glucose conditions upregulate chemoresistance through elevated transcriptional activity of the Hedgehog/GLI pathway. Interfering in O-GlcNAcylation of the GLI transcription factors may be a novel target in controlling cancer progression and drug resistance of breast cancer.

Neurofibromin (NF1) genetic variant structure-function analyses using a full-length mouse cDNA.

Neurofibromatosis type 1 (NF1) is caused by pathogenic variants or mutations in the NF1 gene that encodes neurofibromin. We describe here a new approach to determining the functional consequences of NF1 genetic variants. We established a heterologous cell culture expression system using a full-length mouse Nf1 cDNA (mNf1) and human cell lines. We demonstrate that the full-length murine cDNA produces a > 250 kDa neurofibromin protein that is capable of modulating Ras signaling. We created mutant cDNAs representing NF1 patient variants with different clinically relevant phenotypes, and assessed their ability to produce mature neurofibromin and restore Nf1 activity in NF1-/- cells. These cDNAs represent variants in multiple protein domains and various types of clinically relevant predicted variants. This approach will help advance research on neurofibromin structure and function, determine pathogenicity for missense variants, and allow for the development of activity assays and variant-directed therapeutics.

Loss of Merlin induces metabolomic adaptation that engages dependence on Hedgehog signaling.

The tumor suppressor protein Merlin is proteasomally degraded in breast cancer. We undertook an untargeted metabolomics approach to discern the global metabolomics profile impacted by Merlin in breast cancer cells. We discerned specific changes in glutathione metabolites that uncovered novel facets of Merlin in impacting the cancer cell metabolome. Concordantly, Merlin loss increased oxidative stress causing aberrant activation of Hedgehog signaling. Abrogation of GLI-mediated transcription activity compromised the aggressive phenotype of Merlin-deficient cells indicating a clear dependence of cells on Hedgehog signaling. In breast tumor tissues, GLI1 expression enhanced tissue identification and discriminatory power of Merlin, cumulatively presenting a powerful substantiation of the relationship between these two proteins. We have uncovered, for the first time, details of the tumor cell metabolomic portrait modulated by Merlin, leading to activation of Hedgehog signaling. Importantly, inhibition of Hedgehog signaling offers an avenue to target the vulnerability of tumor cells with loss of Merlin.

Loss of tumor suppressor Merlin results in aberrant activation of Wnt/ß-catenin signaling in cancer.

The expression of the tumor suppressor Merlin is compromised in nervous system malignancies due to genomic aberrations. We demonstrated for the first time, that in breast cancer, Merlin protein expression is lost due to proteasome-mediated elimination. Immunohistochemical analysis of tumor tissues from patients with metastatic breast cancer revealed characteristically reduced Merlin expression. Importantly, we identified a functional role for Merlin in impeding breast tumor xenograft growth and reducing invasive characteristics. We sought to determine a possible mechanism by which Merlin accomplishes this reduction in malignant activity. We observed that breast and pancreatic cancer cells with loss of Merlin show an aberrant increase in the activity of ß-catenin concomitant with nuclear localization of ß-catenin. We discovered that Merlin physically interacts with ß-catenin, alters the sub-cellular localization of ß-catenin, and significantly reduces the protein levels of ß-catenin by targeting it for degradation through the upregulation of Axin1. Consequently, restoration of Merlin inhibited ß-catenin-mediated transcriptional activity in breast and pancreatic cancer cells. We also present evidence that loss of Merlin sensitizes tumor cells to inhibition by compounds that target ß-catenin-mediated activity. Thus, this study provides compelling evidence that Merlin reduces the malignant activity of pancreatic and breast cancer, in part by suppressing the Wnt/ß-catenin pathway. Given the potent role of Wnt/ß-catenin signaling in breast and pancreatic cancer and the flurry of activity to test ß-catenin inhibitors in the clinic, our findings are opportune and provide evidence for Merlin in restraining aberrant activation of Wnt/ß-catenin signaling.

Nanowire nanocomputer as a finite-state machine.

