Search

Pancreatic cancer is one of the most fatal cancers without druggable molecular targets. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcriptional factor that promotes malignancy in various cancers including pancreatic cancer. Herein, we found that HIF-1 is accumulated in normoxic or moderate hypoxic areas of pancreatic cancer xenografts in vivo and is active even during normoxia in pancreatic cancer cells in vitro. This prompted us to analyze whether the HIF-1 activator Mint3 contributes to malignant features of pancreatic cancer. Mint3 depletion by shRNAs attenuated HIF-1 activity during normoxia and cell proliferation concomitantly with accumulated p21 and p27 protein in pancreatic cancer cells. Further analyses revealed that Mint3 increased transcription of the oncogenic ubiquitin ligase SKP2 in pancreatic cancer cells via HIF-1. This Mint3-HIF-1-SKP2 axis also promoted partial epithelial-mesenchymal transition, stemness features, and chemoresistance in pancreatic cancer cells. Even in vivo, Mint3 depletion attenuated tumor growth of orthotopically inoculated human pancreatic cancer AsPC-1 cells. Database and tissue microarray analyses showed that Mint3 expression is correlated with SKP2 expression in human pancreatic cancer specimens and high Mint3 expression is correlated with poor prognosis of pancreatic cancer patients. Thus, targeting Mint3 may be useful for attenuating the malignant features of pancreatic cancer.

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinib-sensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.

Telomerase is a regulated enzyme and its activity is tightly associated with cell proliferation. The mechanisms of this association are unclear, but specific growth factors may regulate telomerase activity. The present study examines the effect of epidermal growth factor (EGF) on telomerase activity and identifies the signal transduction pathway involved in this process. EGF up-regulated telomerase activity in EGF receptor-positive cells after the activation of telomerase reverse transcriptase (TERT) mRNA expression. This activation was rapid, peaked after 6 or 12 h and was not blocked by the concurrent exposure to cycloheximide, suggesting a direct effect of EGF on TERT transcription. Transient expression assays revealed that EGF activates the hTERT promoter and that the proximal core promoter is responsible for this regulation. The activation of hTERT mRNA expression by EGF was specifically blocked by MEK inhibitor, and in vitro kinase assays demonstrated that ERK is activated in response to EGF. Transient expression assays using mutant reporter plasmids revealed that an ETS motif located in the core promoter of hTERT is required for the EGF-induced transactivation of hTERT. Overexpression of wild type Ets in cells enhanced the EGF effect on hTERT transcription, while that of dominant negative Ets significantly repressed EGF action. These findings suggest that EGF activates telomerase through the direct activation of TERT transcription, in which the Ras/MEK/ERK pathway and Ets factor play major roles. Our data support the notion that growth factors directly regulate telomerase via specific signal transduction pathways.

The phosphotyrosine (pTyr) adapter Shc/ShcA is a major connector in various tyrosine kinase signalings following a variety of stimulation such as growth factor/neurotrophin, as well as in those following calcium influx and integrin activation. As in other tissues, Shc has been implicated in neuronal signalings; however, recent evidence suggests that N-Shc/ShcC and Sck/ShcB would take over most of the roles of Shc in mature central neurons, and switching phenomena between Shc and N-Shc expression were observed in several neuronal paradigms. Little is, however, known as to the signal-output differences between Shc and N-Shc. Here we determined the efficacy of Shc and N-Shc toward Erk activation in NGF-treated PC12 cells, and found that N-Shc transduced Grb2/Sos/Ras-dependent Erk activation less efficiently than Shc. This was mainly because N-Shc has only one high-affinity Grb2-binding site, whereas Shc has two such sites. Phosphopeptide mapping revealed that N-Shc has novel tyrosine-phosphorylation sites at Y259/Y260 and Y286; in vivo-phosphorylation of these tyrosines was demonstrated by site-specific anti-pTyr antibodies. Phosphorylated Y286 bound to several proteins, of which one was Crk. The pY221/pY222 site, corresponding to one of the Grb2-binding sites of Shc, also preferentially bound to Crk. The phosphorylation-dependent interaction between N-Shc and Crk was demonstrated in vitro and in vivo. These results indicate that N-Shc has specific features of signal-output, and further suggest that the switching between Shc and N-Shc during neural development and regeneration would lead to differentiation of downstream signalings.

