Expression of the type III TGF-beta receptor is negatively regulated by TGF-beta

The type III transforming growth factor-beta receptor (TbetaRIII or betaglycan) is a ubiquitously expressed transforming growth factor-beta (TGF-beta) superfamily coreceptor with essential roles in embryonic development. Recent studies have defined a role for TbetaRIII in the pathogenesis of human cancers, with frequent loss of TbetaRIII expression at the message and protein level. Mechanisms for the loss of TbetaRIII expression remain to be fully defined. Advanced human cancers often have elevated circulating levels of TGF-beta1. Here, we define a specific role for TGF-beta1 in negatively regulating TbetaRIII at the message level in breast and ovarian cancer models. TGF-beta1 decreased TbetaRIII message and protein levels in ovarian (Ovca420) and breast cancer (MDA-MB-231) cell lines in both a dose- and time-dependent manner. TGF-beta1-mediated TbetaRIII repression is mediated by the type I TGF-beta receptor/Smad2/3 pathway as the activin receptor-like kinase 5 (ALK5) inhibitor, SB431542, abrogated this effect, while the expression of constitutively active ALK5 was sufficient to repress TbetaRIII expression. Mechanistically, TGF-beta1 does not affect TbetaRIII messenger RNA (mRNA) stability, but instead directly regulates the TbetaRIII promoter. We define alternative promoters for the TGFBR3 gene, a distal and proximal promoter. Although both promoters are active, only the proximal promoter was responsive and negatively regulated by TGF-beta1 and constitutively active ALK5. Taken together, these studies define TGF-beta1-mediated downregulation of TbetaRIII mRNA expression through effects on the ALK5/Smad2/3 pathway on the TGFBR3 gene proximal promoter as a potential mechanism for decreased TbetaRIII expression in human cancers.     

tyk2, prototype of a novel class of non-receptor tyrosine kinase genes

We previously identified a novel protein tyrosine kinase gene, tyk2, by screening a human lymphoid cDNA library with a tyrosine kinase domain specific c-fms restriction fragment under low stringency hybridization conditions. We have now isolated and sequenced a full length tyk2 cDNA clone; demonstrated that this gene is widely expressed in hematopoietic and non-hematopoietic cell lines; and mapped it to chromosome 19p13.2. The cDNA clone is 4176 nucleotides long and codes for a putative protein with a molecular weight of 134 kilodaltons. Hydrophobicity analysis of our sequence does not identify a transmembrane domain, which is found in all members of the receptor class of protein tyrosine kinases; nor can we detect an SH2 domain, found in all previously identified non-receptor protein kinases. We therefore propose that tyk2 is the prototype of a new class of non-receptor protein tyrosine kinases.     

Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.     

Isolation of ret proto-oncogene cDNA with an amino-terminal signal sequence

Our recent studies suggested that the ret proto-oncogene protein is a cell surface receptor with a tyrosine kinase domain. However, the sequence of its cDNA lacked an amino-terminal signal sequence characteristic of membrane proteins. In the present study we have isolated and sequenced an additional cDNA clone. The nucleotide sequence of the cDNA obtained indicated that the 5' terminal 330 nucleotides differs from the published sequence of the ret proto-oncogene. In addition, the sequence contains an open reading frame with an amino-terminal signal sequence.     

Anaplastic lymphoma kinase: Role in cancer and therapy perspective

Anaplastic lymphoma kinase (ALK) is correlated with oncogenesis in different types of cancers, such as anaplastic large cell lymphoma, lung cancer, neuroblastoma, and even breast cancer, by abnormal fusion of ALK or non-fusion ALK activation. ALK is a receptor tyrosine kinase, with a single transmembrane domain, that plays an important role in development. Upon ligand binding to the extracellular domain, the receptor undergoes dimerization and subsequent autophosphorylation of the intracellular kinase domain. In recent years, ALK inhibitors have been developed for cancer treatment. These inhibitors target ALK activity and show effectiveness in ALK-positive non-small cell lung cancer. However, acquired treatment resistance makes the future of this therapy unclear; new strategies are underway to overcome the limitations of current ALK inhibitors.     