Implementation of complex computer circuits assembled from the bottom up and integrated on the nanometer scale has long been a goal of electronics research. It requires a design and fabrication strategy that can address individual nanometer-scale electronic devices, while enabling large-scale assembly of those devices into highly organized, integrated computational circuits. We describe how such a strategy has led to the design, construction, and demonstration of a nanoelectronic finite-state machine. The system was fabricated using a design-oriented approach enabled by a deterministic, bottom-up assembly process that does not require individual nanowire registration. This methodology allowed construction of the nanoelectronic finite-state machine through modular design using a multitile architecture. Each tile/module consists of two interconnected crossbar nanowire arrays, with each cross-point consisting of a programmable nanowire transistor node. The nanoelectronic finite-state machine integrates 180 programmable nanowire transistor nodes in three tiles or six total crossbar arrays, and incorporates both sequential and arithmetic logic, with extensive intertile and intratile communication that exhibits rigorous input/output matching. Our system realizes the complete 2-bit logic flow and clocked control over state registration that are required for a finite-state machine or computer. The programmable multitile circuit was also reprogrammed to a functionally distinct 2-bit full adder with 32-set matched and complete logic output. These steps forward and the ability of our unique design-oriented deterministic methodology to yield more extensive multitile systems suggest that proposed general-purpose nanocomputers can be realized in the near future.

Nonclassical activation of Hedgehog signaling enhances multidrug resistance and makes cancer cells refractory to Smoothened-targeting Hedgehog inhibition.

The Hedgehog (Hh) pathway is critical in normal development. However, it has been reported to be up-regulated in numerous cancers and implicated in tumorigenicity and metastasis. Classical activation of Hh signaling initiated by Hh ligands results in activation of Smoothened (SMOH) and culminates in the activation of the GLI transcription factors. Classical Hh signaling is autocrine or paracrine (involving interaction between tumor cells and their stroma/microenvironment). The tumor milieu is rich in inflammatory cytokines that can modulate tumor cell behavior. Here, we show for the first time that the Hh pathway can be nonclassically up-regulated by the inflammatory cytokine, osteopontin (OPN). OPN-initiated Akt-GSK3ß signaling mediates the subcellular distribution and activation of GLI1 resulting in the modulation of epithelial mesenchymal plasticity and drug resistance. Interestingly, the SMOH inhibitor cyclopamine was unable to uncouple the effects of OPN on Hh signaling, indicating that OPN nonclassically activates GLI-mediated transcription. Given the fact that OPN is itself transcriptionally activated upon Hh signaling, our current findings highlight the possibility of a feedforward vicious cycle such that the Hh pathway might be turned on nonclassically by stimuli from the tumor milieu. Thus, drugs that target the classical Hh ligand-mediated activation of Hh signaling may be compromised in their ability to interfere with the functioning of the pathway.

Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh) pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

The hedgehog pathway conditions the bone microenvironment for osteolytic metastasis of breast cancer.

The microenvironment at the site of tumor metastasis plays a key role in determining the fate of the metastasizing tumor cells. This ultimately has a direct impact on the progression of cancer. Bone is the preferred site of metastasis of breast cancer. Painful, debilitating osteolytic lesions are formed as a result of crosstalk between breast cancer cells and cells in the bone, predominantly the osteoblasts and osteoclasts. In this paper, we have discussed the temporal and spatial role of hedgehog (Hh) signaling in influencing the fate of metastatic breast cancer cells in bone. By virtue of its secreted ligands, the Hh pathway is capable of homotypic and heterotypic signaling and consequently altering the microenvironment in the bone. We also have put into perspective the therapeutic implications of using Hh inhibitors to prevent and/or treat bone metastases of breast cancer.

Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation.

Unlike malignancies of the nervous system, there have been no mutations identified in Merlin in breast cancer. As such, the role of the tumor suppressor, Merlin, has not been investigated in breast cancer. We assessed Merlin expression in breast cancer tissues by immunohistochemistry and by real-time PCR. The expression of Merlin protein (assessed immunohistochemically) was significantly decreased in breast cancer tissues (although the transcript levels were comparable) simultaneous with increased expression of the tumor-promoting protein, osteopontin (OPN). We further demonstrate that the loss of Merlin in breast cancer is brought about, in part, due to OPN-initiated Akt-mediated phosphorylation of Merlin leading to its proteasomal degradation. Restoring expression of Merlin resulted in reduced malignant attributes of breast cancer, characterized by reduced invasion, migration, motility, and impeded tumor (xenograft) growth in immunocompromised mice. The possibility of developing a model using the relationship between OPN and Merlin was tested with a logistic regression model applied to immunohistochemistry data. This identified consistent loss of immunohistochemical expression of Merlin in breast tumor tissues. Thus, we demonstrate for the first time a role for Merlin in impeding breast malignancy, identify a novel mechanism for the loss of Merlin protein in breast cancer, and have developed a discriminatory model using Merlin and OPN expression in breast tumor tissues.