Recently, we have shown that an EGF-R-mutant lacking the autophosphorylation sites phosphorylates Shc and retains mitogenic activity. In this report, we have shown that in these cells, in response to EGF, Ras is fully activated with formation of the tyrosine-phosphorylated Shc-Grb2-mSOS complex without the receptor. This pointed out the importance of Shc in EGF-induced Ras activation. To investigate the mechanism of tyrosine phosphorylation of Shc by EGF-R, we carried out in vitro kinase assays using immunoprecipitated EGF-R and bacterially-expressed Shc proteins as substrates. The EGF-R phosphorylated Shc, but not the Shc SH2 mutant, lacking binding ability for phosphotyrosine. This suggests that intact Shc SH2 is essential for the full-length Shc to become phosphorylated, probably by inducing a conformational change in Shc. Thus a Shc SH2 peptide may inhibit competitively Shc phosphorylation. We microinjected the Shc SH2 domain into NIH3T3 cells overexpressing the EGF-R. Microinjected Shc SH2 greatly suppressed EGF-induced DNA synthesis. But microinjection of neither the Shc SH2 mutant nor PLC-gamma 1 SH2 had any effect. This suppressing effect was rescued by comicroinjection of the full-length Shc, suggesting Shc SH2 specifically suppressed the Shc pathway. Thus we concluded Shc phosphorylation is crucial, whereas receptor autophosphorylation is dispensable, in EGF-induced mitogenesis.

Flt-1 (fms-like tyrosine kinase-1), a receptor-type tyrosine kinase of sharing similar features with two other flt-family encoded proteins KDR/Flk-1 and Flt-4, has been recently identified as a receptor for Vascular Endothelial Growth Factor (VEGF) known to induce the proliferation of vascular endothelial cells. In this study, we demonstrate that Flt-1 encodes for a 180 kDa glycoprotein, binds VEGF with high affinity, undergoes autophosphorylation but does not generate any mitogenic response in transfected NIH3T3 fibroblasts. Interestingly, the immediate early gene c-myc was not induced, whereas the c-fos was induced very weakly in Flt-1 expressing NIH3T3 cells. A comparative analysis of the Flt-1 signal cascade in the environment of endothelial cells with that of Flt-1 expressing NIH3T3 cells showed that VEGF induced phosphorylation of PLC gamma and GAP complex on tyrosine in both type of cells. However, a strong activation of MAP kinases was observed only in endothelial cells. Further, different from many other receptor tyrosine kinases, tyrosine phosphorylation of Shc protein, an important adaptor for signal transduction from many receptor kinases, was very weak in both Flt-1-NIH3T3 cells and endothelial cells. These results suggest that Flt-1 kinase utilizes a unique signal transduction system in endothelial cells, and the activation of the Flt-1 kinase is insufficient to trigger a mitogenic response in NIH3T3 fibroblasts.

Hepatocyte Growth Factor (HGF)/Scatter Factor secreted from sinusoidal endothelial cells and Kupffer cells in liver activates the c-Met tyrosine kinase receptor expressed on hepatocytes. Here we report yet another possible communication system through a different ligand and tyrosine kinase receptor in an opposite direction. We isolated and determined the primary structure of the entire coding region of rat flt-1 (fms-like tyrosine kinase), a receptor for Vascular Endothelial Growth Factor (VEGF). Using rat flt-1 cDNA as a probe we found that the flt-1 mRNA was expressed at very high levels in sinusoidal endothelial cells in normal rat liver, but was hardly detectable in hepatocytes. The transcripts of another VEGF receptor KDR/Flk-1 structurally related to Flt-1 was also expressed specifically in sinusoidal endothelial cells. On the other hand, VEGF mRNA was expressed weakly in hepatocytes, but not in the nonparenchymal cell fraction. Furthermore, in an in vitro culture system, VEGF demonstrated a remarkably specific growth-stimulatory activity as well as maintenance activity on the sinusoidal endothelial cells. These results suggest that hepatocytes regulate the proliferation and survival of the sinusoidal endothelial cells in liver in a paracrine manner. Therefore two reciprocal communication systems, VEGF-Flt receptor family and HGF-Met receptor, may exist in hepatic tissue.