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Insulin receptor substrate-1 (IRS-1) mediates signaling from the insulin-like growth factor type-I receptor. We found that all-trans retinoic acid (RA) decreases IRS-1 protein levels in MCF-7, T47-D, and ZR75.1 breast cancer cells, which are growth arrested by RA, but not in the RA-resistant MDA-MB-231 and MDA-MB-468 cells. Based on prior reports of ubiquitin-mediated degradation of IRS-1, we investigated the ubiquitination of IRS-1 in RA-treated breast cancer cells. Two proteasome inhibitors, MG-132 and lactacystin, blocked the RA-mediated degradation of IRS-1, and RA increased ubiquitination of IRS-1 in the RA-sensitive breast cancer cells. In addition, we found that RA increases serine phosphorylation of IRS-1. To elucidate the signaling pathway responsible for this phosphorylation event, pharmacologic inhibitors were used. Two PKC inhibitors, but not a MAPK inhibitor, blocked the RA-induced degradation and serine phosphorylation of IRS-1. We demonstrate that RA activates PKC-din the sensitive, but not in the resistant cells, with a time course that is consistent with the RA-induced decrease of IRS-1. We also show that: (1) RA-activated PKC-dphosphorylates IRS-1 in vitro, (2) PKC-dand IRS-1 interact in RA-treated cells, and (3) mutation of three PKC-dserine sites in IRS-1 to alanines results in no RA-induced in vitro phosphorylation of IRS-1. Together, these results indicate that RA regulates IRS-1 levels by the ubiquitin-proteasome pathway, involving a PKC-sensitive mechanism.Oncogene (2004) 23, 9269-9279. doi:10.1038/sj.onc.1208104 Published online 1 November 2004 [ABSTRACT FROM AUTHOR]

Insulin receptor substrate 1 (IRS-1) is a major signaling molecule activated by the insulin and insulin-like growth factor I receptors. Recent data obtained in different cell models suggested that in addition to its conventional role as a cytoplasmic signal transducer, IRS-1 has a function in the nuclear compartment. However, the role of nuclear IRS-1 in breast cancer has never been addressed. Here we report that in estrogen receptora(ERa)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-ß-estradiol (E2) treatment; (2) E2-dependent nuclear translocation of IRS-1 was blocked with the antiestrogen ICI 182,780; (3) nuclear IRS-1 colocalized and co-precipitated with ERa; (4) the IRS-1:ERacomplex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERa-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERa. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERaactivity at estrogen-responsive element-containing DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERaand that this interaction might influence ERatranscriptional activity.Oncogene (2004) 23, 7517-7526. doi:10.1038/sj.onc.1208014 Published online 16 August 2004 [ABSTRACT FROM AUTHOR]

Communication between cells is often mediated by secreted signaling molecules that bind cell surface receptors and modulate the activity of specific intracellular effectors. The Wnt family of secreted glycoproteins is one group of signaling molecules that has been shown to control a variety of developmental processes including cell fate specification, cell proliferation, cell polarity and cell migration. In addition, mis-regulation of Wnt signaling can cause developmental defects and is implicated in the genesis of several human cancers. The importance of Wnt signaling in development and in clinical pathologies is underscored by the large number of primary research papers examining various aspects of Wnt signaling that have been published in the past several years. In this review, we will present a synopsis of current research with particular attention paid to molecular mechanism of Wnt signal transduction and how the mis-regulation of Wnt signaling leads to cancer.

All-trans retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. NT2/D1 cells basally express transforming growth factor alpha (TGF-alpha) mRNA and secreted protein. After RA-treatment TGF-alpha expression is markedly reduced. This decline in TGF-alpha expression accompanies the induction of the neuronal phenotype and a marked reduction of tumorigenicity in athymic mice. This suggested a causal link between reduced TGF-alpha expression and the induced differentiation or loss of tumorigenicity of these RA-treated TC cells. To evaluate this possibility, an RA-refractory NT2/D1 subclone was analysed. This subclone, designated NT2/D1-R1, failed to induce differentiation or to decrease TGF-alpha expression despite RA treatment. To further explore the relationship between TGF-alpha expression and RA actions in this human TC cell, a TGF-alpha cDNA was stably transfected and expressed in NT2/D1 cells. RA-treatment of independently obtained TGF-alpha over-expressing clones and a representative control transfectant only expressing the neomycin resistance gene produced a neuronal phenotype similar to parental NT2/D1 cells as assessed by morphologic, immunophenotypic, and gene expression markers of differentiation. RA-treatment of these clones also induced a G1 arrest similar to parental cells. However, only the TGF-alpha over-expressing clones that secreted high levels of TGF-alpha protein into the conditioned media before and after RA treatment still developed tumors in athymic mice despite prior exposure to these cells to RA. This finding demonstrates that TGF-alpha can inhibit the anti-tumorigenic effects of RA in human TCs. Thus, over-expression of a single growth factor that normally declines with RA treatment antagonizes the anti-tumorigenic but not the differentiation actions of RA in this human tumor cell.

We examined eight human germ cell cancer lines (GCCLs) for cytogenetic abnormalities and found an isochromosome 12p, i(12p), marker in all seven male nonseminoma GCCLs, but not in the single female teratocarcinoma cell line. Southern blot analysis of these cell lines showed increased copy number for c-ki-ras2, a gene located on 12p, in all the male GCCLs. The comparison of Southern blot analysis for a restriction fragment length polymorphism (RFLP) probe localized to 12p to a probe for int-1, which maps to 12q, indicates that the increased copy number for c-ki-ras2 is primarily from the greater numbers of 12p relative to 12q. Although Northern analysis revealed enhanced mRNA expression for c-ki-ras2 in the GCCLs with an i(12p), hybridization of specific end-labelled oligonucleotides to the polymerase chain reaction products of c-ki-ras2 codons 12, 13, or 61 did not identify c-ki-ras2 mutations of these codons in these cells. Thus, c-ki-ras2 activation through point mutation is an infrequent event in GCCLs. These data further suggest that increased 12p copy number is a common event in the transformation process leading to male germ cell cancer. We conclude that determination of 12p copy number by cytogenetic analysis or Southern blotting is useful in the diagnostic evaluation of human germ cell cancer.

The human teratocarcinoma cell NTERA-2 cl. D1 (NT2/D1) is a cloned embryonal cancer cell line that differentiates into a neuronal phenotype and other cellular lineages after treatment with retinoic acid (RA). We examined the regulated expression of growth factors and proto-oncogenes in NT2/D1 cells. We studied RNA levels after six days of RA treatment to assess gene expression coincident with observed morphologic differentiation. Three growth factors were markedly down-regulated following RA treatment: Hst-1/kFGF and TGF-alpha expression became undetectable by Northern analysis and bFGF expression was substantially reduced. Minimal decline was seen for c-myc, N-myc, c-fos, and c-myb. Increased expression with differentiation was seen for the human homeotic genes Hox 2.1 and Hox 2.2. Assay of RNA levels daily after one to six days of RA treatment showed that the growth factor down-regulation inversely correlated with the homeotic gene up-regulation. Nuclear run-on studies showed low transcriptional rates for these homeotic genes, Hst-1/kFGF, and TGF-alpha that did not measurably change with RA treatment. To explore whether these regulated genes in NT2/D1 play a role in human testicular cancer, we examined RNA levels in a panel of human germ cell cancer lines. Hst-1/kFGF and bFGF are commonly expressed in five of seven male germ cell cancer lines. These data show that specific proto-oncogenes and growth factors are down-regulated with RA treatment of the NT2/D1 cell and that some of these regulated genes are often expressed in human germ cell cancer lines.