Programmable nanowire circuits for nanoprocessors.

A nanoprocessor constructed from intrinsically nanometre-scale building blocks is an essential component for controlling memory, nanosensors and other functions proposed for nanosystems assembled from the bottom up. Important steps towards this goal over the past fifteen years include the realization of simple logic gates with individually assembled semiconductor nanowires and carbon nanotubes, but with only 16 devices or fewer and a single function for each circuit. Recently, logic circuits also have been demonstrated that use two or three elements of a one-dimensional memristor array, although such passive devices without gain are difficult to cascade. These circuits fall short of the requirements for a scalable, multifunctional nanoprocessor owing to challenges in materials, assembly and architecture on the nanoscale. Here we describe the design, fabrication and use of programmable and scalable logic tiles for nanoprocessors that surmount these hurdles. The tiles were built from programmable, non-volatile nanowire transistor arrays. Ge/Si core/shell nanowires coupled to designed dielectric shells yielded single-nanowire, non-volatile field-effect transistors (FETs) with uniform, programmable threshold voltages and the capability to drive cascaded elements. We developed an architecture to integrate the programmable nanowire FETs and define a logic tile consisting of two interconnected arrays with 496 functional configurable FET nodes in an area of ∼960 µm(2). The logic tile was programmed and operated first as a full adder with a maximal voltage gain of ten and input-output voltage matching. Then we showed that the same logic tile can be reprogrammed and used to demonstrate full-subtractor, multiplexer, demultiplexer and clocked D-latch functions. These results represent a significant advance in the complexity and functionality of nanoelectronic circuits built from the bottom up with a tiled architecture that could be cascaded to realize fully integrated nanoprocessors with computing, memory and addressing capabilities.

Hedgehog signaling induced by breast cancer cells promotes osteoclastogenesis and osteolysis.

Bone integrity is maintained by a dynamic equilibrium between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteolytic lesions are a painful consequence of metastasis of breast cancer cells to bone in an overwhelming majority of breast cancer patients. Factors secreted by breast cancer cells propel a cascade of events that trigger osteoclastogenesis and elevated bone resorption. In the present study, we show that the Hedgehog (Hh) ligands secreted by breast cancer cells promote osteoclast differentiation and potentiate the activity of mature osteoclasts. Paracrine Hh signaling induced by breast cancer cells mediates a detrimental chain of events by the up-regulation of osteopontin (OPN), which in turn enhances osteoclastic activity by up-regulating cathepsin K and MMP9. Hh signaling is essential for osteoclasts because blocking the Hh pathway using the pharmacological Hh inhibitor, cyclopamine, results in an overall decrease in osteoclastogenesis and resorptive activity. Our studies suggest that inhibiting Hh signaling interferes with the ability of pre-osteoclasts to respond to the stimulatory effects of the breast cancer cells, indicating that Hh signaling is vital to osteoclast activity.

Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7).

Background. Vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We studied the effect of ATRA on MMP-2 in MCF-7, human breast cancer cells, and the probable signaling pathways which are affected by ATRA on regulating pro-MMP-2 activity and expression. Methods. Gelatin zymography, RT-PCR, ELISA, Western blot, Immunoprecipitation, and Cell adhesion assay are used. Results. Gelatin zymography showed that ATRA caused a dose-dependent inhibition of pro-MMP-2 activity. ATRA treatment downregulates the expression of MT1-MMP, EMMPRIN, FAK, NF-kB, and p-ERK. However, expression of E-cadherin, RAR, and CRABP increased upon ATRA treatment. Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment. Conclusions. The experimental findings clearly showed the inhibition of MMP-2 activity upon ATRA treatment. This inhibitory effect of ATRA on MMP-2 activity in human breast cancer cells (MCF-7) may result due to its inhibitory effect on MT1-MMP, EMMPRIN, and upregulation of TIMP-2. This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

The hedgehog pathway transcription factor GLI1 promotes malignant behavior of cancer cells by up-regulating osteopontin.

