IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

The peroxidase PRDX1 inhibits the activated phenotype in mammary fibroblasts through regulating c-Jun N-terminal kinases.

BACKGROUND: Reactive oxygen species (ROS), including hydrogen peroxide, drive differentiation of normal fibroblasts into activated fibroblasts, which can generate high amounts of hydrogen peroxide themselves, thereby increasing oxidative stress in the microenvironment. This way, activated fibroblasts can transition into cancer-associated fibroblasts (CAFs). METHODS: Mammary fibroblasts from either female 8 weeks old PRDX1 knockout and wildtype mice or Balb/c mice were studied for characteristic protein expression using immunofluorescence and immunoblotting. Cancer-associated fibroblasts was examined by transwell migration and invasion assays. The binding of PRDX1 to JNK1 was assessed by co-immuneprecipitation and JNK regulation of CAF phenotypes was examined using the JNK inhibitor SP600125. Extracellular hydrogen peroxide levels were measured by chemiluminescence via the reaction between hypochlorite and luminol. Statistical analyses were done using Students t-test. RESULTS: We show here PRDX1 activity as an essential switch in regulating the activated phenotype as loss of PRDX1 results in the development of a CAF-like phenotype in mammary fibroblasts. We also show that PRDX1 regulates JNK kinase signaling thereby inhibiting CAF-like markers and CAF invasion. Inhibition of JNK activity reduced these behaviors. CONCLUSIONS: These data suggest that PRDX1 repressed the activated phenotype of fibroblasts in part through JNK inhibition which may present a novel therapeutic option for CAF-enriched cancers such as breast cancer.

An ultra-stable redox-controlled self-assembling polypeptide nanotube for targeted imaging and therapy in cancer.

We introduce a self-assembling polypeptide-based nanotube system having the ability to specifically target cancer cells. The nanotubes target the cancer cell surface through integrin engagement with the help of multiple RGD units present along their surface. While the nanotubes are non-toxic towards cells in general, they can be loaded with suitable drugs to be released in a sustained manner in cancer cells. In addition, the nanotubes can be utilized for cellular imaging using any covalently tagged fluorescent dye. They are stable over a wide range of temperature due to intermolecular disulphide bonds formed during the self-assembly process. At the same time, presence of disulphide bonds provides a redox molecular switch for their degradation. Taken together this system provides a unique avenue for multimodal formulation in cancer therapy.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2.

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K(D) approximately 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Escherichia coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP.

The Continuing Evolution of Insulin-like Growth Factor Signaling.

The insulin-like growth factors (IGFs; IGF1/IGF2), known for their regulation of cell and organismal growth and development, are evolutionarily conserved ligands with equivalent peptides present in flies ( D. melanogaster), worms ( C. elegans) among others. Two receptor tyrosine kinases, the IGF1 receptor and the insulin receptor mediate the actions of these ligands with a family of IGF binding proteins serving as selective inhibitors of IGF1/2. This treatise reviews recent findings on IGF signaling in cancer biology and central nervous system function. This includes overexpression of IGF1 receptors in enhancing tumorigenesis, acquired resistance and contributions to metastasis in multiple cancer types. There is accumulating evidence that insulin resistance, a hallmark of type 2 diabetes, occurs in the central nervous system, independent of systemic insulin resistance and characterized by reduced insulin and IGF1 receptor signaling, and may contribute to dementias including Alzheimer's Disease and cognitive impairment. Controversy over the role(s) of IGF signaling in cancer and whether its inhibition would be of benefit, still persist and extend to IGF1's role in longevity and central nervous system function.

An assessment of racial differences in epidemiological, clinical and psychosocial factors among head and neck cancer patients at the time of surgery.

