Background-The serine-threonine kinase Akt is activated by several ligand-receptor systems previously shown to be cardioprotective. Akt activation reduces cardiomyocyte apoptosis in models of transient ischemia. Its role in cardiac dysfunction or infarction, however, remains unclear. Methods and Results-We examined the effects of a constitutively active Akt mutant (myr-Akt) in a rat model of cardiac ischemia-reperfusion injury. In vivo gene transfer of myr-Akt reduced infarct size by 64% and the number of apoptotic cells by 84% (PϽ0.005 for each). Ischemia-reperfusion injury decreased regional cardiac wall thickening as well as the maximal rate of left ventricular pressure rise and fall (ϩdP/dt and ϪdP/dt). Akt activation restored regional wall thickening and ϩdP/dt and ϪdP/dt to levels seen in sham-operated rats. To better understand this benefit, we examined the effects of myr-Akt on hypoxic cardiomyocyte dysfunction in vitro. myr-Akt prevented hypoxia-induced abnormalities in cardiomyocyte calcium transients and shortening. Akt activation also enhanced sarcolemmal expression of Glut-4 in vivo and increased glucose uptake in vitro to the level seen with insulin treatment. Conclusions-Akt activation exerts a powerful cardioprotective effect after transient ischemia that probably reflects its ability to both inhibit cardiomyocyte death and improve function of surviving cardiomyocytes. Akt may represent an important nodal target for therapy in ischemic and other heart disease.

Akt is a serine-threonine kinase that mediates a variety of cellular responses to external stimuli. During postnatal development, Akt signaling in the heart was up-regulated when the heart was rapidly growing and was down-regulated by caloric restriction, suggesting a role of Akt in nutrient-dependent regulation of cardiac growth. Consistent with this notion, reductions in Akt, 70-kDa S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1 phosphorylation were observed in mice with cardiac-specific deletion of insulin receptor gene, which exhibit a small heart phenotype. In contrast to wild type animals, caloric restriction in these mice had little effect on Akt phosphorylation in the heart. Furthermore, forced expression of Akt1 in these hearts restored 70-kDa S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 phosphorylation to normal levels and rescued the small heart phenotype. Collectively, these results indicate that Akt signaling mediates insulin-dependent physiological heart growth during postnatal development and suggest a mechanism by which heart size is coordinated with overall body size as the nutritional status of the organism is varied.

Generation of arachidonic acid by the ubiquitously expressed cytosolic phospholipase A2 (PLA2) has a fundamental role in the regulation of cellular homeostasis, inflammation and tumorigenesis. Here we report that cytosolic PLA2 is a negative regulator of growth, specifically of striated muscle. We find that normal growth of skeletal muscle, as well as normal and pathologic stress-induced hypertrophic growth of the heart, are exaggerated in Pla2g4a-/- mice, which lack the gene encoding cytosolic PLA2. The mechanism underlying this phenotype is that cytosolic PLA2 negatively regulates insulin-like growth factor (IGF)-1 signaling. Absence of cytosolic PLA2 leads to sustained activation of the IGF-1 pathway, which results from the failure of 3-phosphoinositide-dependent protein kinase (PDK)-1 to recruit and phosphorylate protein kinase C (PKC)-zeta, a negative regulator of IGF-1 signaling. Arachidonic acid restores activation of PKC-zeta, correcting the exaggerated IGF-1 signaling. These results indicate that cytosolic PLA2 and arachidonic acid regulate striated muscle growth by modulating multiple growth-regulatory pathways.

Considerable evidence has demonstrated that transforming growth factor β (TGF-β) plays a key role in hepatic fibrosis, the final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have reported that blocking TGF-β with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no evidence has been provided that TGF-β antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to examine the effects of a murine neutralizing TGF-β monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11 dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-β1 mRNA, tissue hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-β antagonist can diminish and potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas.

Somatic gene transfer of IGF-I may offer strategic advantages over both systemic delivery of IGF-I peptide and expression of cell autonomous cardioprotective transgenes such as Akt by mediating autocrine and paracrine cardiomyocyte protection without elevating serum [IGF-I] levels.

TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Previously we have demonstrated that neutralizing TGFβ with the antibody 1D11 significantly inhibited metastasis in preclinical models of breast cancer and melanoma, with minimal effects on primary tumor growth. Neutralizing the protumorigenic effects that TGFβ has on the immune system and tumor microenvironment could, hypothetically, enhance the direct anti-tumor effects of chemotherapeutic agents. To test this hypothesis, several studies were conducted testing combinatorial therapies with 1D11 and chemotherapeutics against subcutaneous (SQ) 4T1, a murine model of triple negative breast cancer (TNBC), with subsequent metastasis to the lung. Paclitaxel is currently the front-line therapy for TNBC in the clinic. Combining 1D11 with paclitaxel had little effect on enhancing the efficacy of paclitaxel alone against primary tumor growth. However, the combinatorial therapy of 1D11 and paclitaxel inhibited spontaneous metastasis from the primary SQ tumor to the lung to a greater extent than either agent alone. In the clinic TNBC typically becomes resistant to taxane therapy and another treatment that has shown some promise in taxane-refractory TNBC is the combinatorial therapy of capecitabine and ixabepilone. In the 4T1 preclinical model, both capecitabine and ixabepilone inhibited the growth of SQ 4T1 tumors as single agents, and the combination of the two therapeutics resulted in slightly greater efficacy. However, the addition of 1D11 to capecitabine and ixabepilone resulted in even greater efficacy than with the combination alone. Currently, the effects of neutralizing TGFβ with 1D11 during the course of capecitabine/ixabepilone therapy on metastasis in the 4T1 model are being investigated. Additionally, mechanism of action studies are being conducted to determine the mechanisms by which 1D11 is enhancing the efficacy of paclitaxel and the combinatorial therapy. The data from these chemo-combo studies with 1D11 suggests that therapies that target TGFβ may enhance the efficacy of paclitaxel against TNBC metastasis and may potentiate the efficacy of the combinatorial therapy of capecitabine/ixabepilone in taxane-refractory TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3826.

TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Preclinical studies were conducted to determine the effect of neutralizing TGFβ on both primary tumor growth and metastases in in vivo models of experimental and spontaneous metastasis. Treatment with the pan-neutralizing TGFβ antibody, 1D11, as a single agent had limited effect on the growth of established, primary, syngeneic tumors (4T1 mammary carcinomas or B16-F10 melanomas) and did not enhance the efficacy of chemotherapeutic agents against these primary tumors. However, TGFβ antagonism was shown to have a significant effect on inhibiting metastasis to the bone following intracardiac injection of 4T1 cells. In addition, it was demonstrated that neutralization of TGFβ in B16-F10 melanoma models inhibited experimental pulmonary metastases following injection of cells into the tail vein, and inhibited spontaneous metastases from a subcutaneous primary tumor to the draining lymph node. Studies aimed at elucidating the mechanism of action of 1D11 in mice lacking specific immune effector cells suggest that the activity of cytotoxic T lymphocytes is important in the inhibition of metastases. The role of natural killer (NK) cells is less clear; NK cell activity is not required for inhibition of experimental metastasis to the lung, but is critical for the ability of 1D11 to inhibit spontaneous metastasis from a primary tumor to the draining lymph node. Investigation into the effects of neutralizing TGFβ on innate immunity and the tumor microenvironment are currently ongoing. Together these data suggest that neutralization of TGFβ significantly inhibits metastasis and anti-TGFβ strategies may be effective at treating metastatic disease in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B244.

The authors unanimously wish to retract this paper because of several incorrect statements and erroneous presentation of primary data, results and conclusions.