The Tumor Suppressive Effects of HPP1 Are Mediated Through JAK-STAT-Interferon Signaling Pathways

Abstract

1HPP1, a novel tumor suppressive epidermal growth factor (EGF)-like ligand, mediates its effects through signal transducer and activators of transcription (STAT) activation. We previously demonstrated the importance of STAT1 activation for HPP1 function; however the contribution of STAT2 remains unclear. We sought to delineate the components of JAK-STAT-interferon (IFN) signaling specifically associated with HPP1s biological effects. Using stable HPP1-HCT116 transfectants, expression analyses were performed by polymerase chain reaction (PCR)/western blotting while expression knockdowns were achieved using siRNA. Growth parameters evaluated included proliferation, cell cycle distribution, and anchorage-independent growth. STAT dimerization, translocation, and DNA binding were examined by reporter assays, fluorescent microscopy, and chromatin immunoprecipitation (ChIP), respectively. Forced expression of HPP1 in colon cancer cell lines results in the upregulation of total and activated levels of STAT2. We have also determined that JAK1 and JAK2 are activated in response to HPP1 overexpression, and are necessary for subsequent STAT activation. Overexpression of HPP1 was associated with significant increases in STAT1:STAT1 ( p = 0.007) and STAT1:STAT2 ( p = 0.036) dimer formation, as well as subsequent nuclear translocation. By ChIP, binding of activated STAT1 and STAT2 to the interferon-signaling regulatory element promoter sites of the selected genes, protein kinase RNA-activated (PKR), IFI44, and OAS1 was demonstrated. STAT2 knockdown resulted in partial abrogation of HPP1s growth suppressive activity with increased proliferation ( p < 0.0001), reduced G1/G0 phase cell cycle fraction, and a restoration of growth potential in soft agar ( p < 0.01). Presumably as a consequence of upregulation of IFN signaling elements, HPP1 overexpression resulted in an acquisition of exogenous IFN sensitivity. Physiologic doses of IFN-a resulted in a significant reduction in proliferation ( p < 0.001) and increase in G1/G0 cell cycle arrest in HPP1 transfectants. STAT2 is necessary for HPP1-associated growth suppression, and mediates these effects through activation of IFN-a pathways. Given the interest in therapeutic targeting of oncogenic erbB proteins, further understanding of HPP1s role as a tumor suppressive EGF-like ligand is warranted.

TMEFF2 Promoter Hypermethylation Is an Unfavorable Prognostic Marker in Gliomas

“…Taken together, low TMEFF2 expression in gliomas may be due to the methylation regulation of its promoter. It was demonstrated that histone deacetylases as well as c-Myc, STAT1 and STAT3 may contribute independently to the transcriptional suppression of TMEFF2 in colon cancer, prostate cancer and gastric cancer [29][30][31][32]. However, more detailed studies need to be undertaken to better understand the regulatory mechanism of promoter methylation and transcription of TMEFF2 in gliomas.…”

TMEFF2 Promoter Hypermethylation Is an Unfavorable Prognostic Marker in Gliomas

“…Taken together, these results indicate that low TMEFF2 expression in gliomas may be due to methylation regulation of its promoter. It was demonstrated that histone deacetylases as well as c-Myc, STAT1 and STAT3 may contribute independently to the transcriptional suppression of TMEFF2 in colon cancer, prostate cancer and gastric cancer [29][30][31][32]. However, more detailed studies need to be performed to better understand the regulatory mechanism of TMEFF2 promoter methylation and transcription in gliomas.…”

TMEFF2: A Transmembrane Proteoglycan with Multifaceted Actions in Cancer and Disease

“…STAT phosphorylation is mediated by four kinases of the Janus family (JAK1, JAK2, JAK3 and TYK2), and TMEFF2 overexpression resulted in the upregulation of JAK1 and JAK2 but not the other two kinases. The HCT-116 cell line is resistant to interferon therapy (like several other cancers), and an interesting observation from this study [ 56 ] was that TMEFF2 overexpression marginally sensitized HCT-116 cells to INF-α but not INF-γ. In their latest study [ 68 ], David Shibata’s group observed the onco-suppressive and STAT1-inducing actions of TMEFF2 in HCT-116 cells to be dependent upon its proteolytic shedding (ADAM17 was identified as TMEFF2 shedase in HCT-116 cells).…”

“…The treatment of HCT-116 cells with STAT1 siRNA reversed TMEFF2-induced reduction of proliferation both on 2D and 3D cultures. In a subsequent study by the same group [ 56 ], it was reported that the TMEFF2 overexpression in HCT-116 cells led to upregulation of genes with both GAS and ISRE harbouring promoters (GAS to a higher degree than ISRE). Additionally, phosphorylation and subsequent nuclear localization of both STAT1 and STAT2 were observed in stably transfected TMEFF2 overexpressing HCT-116 cells.…”