Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.

Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. Here, we describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase I dose escalation study.

In rat liver epithelial cells constitutively expressing transforming growth factor ␣ (TGF␣), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGF␣ and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR) Growth factor signal pathways are one of the main regulators of cell proliferation, differentiation, and apoptosis. Overexpression of receptors, aberrant expression of growth factor receptors such as truncated receptors, and co-expression of both growth factor ligands and their specific receptors within the same cell contribute to tumorigenesis. Transgenic mice which overexpress transforming growth factor ␣ developed hepatocellular carcinomas, epithelial hyperplasia, pancreatic metaplasia, and breast carcinoma (1-3) and autocrine expression of TGF␣ 1 is demonstrated in various human tumors and cell lines including cancers of the head and neck, lung, stomach, breast, ovary, uterine cervix, kidney, and liver (4 -7).,TGF␣ has a similar structure and function to epidermal growth factor (EGF). Both TGF␣ and EGF bind and activate the same receptor, EGFR, and thus induce a mitogenic and motogenic response in many cell types. EGFR is a transmembrane tyrosine kinase which is encoded by the proto-oncogene c-erb B and is expressed on most cell types. The activated EGFR dimerizes and autophosphorylates tyrosine residues and leads to a cascade of intracellular signaling pathways including the phosphoinositide 3-kinase (PI-3K) pathway, Ras-Raf-MAPK pathway, and phospholipase C pathway (8).Scatter factor/hepatocyte growth factor (HGF) was originally described as a mitogenic factor of hepatocytes during liver regeneration (9, 10), but HGF has a variety of additional biological activities including motogenesis (10) and morphogenesis (11) in many epithelial cells. HGF is essential for normal embryological development (12) and liver regeneration (13,14). The receptor of HGF, c-Met, is also a tyrosine kinase receptor (15), and the binding of its ligand to c-Met leads to the activation of the PI-3K pathway which activates motogenesis (16,17) and the Ras-Raf-MAPK pathway which is involved in mitogenesis (18). HGF pathway has also been found to activate both the STAT (19) and phospholipase C pathway. Spontaneously transformed tumor cell lines which express both HGF and c-Met show increased autonomous proliferation and motility (20). Also, overexpression of c-Met and its activation by autocrine HGF expression is found in a variety of human tumors indicating co-expression of HGF and c-Met may be involved in tumor metastasis (21,22).Previously, we reported that c-Met was phosphorylated in the absence of its ligand in TGF␣ transfectants of rat liver epithelial cells, and tumor clones derived from rat liver epithelial cells TGF␣ transfectants (23). We proposed that EGFR constitutively activated by autocrine TGF␣ expression results in cross-talk between these growth factor receptor systems resulting in constitutive phosphorylation of c-Met. Cross-tal...

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. In this study, we show that breast cancer cells cultured in 1.0% O2 demonstrate changes consistent with epithelial–mesenchymal transition (EMT). Snail translocates to the nucleus, and E-cadherin is lost from plasma membranes. Vimentin expression, cell migration, Matrigel invasion, and collagen remodeling are increased. Hypoxia-induced EMT is accompanied by increased expression of the urokinase-type plasminogen activator receptor (uPAR) and activation of cell signaling factors downstream of uPAR, including Akt and Rac1. Glycogen synthase kinase-3β is phosphorylated, and Snail expression is increased. Hypoxia-induced EMT is blocked by uPAR gene silencing and mimicked by uPAR overexpression in normoxia. Antagonizing Rac1 or phosphatidylinositol 3-kinase also inhibits development of cellular properties associated with EMT in hypoxia. Breast cancer cells implanted on chick chorioallantoic membranes and treated with CoCl2, to model hypoxia, demonstrate increased dissemination. We conclude that in hypoxia, uPAR activates diverse cell signaling pathways that cooperatively induce EMT and may promote cancer metastasis.

Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT.In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O 2 , uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O 2 decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O 2 ). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer. Epithelial-mesenchymal transition (EMT)2 is a well recognized process in embryonic development (1). To facilitate migration of neural crest cells out of the neuroectoderm, N-cadherin-based cell adhesions are lost. Endocardial cells adopt a mesenchymal cell phenotype during formation of cardiac septa and valves. EMT also may be involved in metastasis of epithelial cell malignancies (2). In cancer cell EMT, E-cadherin protein and activity are decreased, causing disruption of cell-cell junctions and loss of cell polarity. At the same time, cancer cells that undergo EMT demonstrate increased expression of mesenchymal cell proteins such as vimentin. Loss of E-cadherin, in human malignancies, may reflect mutation of the E-cadherin gene or E-cadherin promoter hypermethylation (3-5). E-cadherin expression also may be repressed at the transcriptional level by Slug, Snail, or Twist (6 -9). Experimental regulation of E-cadherin expression controls cancer progression in mouse model systems (10 -13).Activation of the phosphatidylinositol 3-kinase (PI3K) and Akt promotes EMT in cancer cells (14). Akt phosphorylates and thereby inactivates glycogen synthase kinase-3␤ (GSK-3␤), increasing Snail activity by regulating Snail expression, degradation, and nuclear localization (6,7,15,16). Wnt signaling also regulates GSK-3␤ and thereby supports EMT by downstream effects on Snail and Slug (17). Extracellular signal-regulated kinase (ERK1/2) increases expression of Snail (18). Rac1 and its alternatively spliced variant, Rac1b, control Snail expression and activity, through a pathway that involves reactive oxygen species and NF-B...

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.

BackgroundThe Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin’s lymphoma population, primarily consisting of patients with DLBCL.MethodsPatients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells.ResultsThirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance.ConclusionsAZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing.Trial registrationRegistered at ClinicalTrials.gov: NCT01563302. First submitted 2/13/2012.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0436-5) contains supplementary material, which is available to authorized users.

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O 2 ) for 3 h, the Epo mRNA level increased 2.4 ؎ 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 ؎ 0.1-fold. Soluble EpoR and Eponeutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration. Erythropoietin (Epo)2 is a 34-kDa member of the class I cytokine family that initiates cell signaling by forming a trimeric complex with two molecules of the Epo receptor (EpoR) (1). Epo binding induces a conformational change in EpoR so that receptor-associated Janus kinase-2 is activated (2). This leads to phosphorylation of tyrosine residues in EpoR and recruitment of Src homology 2 domain-containing proteins. Signaling proteins activated downstream of EpoR and Janus kinase-2 include phosphatidylinositol 3-kinase, protein kinase C, and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) (3, 4).In adults, Epo is produced mainly by peritubular fibroblasts in the kidney; however, diverse cells in multiple organs also express Epo (5). Epo expression is stimulated by hypoxia as a result of activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1␣) (6). Epo is well characterized for its ability to promote proliferation and differentiation of erythroid progenitor cells and also inhibit apoptosis in these cells (7); however, Epo is active outside the hematopoietic system as well. In the nervous system, Epo inhibits neuronal apoptosis resulting from hypoxia or ischemia (8 -10) and stimulates Schwann cell proliferation (11). Epo also regulates angiogenesis by multiple mechanisms, including stimulation of endothelial cell proliferation and migration (12).The function of Epo in cancer is not well understood. EpoR is expressed by several human tumor types, including breast carcinoma, renal cell carcinoma, melanoma, endometrial carcinoma, and gastric carcinoma (13-17). In breast cancer, EpoR is observed at high levels by immunohistochemistry, whereas normal mammary tissue is typically EpoR negative (18). Many tumors are also strongly Epo positive (13). Furthermore, in at least one study, Epo and EpoR were observed at increased levels in hypoxic regions of breast cancers (13). Lai et al. (19) demonstrated increased Matrigel invasion by...