Overexpression of the pp32r1 (ANP32C) oncogene or its functional mutant pp32r1Y140H confers enhanced resistance to FTY720 (Finguimod).

pp32r1 (ANP32C) is oncogenic and has been shown to be overexpressed in tumors of the breast, prostate, and pancreas. In this work we show that pp32 family proteins are able to bind to the sphingosine analog FTY720 (Finguimod). Molecular docking studies highlight that a conserved residue F136 is likely to be a key determinant of the FTY720 binding site on the pp32 leucine-rich repeat domain. Transduction of the renal carcinoma cell line ACHN or cervical cancer cell line HeLa with lentivirus expressing the oncogenic family member pp32r1 or a pp32r1Y140H functional mutant illustrated an enhanced resistance to FTY720 induced apoptosis. These findings highlight that certain cancers overexpressing pp32r1 or pp32r1 mutants are likely to demonstrate enhanced resistance to FTY720 treatment.

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7.

BACKGROUND: CD7 expression is found on ~30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7+) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation. METHODS: KG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7+, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCßII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA. RESULTS: SECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCßII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression. CONCLUSION: These findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis. GENERAL SIGNIFICANCE: This work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.

Dysregulation of cell cycle control caused by overexpression of the oncogene pp32r1 (ANP32C) and the Tyr>His mutant pp32r1Y140H.

The pp32 (ANP32A) gene acts as a tumor suppressor while its closely related homologue pp32r1 (ANP32C) is oncogenic and is overexpressed in breast, prostate and pancreatic tumors. The transduction of p53wt cell lines (ACHN and HeLa) with pp32r1 or pp32r1Y140H lentivirus increased the proliferation of p53wt cell lines compared to the untransduced control cells while transduction of the p53(R248W) MiaPaCa2 cell line had no effect. Cell cycle analysis of transduced ACHN cells by PI staining and BrdU incorporation illustrated a pronounced shift toward the S-phase of the cell cycle in cells overexpressing the pp32r1 and pp32r1Y140H proteins. Confocal microscopy and western blotting demonstrated that pp32r1 and the pp32r1Y140H mutant protein reside predominantly in the cytoplasm in constrast to pp32 which is a nuclear/cytoplasmic shuttling protein. To determine the effects of pp32r1 or pp32r1Y140H overexpression at the proteomic level we performed a comprehensive proteome analysis on ACHN, ACHN-pp32r1 and ACHN-pp32r1Y140H cell lysates using the isotope-coded protein label (ICPL) method. Among those proteins with >40% regulation were Macrophage Capping protein (CAPG) and Chromodomain Helicase DNA binding protein 4 (CHD4) proteins which were significantly upregulated by pp32r1 and pp32r1Y140H overexpression. This increase in CHD4 also appears to influence a number of cell cycle regulator genes including; p53, p21 and cyclinD1 as judged by western blotting. Silencing of CHD4 in ACHN-pp32r1Y140H cells using specific shRNA reverted the cell cycle dysregulation caused by pp32r1Y140H expression to that of the untransduced ACHN cell line, suggesting that CHD4 is the prominent effector of the pp32r1/pp32r1Y140H phenotype.

The T/NK cell co-stimulatory molecule SECTM1 is an IFN "early response gene" that is negatively regulated by LPS in human monocytic cells.

BACKGROUND: SECTM1 is a T/NK cell "co-stimulatory" molecule that is expressed in the peripheral blood by neutrophils and monocytes. METHODS: We used qRT-PCR to investigate the mRNA expression of SECTM1 in human monocytic cells after stimulation with interferons and LPS and confirmed the protein expression by flow cytometry. RESULTS: The kinetics of interferon induced SECTM1 mRNA expression in MM6 cells are time dependent occurring rapidly within 3h of stimulation and reaching a maximal level at ~6h for IFN-α and ~12h for IFN-ß and IFN-γ. Co-treatment of MM6 cells with IFN-γ and cycloheximide caused a superinduction of SECTM1 mRNA expression while cycloheximide alone had no effect, illustrating that de novo protein synthesis is not required for IFN-γ enhanced expression of SECTM1 mRNA, a characteristic of IFN early response genes. The kinetics of IFN induced SECTM1 mRNA expression in primary monocytes is comparable although it occurs much quicker with rapid induction by IFN-α, IFN-ß and IFN-γ and maximal levels reached in <6h. Human monocytic cells also displayed a pronounced negative regulation of SECTM1 mRNA expression by LPS, while at the protein level SECTM1 expression was also shown to be regulated by IFN and LPS. Bioinformatic analysis of the SECTM1 promoter region identified STAT1α/GAS, STAT3, ISRE, NFκB and putative p63 binding sites suggesting a complex transcriptional control. This tight regulation of SECTM1 gene expression and rapid upregulation highlights its relevance in the innate immune response. CONCLUSION: Human monocytes produce SECTM1 in response to interferon stimuli that is negatively regulated by LPS. GENERAL SIGNIFICANCE: The level of SECTM1 expression is likely to be a key factor in innate immune responses and in the immune tolerance of cancerous cells.