Steven A. Rosenzweig

Oleic Acid–Induced Mitogenic Signaling in Vascular Smooth Muscle Cells A Role for Protein Kinase C

As an initial step in testing the hypothesis that high oleic acid concentrations contribute to vascular remodeling in obese hypertensive patients by activating protein kinase C (PKC), the effects of oleic acid on primary cultures of rat aortic smooth muscle cells (RASMCs) were studied. Oleic acid, an 18-carbon cis-monounsaturated fatty acid (18:1 [cis]), from 25 to 200 mumol/L significantly increased [3H]thymidine uptake in RASMCs with an EC50 of 41.0 mumol/L and a maximal response of 196 +/- 15% of control (P < .01). Oleic acid from 25 to 200 mumol/L caused a concentration-dependent increase in the number of RASMCs in culture at 6 days, reaching a maximum of 210 +/- 13% of control at 100 mumol/L (P < .001). PKC inhibition with 4 mumol/L bisindolyImaleimide I and PKC depletion (alpha, mu, iota, and zeta) with 24-hour exposure to 200 nmol/L phorbol 12-myristate 13-acetate in RASMCs eliminated the mitogenic effects of oleic acid but did not reduce responses to 10% FBS. Stimulation of intact cells with oleic acid induced a peak increase of cytosolic PKC activity, reaching 328 +/- 8% of control (P < .001), but did not enhance PKC activity in the membrane fraction (105 +/- 4%, P = NS). The oleic acid-induced increase of PKC activity in cell lysates was similar in the presence and absence of Ca2+, phosphatidylserine, and diolein (maximum response, 360 +/- 4% versus 342 +/- 9% of control, P = NS). Unlike phorbol 12-myristate 13-acetate, oleic acid over 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs. Oleic acid treatment activated mitogen-activated protein (MAP) kinase. PKC depletion in RASMCs eliminated the rise in thymidine uptake, activation of PKC, and activation of MAP kinase in response to oleic acid. In contrast to oleic acid, 50 to 200 mumol/L stearic (18:0) and elaidic (18:1 [trans]) acids, which are less effective activators of PKC than oleic acid, did not enhance thymidine uptake. These data suggest that oleic acid induces proliferation of RASMCs by activating PKC, particularly one or more of the Ca(2+)-independent isoforms, and raise the possibility that the higher oleic acid concentrations observed in obese hypertensive patients may contribute to vascular remodeling.

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.

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.

ACTIVATION OF C-JUN N-TERMINAL KINASE/STRESS-ACTIVATED PROTEIN KINASE IN PRIMARY GLIAL CULTURES

Glial cells in the mammalian CNS are subject to environmental stress resulting from a variety of neuropathological conditions. In this study, we have examined the activation of a stress signal responsive kinase, i.e., stress‐activated protein kinase (SAPK) or c‐Jun N‐terminal kinase (JNK), in primary cultures of rat brain glial cells (i.e., astrocytes and oligodendrocytes) and an oligodendrocyte progenitor cell line, CG4, in response to cytokines and other stress inducers. JNK/SAPK activity was measured by an immune complex kinase assay using polyclonal anti‐JNK antibodies along with GST c‐Jun (1‐79) as the substrate. Among the cytokines tested, TNF‐α had the strongest effect on JNK activation followed by TNF‐β in both the glial cell types while a substantial level of kinase activation was observed in response to IL‐1 in astrocytes. JNK activation by TNF‐α in astrocytes, but not in oligodendrocytes, showed a biphasic response. An in‐gel kinase assay of cell extracts and immunoprecipitated JNK confirmed the activation of JNK1 in cells treated with TNF‐α. JNK was also activated by several other stress‐inducing factors including UV light, heat shock, inhibitors of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase and a cell‐permeable ceramide stimulated JNK activity, suggesting that the ceramide pathway may play a role in JNK activation, although the time course of activation did not correspond to that of TNF‐α. The results are discussed in terms of possible roles of JNK activation in signaling for gliosis in astrocytes and as a protective/toxic response in oligodendrocytes.

Hypoxia-Inducible Factor-1-Dependent and -Independent Regulation of Insulin-Like Growth Factor-1-Stimulated Vascular Endothelial Growth Factor Secretion

Hypoxia-induced stress plays a central role in retinal vascular disease and cancer. Increased hypoxia-inducible factor-1α (Hif-1α) expression leads to HIF-1 formation and the production of vascular endothelial growth factor (VEGF). Cytokines, including insulin-like growth factor-1 (IGF-1), also stimulate VEGF secretion. In this study, we examined the relationship between IGF-1 signaling, HIF-1α protein turnover and VEGF secretion in the ARPE-19 retinal pigment epithelial cell line. Northern analysis revealed that IGF-1 stimulated Hif-1α message expression, whereas the hypoxia-mimetic CoCl2 did not. CoCl2 treatment increased Hif-1α protein accumulation to a greater extent than IGF-1 treatment. However, IGF-1 stimulated a more significant increase in VEGF secretion. IGF-1-stimulated VEGF promoter activity was phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent, whereas VEGF secretion was only partially reduced by inhibition of PI3K/Akt/mTOR and HIF-1 activities. Analysis of VEGF promoter truncation mutants indicated that sensitivity to CoCl2 was hypoxia response element (HRE)-dependent with the region upstream of the HRE conferring IGF-1 sensitivity. In conclusion, IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways.

Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent.

We previously reported a vascular endothelial growth factor (VEGF) autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach, we screened the HNSCC cell line, squamous cell carcinoma-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation 1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion and matrix metalloproteinase (MMP) expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix-coated coverslips showed enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared with a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also show for the first time that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1s recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.

Oleic Acid and Angiotensin II Induce a Synergistic Mitogenic Response in Vascular Smooth Muscle Cells

Oleic acid and angiotensin II (Ang II) are elevated and may interact to accelerate vascular disease in obese hypertensive patients. We studied the effects of oleic acid and Ang II on growth responses of rat aortic smooth muscle cells (VSMCs). Oleic acid (50 micromol/L) raised thymidine incorporation by 50% at 24 hours and cell number by 55% at 6 days (P<.05). Ang II (10(-11) to 10(-6) mol/L) did not significantly increase thymidine incorporation or VSMC number. Combining Ang II and 50 micromol/L oleic acid doubled thymidine incorporation and VSMC number. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked the synergistic interaction between Ang II and oleic acid, whereas the AT2 receptor antagonist PD 123319 did not. Protein kinase C inhibition and downregulation, as well as inhibition of extracellular signal-regulated kinase (ERK) activation by PD 98059, eliminated the rise of thymidine incorporation in response to oleic acid and the synergistic interaction with Ang II. However, the response to 10% fetal bovine serum was unaffected. An antisense oligodeoxynucleotide to ERK-1 and ERK-2 reduced ERK protein expression and activation by 83% and 75%, respectively. Antisense prevented the rise of thymidine incorporation in response to oleic acid and the synergy with Ang II. Antisense reduced but did not prevent increased thymidine incorporation in response to serum. The data indicate that oleic acid and Ang II exert a synergistic mitogenic effect in VSMCs and suggest an important role for the AT1 receptor, PKC, and ERK in this synergy. The observations raise the possibility that a synergistic mitogenic interaction between oleic acid and Ang II accelerates vascular remodeling in obese hypertensive patients.

Elevated glucose increases mesangial cell sensitivity to insulin-like growth factor I.

To determine the effects of glucose on insulin-like growth factor I (IGF-I)-induced mesangial cell (MC) proliferation, we have examined the relationships between IGF binding protein 2 (IGFBP-2) secretion and proliferation in murine MCs (MMCs). MMCs incubated in high glucose (HG, 25 mM) exhibited a 25-30% reduction in IGFBP-2 secretion compared with cells in normal glucose (NG, 5.6 mM). This loss was not due to cell surface binding; it correlated with a 3.1-fold decrease in IGFBP-2 mRNA. IGFBP-2 secretion was stimulated by IGF-I in NG but was unaltered in HG. Insulin treatment yielded similar results at 10-fold higher doses, indicating that this response is IGF-I receptor dependent. MMCs in HG displayed increased IGF-I-stimulated insulin receptor substrate-1/2 phosphorylation and activator protein-1 transcriptional activity compared with NG controls. Accordingly, although IGF-I was not proliferative in NG, it increased [3H]thymidine incorporation and cell number in HG to an extent proportional to the decrease in IGFBP-2. Thus hyperglycemia, as seen in diabetes, may increase MC IGF-I sensitivity by reducing IGFBP-2 expression, in turn increasing its proliferative and secretory responses and contributing to the development of diabetic glomerulosclerosis.

Secretion of vascular endothelial growth factor by oral squamous cell carcinoma cells skews endothelial cells to suppress T-cell functions

Patients with oral squamous cell carcinoma (OSCC) have severe defects in antitumor immune function. Endothelial cells are potential regulators of immune cell function and have therefore been examined to determine their role in tumor-induced immune suppression. The present studies demonstrated that supernatants from endothelial cells exposed to OSCC-conditioned media (endo(OSCC-sup)) exhibited elevated levels of the immune suppressive products prostaglandin E(2) (PGE(2)) and vascular endothelial growth factor (VEGF) compared with supernatants from endothelial cells treated with medium alone (endo(medium)) or with keratinocyte-conditioned medium (endo(ker-sup)). Antibody neutralization of OSCC-derived VEGF prevented tumor-conditioned media from inducing endothelial cells to increase production of PGE(2)and VEGF. Furthermore, treatment of T-cells with supernatants from endo(OSCC-sup) resulted in diminished T-cell proliferation and decreased interferon-gamma (IFN-gamma) production compared with T-cells treated with medium or supernatants from endo(medium) or endo(ker-sup) controls. T-cell levels of granzyme B and perforin were reduced after treatment with supernatant from endo(OSCC-sup) compared with control treatments. The addition of VEGF neutralizing antibody to the OSCC-conditioned medium prevented endothelial cells from being skewed to downregulate T-cell proliferation and production of IFN-gamma, perforin, and granzyme B. Taken together, these studies provide support for the use of VEGF-targeting therapies as an immunotherapeutic agent to block induction of immune suppressive endothelial cells in patients with OSCC.

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.