Kevin F. Dyer

Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth

The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers including squamous cell carcinoma of the head and neck (SCCHN). Previous investigations have demonstrated that activated Stat3 contributes to a loss of growth control and transformation. To investigate the therapeutic potential of blocking Stat3 in cancer cells, we developed a transcription factor decoy to selectively abrogate activated Stat3. The Stat3 decoy was composed of a 15-mer double-stranded oligonucleotide, which corresponded closely to the Stat3 response element within the c-fos promoter. The Stat3 decoy bound specifically to activated Stat3 and blocked binding of Stat3 to a radiolabeled Stat3 binding element. By contrast, a mutated version of the decoy that differed by only a single base pair did not bind the activated Stat3 protein. Treatment of head and neck cancer cells with the Stat3 decoy inhibited proliferation and Stat3-mediated gene expression, but did not decrease the proliferation of normal oral keratinocytes. Thus, disruption of activated Stat3 by using a transcription factor decoy approach may serve as a novel therapeutic strategy for cancers characterized by constitutive Stat3 activation.

Interleukin-6 induces prostate cancer cell growth accompanied by activation of Stat3 signaling pathway

Interleukin‐6 (IL‐6) is a pleiotropic cytokine that regulates growth and differentiation of various types of malignant tumors, including prostate carcinomas. The levels of IL‐6 are elevated in sera of patients with metastatic prostate cancer. In this study, we evaluate the role of IL‐6 in the growth regulation of prostate cancer cells.

Distinct effects of systemic infusion of G-CSF vs. IL-6 on lung and liver inflammation and injury in hemorrhagic shock

Production of pro-inflammatory cytokines, such as granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) occurs at multiple tissue sites in hemorrhagic shock (HS), resulting in elevated circulating plasma levels. The current study was designed to test the hypothesis that circulating G-CSF and IL-6 contribute to polymorphonuclear neutrophilic granulocyte (PMN)-mediated inflammation and organ injury in HS. Sprague-Dawley rats were subjected to decompensated HS (mean arterial blood pressure = 40 mm Hg for 2.5 h), followed by resuscitation with lactated Ringers solution with or without G-CSF (3 microg/kg) or IL-6 (3 microg/kg). Animals were killed 4 h after resuscitation, and their lungs and livers were assessed quantitatively for PMN infiltration, organ injury, and activation of NF-kappaB and signal transducer and activator or transcription (STAT) 3. Infusion of G-CSF during resuscitation increased PMN infiltration into the lungs by 2.4-fold (P < 0.01) compared with animals resuscitated with lactated Ringers solution alone. Increased PMN infiltration was accompanied by interstitial edema and pneumocyte swelling, resulting in a 42% increase in lung alveolar wall cross-sectional surface area (P < 0.01) and a 3.7-fold increase in Stat3 activity (P < 0.01). G-CSF infusion did not affect PMN infiltration into the liver and was accompanied by a 68% decrease in focal hepatocellular necrosis (P < 0.01). Infusion of IL-6, in contrast, dramatically decreased inflammation and injury in both the lung and liver; the anti-inflammatory effects of IL-6 may be mediated, in part, by down-modulation of nuclear factor (NF)-kappaB activity. Thus, circulating G-CSF and IL-6 have opposing effects on PMN recruitment and injury in the lung in HS while both protect against hepatic necrosis. The beneficial effect of these cytokines on liver injury in HS appears to be independent of PMN recruitment.

Differential function of STAT5 isoforms in head and neck cancer growth control.

Up-regulation of the epidermal growth factor receptor (EGFR) is critical for the loss of growth control in a variety of human cancers, including squamous cell carcinoma of the head and neck (SCCHN). Stimulation of EGFR results in activation of mitogenic signaling pathways including Signal Transducers and Activators of Transcription (STATs). Stat5 activation has been primarily demonstrated in hematopoietic malignancies. Gene disruption studies suggest potentially distinct functions of the Stat5 isoforms, Stat5a and Stat5b, which are encoded by two genes closely linked on human chromosome 17. To determine the function of Stat5 in SCCHN growth control, we studied the expression and constitutive activation of Stat5a and Stat5b in normal and transformed human squamous epithelial cells. Increased constitutive activation of Stat5 was detected in transformed compared with normal squamous cells. Blockade of TGF-α or EGFR, abrogated Stat5 activation. Targeting of Stat5b using antisense oligonucleotides inhibited SCCHN growth. In addition, SCCHN cells stably transfected with dominant negative mutant Stat5b failed to proliferate in vitro. In contrast, targeting of Stat5a using either antisense or dominant negative strategies had no effect on cell growth. These results suggest that TGF-α/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat5b but not Stat5a.