OBJECTIVE: Racial disparities have been well characterized and African American (AA) patients have 30% lower 5-year survival rates than European Americans (EAs) for head and neck squamous carcinoma (HNSCC). This poorer survival can be attributed to a myriad of different factors. The purpose of this study was to characterize AA-EA similarities and differences in sociodemographic, lifestyle, clinical, and psychosocial characteristics in HNSCC patients near the time of surgery. METHODS: Setting: Single tertiary care center. Participants: Thirty-nine newly diagnosed, untreated HNSCC patients (n = 24 EAs,n = 15 AAs) who were to undergo surgery were recruited. Study Design: Cross-sectional study Sociodemographic, lifestyle factors, and disease factors (cancer site, AJCC clinical and pathologic stage, and HPV status)were assessed. Risk factors, leisure time, quality of life and social support were also assessed using validated questionnaires. Exposures: EA and AA patients were similar in the majority of sociodemographic factors assessed. AAs had a higher trend toward pathologically later stage disease compared to EAs and significantly increased time to treatment. RESULTS: EA and AA patients were similar in the majority of sociodemographic factors assessed. AAs had a higher trend toward pathologically later stage disease compared to EAs. AAs also had significantly increased time to treatment (P = 0.05). The majority of AA patients (62%) had later stage pathologic disease. AA were less likely to complete high school or college (P = 0.01) than their EA counterparts. Additionally, AAs were more likely to report having a gap in health insurance during the past decade (37% vs. 15%). CONCLUSIONS: This preliminary study demonstrates a similar profile of demographics, clinical and psychosocial characteristics preoperatively for AAs and EAs. Key differences were AAs tending to have later pathologic stage disease, educational status, delays in treatment initiation, and gaps in health insurance.

Vimentin and Non-Muscle Myosin IIA are Members of the Neural Precursor Cell Expressed Developmentally Down-Regulated 9 (NEDD9) Interactome in Head and Neck Squamous Cell Carcinoma Cells.

Here we demonstrate an interaction between neural precursor cell expressed, developmentally-downregulated 9 (NEDD9) and the cytoskeletal proteins vimentin and non-muscle myosin IIA (NMIIA), based on co-immunoprecipitation and mass spectrometric sequence identification. Vimentin was constitutively phosphorylated at Ser56 but vimentin associated with NEDD9-was not phosphorylated at Ser56. In contrast, NMIIA bound to NEDD9 was phosphorylated on S1943 consistent with its function in invasion and secretion. Treatment of cells with the vimentin-targeting steroidal lactone withaferin A had no effect on vimentin turnover as previously reported, instead causing NEDD9 cleavage and cell death. The NMIIA-selective inhibitor blebbistatin induced cells to form long extensions and attenuated secretion of matrix metalloproteinases (MMPs) 2 and 9. While the site of vimentin interaction on NEDD9 was not defined, NMIIA was found to interact with NEDD9 at its substrate domain. NEDD9 interactions with vimentin and NMIIA are consistent with these proteins having roles in MMP secretion and cell invasion. These findings suggest that a better understanding of NEDD9 signaling is likely to reveal novel therapeutic targets for the prevention of invasion and metastasis.

Acquired Resistance to Drugs Targeting Tyrosine Kinases.

Resistance to chemotherapeutic drugs exemplifies the greatest hindrance to effective treatment of cancer patients. The molecular mechanisms responsible have been investigated for over 50 years and have revealed the lack of a single cause, but instead, multiple mechanisms including induced expression of membrane transporters that pump drugs out of cells (multidrug resistance (MDR) phenotype), changes in the glutathione system, and altered metabolism. Treatment of cancer patients/cancer cells with chemotherapeutic agents and/or molecularly targeted drugs is accompanied by acquisition of resistance to the treatment administered. Chemotherapeutic agent resistance was initially assumed to be due to induction of mutations leading to a resistant phenotype. While this has occurred for molecularly targeted drugs, it is clear that drugs selectively targeting tyrosine kinases (TKs) cause the acquisition of mutational changes and resistance to inhibition. The first TK to be targeted, Bcr-Abl, led to the generation of several drugs including imatinib, dasatinib, and sunitinib that provided a rich understanding of this phenomenon. It became clear that mutations alone were not the only cause of resistance. Additional mechanisms were involved, including alternative splicing, alternative/compensatory signaling pathways, and epigenetic changes. This review will focus on resistance to tyrosine kinase inhibitors (TKIs), receptor TK (RTK)-directed antibodies, and antibodies that inactivate specific RTK ligands. New approaches and concepts aimed at avoiding the generation of drug resistance will be examined. Many RTKs, including the IGF-1R, are dependence receptors that induce ligand-independent apoptosis. How this signaling paradigm has implications on therapeutic strategies will also be considered.

NEDD9 stimulated MMP9 secretion is required for invadopodia formation in oral squamous cell carcinoma.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3-SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.

Insulin-like growth factor-1 receptor and ligand targeting in head and neck squamous cell carcinoma.