A ligand-inducible anaplastic lymphoma kinase chimera is endocytosis impaired

Ligand-induced membrane trafficking of the anaplastic lymphoma kinase (ALK) was studied using a chimeric receptor in which the extracellular and transmembrane domain of ALK was substituted for the corresponding regions of epidermal growth factor receptor (EGFR). Wild-type EGFR, EGFR/ALK and an EGFR/ALK kinase negative mutant were independently expressed in mouse NR6 fibroblasts. The capacity of EGFR/ALK to mediate [125I]-EGF internalization, receptor degradation and downregulation, which has never been previously described, was assayed. The rate of [125I]-EGF-induced internalization mediated by the cytoplasmic domain of ALK was reduced several fold compared with the wild-type EGFR. The low rate of EGF internalization promoted by EGFR/ALK correlated with an impaired degradation and downregulation of the receptor and indicate that ALK is not subject to traditional mechanisms used to regulate receptor tyrosine kinase function. Accordingly, ALK-activated intracellular domain does not associate in vivo with c-cbl and does not undergo ligand-mediated ubiquitination. The current study provides new insight into the function and regulation of ALK suggesting that the relative long membrane residence of activated ALK might confers a more potent and prolonged signaling activity. Indeed NR6-EGFR/ALK cells exhibited a approximately 3-fold increase in a maximal mitogenic response than NR6-EGFR.     

Glioblastoma-secreted factors induce IGFBP7 and angiogenesis by modulating Smad-2-dependent TGF-beta signaling

Insulin-like growth factor-binding protein 7 (IGFBP7) is a selective biomarker of glioblastoma (GBM) vessels, strongly expressed in tumor endothelial cells and vascular basement membrane. IGFBP7 gene regulation and its potential role in tumor angiogenesis remain unclear. Mechanisms of IGFBP7 induction and its angiogenic capacity were examined in human brain endothelial cells (HBECs) exposed to tumor-like conditions. HBEC treated with GBM cell (U87MG)-conditioned media (-CM) exhibited fourfold upregulation of IGFBP7 mRNA and protein compared to control cells. IGFBP7 gene regulation in HBEC was methylation independent. U87MG-CM analysed by enzyme-linked immunosorbent assay contained approximately 5 pM transforming growth factor (TGF)-beta1, a concentration sufficient to stimulate IGFBP7 in HBEC to similar levels as U87MG-CM. Both pan-TGF-beta-neutralizing antibody (1D11) and the TGF-beta1 receptor (activin receptor-like kinase 5, ALK5) antagonist, SB431542, blocked U87MG-CM-induced IGFBP7 expression in HBEC, indicating that TGF-beta1 is an important tumor-secreted effector capable of IGFBP7 induction in endothelial cells. HBEC exposed to either U87MG-CM or IGFBP7 protein exhibited increased capillary-like tube (CLT) formation in Matrigel. Both TGF-beta1- and U87MG-CM-induced Smad-2 phosphorylation and U87MG-CM-induced CLT formation in HBEC were inhibited by the ALK5 antagonist, SB431542. These data suggest that proangiogenic IGFBP7 may be induced in brain endothelial cells by TGF-betas secreted by GBM, most likely through TGF-beta1/ALK5/Smad-2 pathway.     