Differential activation of apoptosis regulatory pathways during monocytic vs granulocytic differentiation: a requirement for Bcl-X L and XIAP in the prolonged survival of monocytic cells

Neutrophils and monocytes/macrophages are derived from common progenitors, but exhibit markedly different lifespans. Differentiated neutrophils are short-lived and die rapidly by apoptosis, while monocytic cells are longer-lived. In this report we used the HL-60 cell line as a model system to identify differences in apoptotic pathways which might account for the differing lifespans of granulocytic vs monocytic cells. We observed that induction of granulocytic differentiation by retinoic acid led to robust activation of the executioner protease caspase-3, and early onset of apoptosis. By contrast, caspase-3 was not appreciably activated during phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation, and apoptosis was delayed in these cells. Since the activation of caspase-3 is inhibited by members of the inhibitor of apoptosis (IAP) and Bcl-2 protein families, we investigated the expression of anti-apoptotic members of these families. Induction of monocytic differentiation led to marked upregulation of the IAP protein XIAP, as well as the Bcl-2 family member Bcl-XL. During granulocytic differentiation the levels of XIAP progressively declined, while Bcl-XL levels remained unchanged. A different IAP protein, survivin, was downregulated during differentiation along either lineage, as was expression of Bcl-2. The upregulation of Bcl-XL during monocytic differentiation coincided with phosphorylation/activation of STAT3, a known activator of bcl-X gene transcription. Moreover, Bcl-XL upregulation was dependent on MEK/ERK signaling. Upregulation of XIAP proceeded in a MEK/ERK-independent fashion. Treatment with antisense Bcl-XL or XIAP oligonucleotides resulted in significant loss of viability in cells differentiating along the monocytic lineage. Together, these findings indicate that the levels of XIAP and Bcl-XL are regulated by distinct pathways during monocytic differentiation, and that upregulation of these proteins contributes to the increased longevity of cells in the monocytic lineage.

Tissue distribution of liposome-mediated epidermal growth factor receptor antisense gene therapy

Despite the widespread use of liposome-mediated gene transfer in cancer therapy protocols, little is known about the tissue distribution of intralesionally administered DNA. We have previously shown that antisense gene therapy targeting the epidermal growth factor receptor (EGFR) inhibited tumor growth in a human head and neck squamous cell carcinoma (HNSCC) xenograft model. Further investigation demonstrated lack of systemic toxicity with intramuscular or intratumoral administration of this liposomal–DNA complex. In the present study, we compared two approaches to determine the presence of exogenous DNA in the plasma and tissues of mice treated with intramuscular injection of EGFR antisense gene therapy. PCR analysis using genomic DNA plus plasmid DNA as template was 83-fold more sensitive than PCR using a mixture of total RNA and plasmid DNA as template. With the more sensitive method (able to detect fewer than 500 molecules of EGFR antisense DNA in 1 μg of genomic DNA), foreign DNA was detected in all organs up to 1 month following a single injection. In contrast, using RNA plus plasmid DNA as template, exogenous DNA was only detected at the injection site at 1 week, and was undetectable at 1 month. Optical imaging studies demonstrated plasmid DNA only at the injection site. Although less sensitive than PCCR, Southern blot hybridization showed no evidence of integration of foreign DNA into the host genome in vitro or in vivo. These results emphasize the importance of defining the assays used to detect foreign DNA and suggest that the ability to detect intralesionally administered liposomal gene therapy, in organs distant from the injection site, is directly correlated with the sensitivity of the method employed.

Induced nitric oxide inhibits IL-6-induced stat3 activation and type II acute phase mRNA expression.

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.