IGF-1 receptor (IGF-1R) signaling is associated with increased tumorigenesis of epithelial cancers. In light of recent epidemiological studies correlating high circulating levels of IGF-1 with increased risk of second primary tumors (SPTs) of the head and neck, we examined IGF system and epidermal growth factor receptor (EGFR) expression in human head and neck squamous cell carcinoma (HNSCC) matched pairs and a cross-section of HNSCC cell lines. Employing the latter, we demonstrated that IGF-1 stimulated S-phase transition in a PI 3-K/Akt and Erk-dependent manner in 5 of 8 cell lines, with Erk activation being dependent upon EGFR kinase activity. IGF-1 stimulated thymidine incorporation was inhibited by treatment with IGFBP-3, the IGF-1R tyrosine kinase inhibitor NVP-AEW541, or the EGFR tyrosine kinase inhibitor AG1478. Combining IGFBP-3 with NVP-AEW541 or AG1478 abrogated IGF-1 responses at 10-fold lower doses than either compound alone. These results demonstrate the potential for co-targeting the IGF system and EGFR in HNSCC.

The tumor suppressor capability of p53 is dependent on non-muscle myosin IIA function in head and neck cancer.

Over 300,000 patients develop squamous cell carcinoma of the head and neck (HNSCC) worldwide with 25-30% of patients ultimately dying from their disease. Currently, molecular biomarkers are not used in HNSCC but several genes have been identified including mutant TP53 (mutp53). Our recent work has identified an approach to stratify patients with tumors harboring high or low risk TP53 mutations. Non-muscle Myosin IIA (NMIIA) was recently identified as a tumor suppressor in HNSCC. We now demonstrate that low MYH9 expression is associated with decreased survival in patients with head and neck cancer harboring low-risk mutp53 but not high-risk mutp53. Furthermore, inhibition of NMIIA leads to increased invasion in cells harboring wildtype p53 (wtp53), which was not observed in high-risk mutp53 cells. This increased invasiveness of wtp53 following NMIIA inhibition was associated with reduced p53 target gene expression and was absent in cells expressing mutp53. This reduced expression may be due, in part, to a decrease in nuclear localization of wtp53. These findings suggest that the tumor suppressor capability of wtp53 is dependent upon functional NMIIA and that the invasive phenotype of high-risk mutp53 is independent of NMIIA.

Low insulin-like growth factor binding protein-2 expression is responsible for increased insulin receptor substrate-1 phosphorylation in mesangial cells from mice susceptible to glomerulosclerosis.

Mesangial cells (MC) isolated from glomerulosclerosis-prone ragged, olygosyndactilism, pintail (ROP) mice retain a stable phenotype after exposure to elevated glucose concentrations, whereas MC from glomerulosclerosis-resistant C57BL/6 (C) mice do not. In NOD and db/db mice, the stable phenotype induced by diabetes consists of autocrine activation of the IGF-I signaling pathway. We hypothesized that high ambient glucose activates the IGF-I pathway in ROP but not in C MC. MC were propagated in either 6 or 25 mm glucose. Isolated murine glomeruli were used to confirm in vitro experiments. 25 mm glucose induced increased insulin receptor substrate (IRS)-1 phosphorylation in ROP but not C MC. However, IGF-I, IGF-I receptor, and IRS-1 protein levels were induced by exposure to 25 mm glucose in both cell lines. This occurred without a change in IGF-I binding sites, suggesting a role for IGF binding protein (IGFBP). ROP MC and glomeruli expressed less IGFBP-2 than C MC and glomeruli. Addition of exogenous IGFBP-2 partially blunted the effect of 25 mm glucose on IRS-1 phosphorylation in ROP MC. Renal biopsies from patients with diabetic nephropathy also showed markedly decreased IGFBP-2 expression when compared with patients without nephropathy. In summary, glucose induces IRS-1 phosphorylation in MC isolated from ROP mice susceptible to glomerulosclerosis. IGFBP-2 expression was low in ROP MC and glomeruli from patients with diabetic nephropathy, suggesting that this may represent a new marker of susceptibility to diabetic nephropathy. Finally, addition of exogenous IGFBP-2 in ROP MC partially blunted the effect of high glucose on IRS-1 phosphorylation and might have a protective role.

Paradoxical effects of the phage display-derived peptide antagonist IGF-F1-1 on insulin-like growth factor-1 receptor signaling.