A dominant negative mutation of transforming growth factor-beta receptor type II gene in microsatellite stable oesophageal carcinoma

Recent investigations revealed microsatellite instability in colon cancers are associated with mutations of the transforming growth factor-beta receptor type II gene (TGF-beta RII) that encodes a transmembrane protein containing an intracellular serine/threonine kinase domain. Activation of TGF-beta receptor type I (RI) and RII by TGF-beta induces nuclear translocation of Smad proteins including Smad2 and Smad4 that have been originally identified as tumour suppressor genes. We have previously reported six cases with microsatellite instability in 32 oesophageal carcinomas. In this study, we analysed genetic mutations of TGF-beta RII, Smad2 and Smad4 in these oesophageal carcinoma tissues and established 16 cell lines. No genetic mutation was detected in any tissues or cell lines except one tissue sample of microsatellite stable oesophageal carcinoma, that is, a mis-sense mutation of glutamic acid to glutamine at codon 526 (E526Q) in the TGF-beta RII serine/threonine kinase domain. Interestingly, the mutant TGF-beta RII E526Q can completely inhibit TGF-beta-induction of nuclear translocation of Smad4 protein in oesophageal carcinoma cells. This mutation of TGF-beta RII that is not associated with microsatellite instability might make a dominant negative effect on TGF-beta signal transduction in oesophageal carcinoma.     

The proto-oncogene c-ros codes for a transmembrane tyrosine protein kinase sharing sequence and structural homology with sevenless protein of Drosophila melanogaster

Our earlier study predicted that proto-oncogene c-ros codes for a receptor-like tyrosine protein kinase (TPK). To further understand its protein structure and physiological function, we have analysed its expression in various tissues of chicken and have isolated and sequenced cDNA clones containing the entire coding region of the gene. Confirming our earlier study, we found that kidney is the organ that expresses the highest level of c-ros mRNA, in addition, we found a lower level of expression in gonad, thymus, bursa and brain. A distinctive 8.3 kb c-ros mRNA is present in kidney. No detectable amount of c-ros mRNA was found in the rest of tissues examined. Nucleotide sequence of the c-ros cDNA predicts that it codes for a transmembrane (TM) TPK molecule of 2311 amino acids (aa). The extracellular domain consists of 1873 amino acids which share 20 to 43% homology with that of the Drosophila sevenless protein and TPK domains of the two genes have 58 to 74% homology. The extracellular domain containing 37 potential N-linked glycosylation sites is preceded by a 5' hydrophobic sequence resembling a typical signal peptide. An internal hydrophobic domain of 26 amino acids, the presumed transmembrane domain, is followed by a spacer sequence of 58 amino acids, a TPK domain of 270 amino acids and a carboxyl tail of 84 amino acids. Overall, our result indicates that c-ros codes for a glycosylated transmembrane TPK molecule which shares a remarkable sequence and structural homology with that of Drosophila sevenless protein.     

The Hsp90 inhibitor IPI-504 rapidly lowers EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models

Heat shock protein 90 (Hsp90) is an emerging target for cancer therapy due to its important role in maintaining the activity and stability of key oncogenic signaling proteins. We show here that the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion protein, presumed to be the oncogenic driver in about 5% of patients with non-small cell lung cancer (NSCLC), is associated with Hsp90 in cells and is rapidly degraded upon exposure of cells to IPI-504. We find EML4-ALK to be more sensitive to Hsp90 inhibition than either HER2 or mutant epidermal growth factor receptor (EGFR) with an inhibitory concentration (IC)(50) for protein degradation in the low nanomolar range. This degradation leads to a potent inhibition of downstream signaling pathways and to the induction of growth arrest and apoptosis in cells carrying the EML4-ALK fusion. To generate a causative link between the expression of EML4-ALK and sensitivity to IPI-504, we introduced an EML4-ALK cDNA into HEK293 cells and show that the expression of the fusion protein sensitizes cells to IPI-504 both in vitro and in vivo. In a xenograft model of a human NSCLC cell line containing the ALK rearrangement, we observe tumor regression at clinically relevant doses of IPI-504. Finally, cells that have been selected for resistance to ALK kinase inhibitors retain their sensitivity to IPI-504. We have recently observed partial responses to administration of IPI-504 as a single agent in a phase 2 clinical trial in patients with NSCLC, specifically in patients that carry an ALK rearrangement. This study provides a molecular explanation for these clinical observations.