The role of Hedgehog (Hh) signaling as a developmental pathway is well established. Several recent studies have implicated a role for this pathway in multiple cancers. In this study we report that expression of GLI1 and osteopontin (OPN) increase progressively with the progression of melanoma from primary cutaneous cancer to metastatic melanoma in clinically derived specimens. We have further determined that OPN is a direct transcriptional target of GLI1. We have observed that OPN expression is stimulated in the presence of Hh ligands and inhibited in the presence of the Smoothened (SMO) inhibitor, cyclopamine. Transcriptional silencing of GLI1 negatively impacts OPN expression and compromises the ability of cancer cells to proliferate, migrate, and invade in vitro and interferes with their ability to grow as xenografts and spontaneously metastasize in nude mice. These altered attributes could be rescued by re-expressing OPN in the GLI1-silenced cells, suggesting that OPN is a critical downstream effector of active GLI1 signaling. Our observations lead us to conclude that the GLI1-mediated up-regulation of OPN promotes malignant behavior of cancer cells.

Performance simulation and analysis of a CMOS/nano hybrid nanoprocessor system.

This paper provides detailed simulation results and analysis of the prospective performance of hybrid CMOS/nanoelectronic processor systems based upon the field-programmable nanowire interconnect (FPNI) architecture. To evaluate this architecture, a complete design was developed for an FPNI implementation using 90 nm CMOS with 15 nm wide nanowire interconnects. Detailed simulations of this design illustrate that critical design choices and tradeoffs exist beyond those specified by the architecture. This includes the selection of the types of junction nanodevices, as well as the implementation of low-level circuits. In particular, the simulation results presented here show that only nanodevices with an 'on/off' current ratio of 200 or more are suitable to produce correct system-level behaviour. Furthermore, the design of the CMOS logic gates in the FPNI system must be customized to accommodate the resistances of both 'on'-state and 'off'-state nanodevices. Using these customized designs together with models of suitable nanodevices, additional simulations demonstrate that, relative to conventional 90 nm CMOS FPGA systems, performance gains can be obtained of up to 70% greater speed or up to a ninefold reduction in energy consumption.

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7.

AIMS: The tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7. MAIN METHODS: MCF-7 was treated with EGCG (20 muM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay. KEY FINDINGS: EGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors alpha5, beta1, alphav and beta3 due to EGCG treatment. SIGNIFICANCE: Down regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.

Culture of human A375 melanoma cells in the presence of fibronectin causes expression of MMP-9 and activation of MMP-2 in culture supernatants.

UNLABELLED: Interactions between tumor cell surface integrin receptors and extracellular matrix (ECM) ligands play an important role in tumor development, affecting cell survival, proliferation, and migration. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen-activated protein kinase (MAPK) pathways. It has been reported that integrins also regulate expression and function of matrix metalloproteinases (MMPs). In this present work, we cultured human A375 melanoma cells in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: A375 cells were cultured in serum-free culture medium (SFCM) in the presence of fibronectin (25 microg/0.75 ml), SFCM was collected and gelatin zymography was performed. Western blot and RT-PCR were performed with A375 cells cultured in the presence of fibronectin. RESULTS: Culture of A375 cells in the presence of fibronectin led to expression of MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) or PI-3K inhibitor (LY294002) and grown in the presence of fibronectin, MMP-9 expression and MMP-2 activation was inhibited. Tyrosine phosphorylation of FAK and ERK were increased in A375 cells grown in the presence of fibronectin. Increased MMP-9 mRNA expression and processing of MT1-MMP were also observed in A375 cells grown in the presence of fibronectin. CONCLUSIONS: Our findings indicate culture of A375 cells in SFCM in the presence of fibronectin perhaps generates a signaling cascade that leads to expression of MMP-9 and activation of MMP-2 in culture supernatants within 2 h. The signaling pathway activated is probably the FAK/ERK pathway.

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium.

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.

Cutting Edge: Stimulation of dopamine D4 receptors induce T cell quiescence by up-regulating Kruppel-like factor-2 expression through inhibition of ERK1/ERK2 phosphorylation.

The neurotransmitter dopamine (DA) is an important regulator of human T cell functions. Although it has been observed that DA, by acting through the D1/D5, D2, and D3 receptors, can activate resting T cells by stimulating the release of cytokines and the expression of surface integrins and also inhibit the proliferation of activated T cells by down-regulating nonreceptor tyrosine kinases, there is not yet a report indicating the functional significance of the D4 DA receptors present in these cells. The present work, for the first time, demonstrates that the stimulation of D4 DA receptors in human T cells induces T cell quiescence by up-regulating lung Krüppel-like factor-2 expression through the inhibition of ERK1/ERK2 phosphorylation. These results reveal a new link between the nervous system and T cell quiescence and indicate that D4 DA receptor agonists may have a therapeutic value in diseases with uncontrolled T cell proliferation.