The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1, (125)I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [(3)H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [(3)H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor.

IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407.

PURPOSE: To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1 alpha and secretion of vascular endothelial growth factor (VEGF) and IGF binding protein (IGFBP)-3 in human retinal pigment epithelial (RPE) cell line D407. METHODS: D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1 alpha expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1 alpha, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na(+)/K(+)-ATPase alpha-1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy. RESULTS: Immunoblot analysis of whole cell lysates from IGF-1-treated D407 cells revealed the upregulation of HIF-1 alpha protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1 alpha expression and nuclear localization, VEGF and IGFBP-3 synthesis and export, and IGF-1 action. Western and ligand blot analyses of RPE cell-conditioned medium indicated that IGF-1 induced increases in VEGF and IGFBP-3 secretion. Cells grown on Transwell inserts exhibited constitutive apical secretion of VEGF and IGFBP-3, which increased on apical or basolateral treatment with IGF-1. Confocal analysis of Transwell-cultured D407 cells confirmed the apical localization of the Na(+)/K(+)-ATPase alpha-1 subunit, characteristic of polarized RPE, with IGF-1 receptor alpha and beta subunits exhibiting a nonpolarized distribution. CONCLUSIONS: IGF-1 stimulates increased HIF-1 alpha expression as well as VEGF and IGFBP-3 secretion in D407 cells. Similar to their in vivo counterparts, D407 cells maintain reversed epithelial polarity. Apical secretion of VEGF and IGFBP-3 increases in response to either apical or basolateral IGF-1 stimulation consistent with the nonpolarized distribution of IGF-1 receptors.

What's new in the IGF-binding proteins?

Since their initial discovery over 25 years ago as IGF carrier proteins, the insulin-like growth factor binding protein (IGFBP) family has grown to six members, ranging in size from 216 to 289 amino acids. The assumption over the years has been that this family of proteins, having higher affinities for IGF-I and IGF-II than does the IGF-IR, serves to block access of these ligands to the receptor. Although the need for such regulatory proteins is consistent with the constitutive secretion of IGFs from many cell types, it is not surprising that additional functions have begun to be uncovered for these proteins. This review will examine new and old actions of the IGFBPs from a biochemical and cell biological perspective.

Role of oxidative stress and the microenvironment in breast cancer development and progression.

Breast cancer is a highly complex tissue composed of neoplastic and stromal cells. Carcinoma-associated fibroblasts (CAFs) are commonly found in the cancer stroma, where they promote tumor growth and enhance vascularity in the microenvironment. Upon exposure to oxidative stress, fibroblasts undergo activation to become myofibroblasts. These cells are highly mobile and contractile and often express numerous mesenchymal markers. CAF activation is irreversible, making them incapable of being removed by nemosis. In breast cancer, almost 80% of stromal fibroblasts acquire an activated phenotype that manifests by secretion of elevated levels of growth factors, cytokines, and metalloproteinases. They also produce hydrogen peroxide, which induces the generation of subsequent sets of activated fibroblasts and tumorigenic alterations in epithelial cells. While under oxidative stress, the tumor stroma releases high energy nutrients that fuel cancer cells and facilitate their growth and survival. This review describes how breast cancer progression is dependent upon oxidative stress activated stroma and proposes potential new therapeutic avenues.

Acquired resistance to drugs targeting receptor tyrosine kinases.

Development of resistance to chemotherapeutic drugs represents a significant hindrance to the effective treatment of cancer patients. The molecular mechanisms responsible have been investigated for over half a century and have revealed the lack of a single cause. Rather, a multitude of mechanisms have been delineated ranging from induction and expression of membrane transporters that pump drugs out of cells (multidrug resistance (MDR) phenotype), changes in the glutathione system and altered metabolism to name a few. Treatment of cancer patients/cancer cells with chemotherapeutic agents and/or molecularly targeted drugs is accompanied by acquisition of resistance to the treatment administered. Chemotherapeutic agent resistance was initially assumed to be due to induction of mutations leading to a resistant phenotype. This has also been true for molecularly targeted drugs. Considerable experience has been gained from the study of agents targeting the Bcr-Abl tyrosine kinase including imatinib, dasatinib and sunitinib. It is clear that mutations alone are not responsible for the many resistance mechanisms in play. Rather, additional mechanisms are involved, ranging from epigenetic changes, alternative splicing and the induction of alternative/compensatory signaling pathways. In this review, resistance to receptor tyrosine kinase inhibitors (RTKIs), RTK-directed antibodies and antibodies that inactivate ligands for RTKs are discussed. New approaches and concepts aimed at avoiding the generation of drug resistance will be examined. The recent observation that many RTKs, including the IGF-1R, are dependence receptors that induce apoptosis in a ligand-independent manner will be discussed and the implications this signaling paradigm has on therapeutic strategies will be considered.

Loss of expression and function of SOCS3 is an early event in HNSCC: altered subcellular localization as a possible mechanism involved in proliferation, migration and invasion.

BACKGROUND: Suppressor of cytokine signaling 3 (SOCS3) is an inducible endogenous negative regulator of signal transduction and activator of transcription 3 (STAT3). Epigenetic silencing of SOCS3 has been shown in head and neck squamous cell carcinoma (HNSCC), which is associated with increased activation of STAT3. There is scarce information on the functional role of the reduction of SOCS3 expression and no information on altered subcellular localization of SOCS3 in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We assessed endogenous SOCS3 expression in different HNSCC cell lines by RT-qPCR and western blot. Immunofluorescence and western blot were used to study the subcellular localization of endogenous SOCS3 induced by IL-6. Overexpression of SOCS3 by CMV-driven plasmids and siRNA-mediated inhibition of endogenous SOCS3 were used to verify the role of SOCS3 on tumor cell proliferation, viability, invasion and migration in vitro. In vivo relevance of SOCS3 expression in HNSCC was studied by quantitative immunohistochemistry of commercially-available tissue microarrays. Endogenous expression of SOCS3 was heterogeneous in four HNSCC cell lines and surprisingly preserved in most of these cell lines. Subcellular localization of endogenous SOCS3 in the HNSCC cell lines was predominantly nuclear as opposed to cytoplasmic in non-neoplasic epithelial cells. Overexpression of SOCS3 produced a relative increase of the protein in the cytoplasmic compartment and significantly inhibited proliferation, migration and invasion, whereas inhibition of endogenous nuclear SOCS3 did not affect these events. Analysis of tissue microarrays indicated that loss of SOCS3 is an early event in HNSCC and was correlated with tumor size and histological grade of dysplasia, but a considerable proportion of cases presented detectable expression of SOCS3. CONCLUSION: Our data support a role for SOCS3 as a tumor suppressor gene in HNSCC with relevance on proliferation and invasion processes and suggests that abnormal subcellular localization impairs SOCS3 function in HNSCC cells.

Defining the pathway to insulin-like growth factor system targeting in cancer.

The insulin-like growth factors (IGFs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the IGF system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies. The IGF-binding proteins (IGFBPs) represent the third component of the IGF system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring IGF-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped "third" class of IGF-1R inhibitors. In this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold.

Tumor secretion of VEGF induces endothelial cells to suppress T cell functions through the production of PGE2.

Endothelial cells are potent regulators of immune cell functions and have therefore been examined to determine their role in tumor-induced immune suppression. Previous studies by our laboratory showed that exposure to Lewis lung carcinoma (LLC)-secreted products induced endothelial cells to suppress T-cell functions in vitro. The current studies examined in vitro and in vivo the mechanism by which tumors induce the formation of suppressor endothelial cells and the means by which suppressor endothelial cells disrupt T-cell functions. In vitro studies demonstrated that inhibition of tumor-derived VEGF with neutralizing antibodies or treatment of endothelial cells with the VEGF receptor tyrosine kinase inhibitor, SU5416, prevented endothelial cells from being induced to suppress T-cell functions. Treatment of tumor-bearing mice with SU5416 blocked the development of endothelial cells that are suppressive to CD4 and CD8 T-cell functions. We next examined the role of suppressor endothelial cell-derived PGE2 in the inhibition of T-cell functions. Abrogation of endothelial cell PGE2 production in vitro with indomethacin prevented tumor-conditioned media from stimulating endothelial cell production of immune inhibitory activity toward T-cell functions. Similar treatment of endothelial cells from lungs of tumor-bearing mice blocked their capacity to produce T-cell-inhibitory mediators. These studies demonstrate that tumor-derived VEGF induces endothelial cells to upregulate production of PGE2 which, in turn, leads to suppression of T-cell functions.